Formulations for protein therapeutics

Abstract

To provide compositions comprising protein therapeutics, including multispecific polypeptides and fusion proteins for intravenous administration.SOLUTION: A composition comprising a multispecific protein, a buffer, an excipient, and a surfactant, wherein (a) the multispecific protein is a dimer of two identical polypeptides, each polypeptide comprising, in amino to carboxyl terminal order or in carboxyl to amino terminal order, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a second binding domain, and (b) the buffer comprises or consists of succinate or a pharmaceutically acceptable salt or acid thereof.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 121,633, filed December 4, 2020, and U.S. Provisional Patent Application No. 62 / 960,562, filed January 13, 2020. Each of these provisional applications is incorporated herein in its entirety into this application. Field of Disclosure

[0002] The present disclosure relates to formulations for protein therapeutics. Specifically, the present disclosure relates to compositions comprising a bispecific or multispecific protein, a buffer, an excipient, and a surfactant. More specifically, the present disclosure relates to formulations for bispecific or multispecific proteins comprising a CD123-binding domain and a CD3-binding domain. The present disclosure also relates to clinical methods including dosing regimens for administering the protein therapeutic to a subject in need thereof.

[0003] Sequence Listing This application contains an electronically submitted Sequence Listing, which is incorporated herein by reference in its entirety. The Sequence Listing was recorded on January 13, 2021, is named APVO_061_02SeqList_ST25.txt, and is approximately 103 kilobytes in size. [Background technology]

[0004] One of the key steps in the successful development of protein therapeutics is the development of formulations that maintain the physical and chemical integrity of the protein during long-term storage, handling by medical professionals, and administration. Protein therapeutics administered intravenously (iv) are often stored frozen in concentrated solutions and diluted in the clinic before use. Furthermore, protein therapeutics such as bispecific and multispecific antibodies, especially those containing one or more scFv domains, can be prone to forming aggregates.

[0005] The development of protein formulations, particularly for administration by the intravenous route, requires careful consideration of many factors, including the properties of the protein, the formulation composition, the choice of diluent, storage temperature, infusion rate, exposure to light, etc. There is a need in the art for stable formulations for protein therapeutics, particularly for administration by the intravenous route.

[0006] Additionally, there is a need for dosing strategies that mitigate the risks associated with the effects of cytokine release in patients treated with bispecific and multispecific therapeutics (i.e., T cell engagers) that act through T cell engagement. This class of therapeutics includes bispecific therapeutics that target CD123 and CD3. The CD123 x CD3 bispecific antibody molecule, mAb14045 (Xencor), being evaluated in patients with relapsed or refractory acute myeloid leukemia and other CD123-expressing hematologic malignancies, was placed on partial clinical hold by the FDA in 2019 following two patient deaths in a phase 1 trial, including one death due to cytokine release syndrome (CRS).

[0007] Administration strategies designed to reduce the potential for serious effects of cytokine release, including cytokine release syndrome, may be ineffective for treatment. Thus, there remains a need for methods of delivering therapeutically effective amounts of T cell engagers (such as CD123 x CD3 therapeutics) to patients in a manner that reduces the risk of toxicity, including cytokine toxicity. Summary of the Invention

[0008] Described herein are compositions containing protein therapeutics, including multispecific polypeptides and fusion proteins, for intravenous administration. The compositions may include, for example, a multispecific protein, a buffer, an excipient, and a surfactant. In some embodiments, the compositions may include a multispecific protein, a succinate buffer, sucrose, and polysorbate 80. In some embodiments, the compositions are for intravenous or subcutaneous administration.

[0009] The present disclosure provides a multispecific polypeptide formulated with a succinate buffer and sucrose. In some embodiments, the multispecific polypeptide comprises two or more scFv binding domains. In some embodiments, the multispecific polypeptide forms a homodimer. In other embodiments, the multispecific polypeptide forms a heterodimer. In some embodiments, the multispecific polypeptide is selected from the group consisting of scFv-Fc-scFv (e.g., ADAPTIR®), quadroma, Kλ-body, dAb, diabody, TandAb, nanobody, DOCK-AND-LOCK® (DNL®), CrossMab Fab, CrossMab VH-VL, strand-exchange engineered domain body (SEEDbody), affibody, Fynomer, Kunitz domain, Albu-dab, two engineered Fv fragments with exchanged VHs (e.g., dual-affinity re-targeting molecule (DART)), scFv x The format is selected from the group consisting of scFv (e.g., BiTE), SVD-IG, Covx-body, peptibody, scFv-Ig, SVD-Ig, dAb-Ig, Knob-in-Holes, IgG1 antibody comprising matched mutation in the CH3 domain (e.g., duobody antibody), and triomab.

[0010] In some embodiments, the present disclosure provides a composition comprising a multispecific protein, a buffer, an excipient, and a surfactant, wherein the multispecific protein is a dimer of two identical polypeptides, each polypeptide comprising, in amino-terminal to carboxyl-terminal order or carboxyl-terminal to amino-terminal order, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a second binding domain; and the buffer comprises succinate or a pharmaceutically acceptable salt or acid thereof. In some embodiments, each polypeptide comprises a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 31.

[0011] In some embodiments, the composition comprises about 1 mM to about 10 mM succinate, or a pharmaceutically acceptable salt or acid thereof, hi some embodiments, the composition comprises about 5 mM succinate, or a pharmaceutically acceptable salt or acid thereof.

[0012] In some embodiments, the excipient comprises or consists of a sugar, such as sucrose. In some embodiments, the composition may comprise about 1% to about 12% weight / volume (w / v) sugar. In some embodiments, the composition comprises about 6.5% w / v sugar.

[0013] In some embodiments, the surfactant comprises or consists of polysorbate 80. In some embodiments, the composition comprises about 0.02% w / v polysorbate 80.

[0014] In some embodiments, the composition comprises about 0.1 mg / ml to about 10 mg / ml of multispecific protein. For example, the composition may comprise about 1 mg / ml to about 5 mg / ml of multispecific protein. In some embodiments, the composition comprises about 2 mg / ml of multispecific protein. In some embodiments, the composition comprises about 5 mM succinate, about 6.5% weight / volume (w / v) sucrose, and about 0.02% w / v polysorbate 80. In some embodiments, the composition has a pH of about 4.0 to about 5.5. In some embodiments, the composition has a pH of about 4.8.

[0015] In some embodiments, the immunoglobulin constant region is a human Fc domain. In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD.

[0016] In some embodiments, the first binding domain is a CD3-binding domain and the second binding domain is a tumor antigen-binding domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD3-binding domain, a hinge region, an immunoglobulin constant region, and a tumor antigen-binding domain. In some embodiments, the first domain is a tumor antigen-binding domain and the second binding domain is a CD3-binding domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a tumor antigen-binding domain, a hinge region, an immunoglobulin constant region, and a CD3-binding domain. In some embodiments, the tumor antigen-binding domain binds to CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0017] In some embodiments, the first binding domain is a 4-1-BB binding domain and the second binding domain is a tumor antigen binding domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a 4-1-BB binding domain, a hinge region, an immunoglobulin constant region, and a tumor antigen binding domain. In some embodiments, the first binding domain is a tumor antigen binding domain and the second binding domain is a 4-1-BB binding domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a tumor antigen binding domain, a hinge region, an immunoglobulin constant region, and a 4-1-BB binding domain. In some embodiments, the tumor antigen binding domain binds to CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0018] In some embodiments, at least one of the first binding domain and the second binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments, at least one of the first binding domain and the second binding domain is a single-chain variable fragment (scFv). In some embodiments, the light chain variable region of the scFv is carboxy-terminal to the heavy chain variable region of the scFv. In some embodiments, the light chain variable region of the scFv is amino-terminal to the heavy chain variable region of the scFv. In some embodiments, the scFv comprises a linker polypeptide. The linker polypeptide may be present, for example, between the light chain variable region and the heavy chain variable region of the scFv. In some embodiments, the linker polypeptide is a Gly4Ser (SEQ ID NO: 128) linker, for example, (Gly4Ser) n where n=1 to 5 (SEQ ID NO: 129).

[0019] In some embodiments, the tumor antigen-binding domain is an anti-CD123 scFv comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12; and an LCDR1 comprising SEQ ID NO: 13, an LCDR2 comprising SEQ ID NO: 14, and an LCDR3 comprising SEQ ID NO: 15. In some embodiments, the tumor antigen-binding domain is an anti-CD123 scFv comprising a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 136, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 134. In some embodiments, the tumor antigen-binding domain is an anti-CD123 scFv, wherein the scFv comprises a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 18.

[0020] In some embodiments, the CD3 binding domain is an anti-CD3 scFv comprising an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HCDR3 comprising SEQ ID NO: 21; and an LCDR1 comprising SEQ ID NO: 22, an LCDR2 comprising SEQ ID NO: 23, and an LCDR3 comprising SEQ ID NO: 24. In some embodiments, the CD3 antigen binding domain is an anti-CD3 scFv comprising a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 384. In some embodiments, the CD3 binding domain is an anti-CD3 scFv comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27.

[0021] In some embodiments, the immunoglobulin constant region comprises one or more mutations to reduce / prevent FcγR binding, ADCC activity, and / or CDC activity. In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A according to the EU numbering system. In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, E318A, K320A, and K322A according to the EU numbering system. In some embodiments, the immunoglobulin comprises the sequence of SEQ ID NO: 131, or a sequence at least 90%, at least 95% identical thereto.

[0022] In some embodiments, the hinge region is derived from an immunoglobulin hinge region.

[0023] In some embodiments, each polypeptide comprises an Fc binding domain linker between the immunoglobulin constant region and the second binding domain. In some embodiments, the Fc binding domain linker comprises the sequence Gly4Ser (SEQ ID NO: 128). In some embodiments, the Fc binding domain linker has the formula (Gly4Ser) n (wherein n=1 to 5) (SEQ ID NO: 129).

[0024] In some embodiments, the composition substantially prevents degradation of the multispecific protein. In some embodiments, the composition is substantially stable at 4° C. for at least 1 year. In some embodiments, the composition substantially resists aggregate formation of the multispecific protein.

[0025] The present disclosure also provides a composition comprising a fusion protein, a buffer, an excipient, and a surfactant, wherein the fusion protein is a dimer of two identical polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus, (i) a first binding domain that specifically binds to CD123, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a second binding domain that specifically binds to CD3; and the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

[0026] The present disclosure also provides a composition comprising a fusion protein, a buffer, an excipient, and a surfactant, wherein the fusion protein comprises a first binding domain that specifically binds to CD123 and a second binding domain that specifically binds to CD3; and the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

[0027] The present disclosure also provides a composition comprising a fusion protein, a buffer, an excipient, and a surfactant, wherein the fusion protein comprises (i) a first binding domain that specifically binds to CD123, the binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15, and (ii) a second binding domain that specifically binds to CD3, the binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO: 24; and the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

[0028] The present disclosure also provides a composition comprising a fusion protein, about 5 mM succinate, about 6.5 weight / volume (w / v)% sucrose, and about 0.02 w / v% polysorbate 80, wherein the fusion protein comprises: (i) a first binding domain that specifically binds to CD123, the first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and and (ii) a second binding domain that specifically binds to CD3, the second binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO: 24.

[0029] The present disclosure also provides a composition comprising a fusion protein, a buffer, an excipient, and a surfactant, wherein the fusion protein comprises (i) a first binding domain that specifically binds to CD123, the binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising SEQ ID NO: 136; and an immunoglobulin light chain variable region (VL) comprising SEQ ID NO: 134, and (ii) a second binding domain that specifically binds to CD3, the binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising SEQ ID NO: 383 or 387; and an immunoglobulin light chain variable region (VL) comprising SEQ ID NO: 384; and the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

[0030] The present disclosure also provides a composition comprising a fusion protein, a buffer, an excipient, and a surfactant, wherein the fusion protein comprises (i) a first binding domain that specifically binds to CD123, the binding domain comprising SEQ ID NO: 18; and (ii) a second binding domain that specifically binds to CD3, the binding domain comprising SEQ ID NO: 27; and the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

[0031] The present disclosure also provides a composition comprising a fusion protein, a buffer, an excipient, and a surfactant, wherein the fusion protein is a dimer of two identical polypeptides, each polypeptide comprising, in amino-terminal to carboxyl-terminal order, or in carboxyl-terminal to amino-terminal order: (i) a first binding domain that specifically binds to CD123, the first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15. domain, (ii) a hinge region of SEQ ID NO: 47, (iii) an immunoglobulin constant region of SEQ ID NO: 33, (iv) an Fc-binding domain linker of SEQ ID NO: 132, and (v) a second binding domain that specifically binds to CD3, the binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO: 24; the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

[0032] The present disclosure also provides a composition comprising a fusion protein, about 5 mM succinate, about 6.5 weight / volume (w / v) % sucrose, and about 0.02 w / v % polysorbate 80, wherein the fusion protein is a dimer of two identical polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus: (i) a first binding domain that specifically binds to CD123, the first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an HCDR3 of SEQ ID NO: 15. (ii) a binding domain comprising an immunoglobulin light chain variable region (VL) comprising LCDR3; (ii) a hinge region of SEQ ID NO: 47; (iii) an immunoglobulin constant region of SEQ ID NO: 33; (iv) an Fc binding domain linker of SEQ ID NO: 132; and (v) a second binding domain that specifically binds to CD3, the binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO: 24.

[0033] The present disclosure also provides a composition comprising a fusion protein, about 5 mM succinate, about 6.5% weight / volume (w / v) sucrose, and about 0.02% w / v polysorbate 80, wherein the fusion protein comprises or consists of SEQ ID NO: 31, the composition comprises about 2 mg / ml of the fusion protein, and the composition has a pH of about 4.8.

[0034] The present disclosure further provides a method for inhibiting the growth of psoriatic plaques in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure.

[0035] The present disclosure further provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure. The cancer may be, for example, a hematological malignancy. For example, the cancer may be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, B-cell acute lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML).

[0036] Also provided is the use of a composition of the present disclosure for the treatment of cancer in a subject. Also provided is the use of a composition of the present disclosure in the manufacture of a medicament for the treatment of cancer. For example, a composition of the present disclosure may be used for the treatment of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). In some embodiments, a composition of the present disclosure may be used for the treatment of high-risk or high-grade MDS. A composition comprising a multispecific protein comprising a CD123-binding domain and a CD3-binding domain may be administered to a subject via IV infusion at a weekly dose of about 0.3, about 1, about 3, about 6, about 9, about 12, about 18, about 20, about 24, about 30, about 36, about 50, about 48, about 60, about 75, or about 100 μg. To reduce the risk of adverse events, the initial dose may be administered to the patient via IV infusion over several hours, for example, about 20-24 hours. In some embodiments, a first dose of the composition is administered over a period of about 20 to 24 hours, a second dose is administered over a period of about 8 hours, a third dose is administered over a period of about 6 hours, and a fourth dose and subsequent doses are administered over a period of about 4 hours. The composition may also be administered to a subject by continuous IV infusion, e.g., continuous IV infusion for up to 72 hours.

[0037] A method for treating a patient in need of treatment can include intravenously administering a multispecific protein comprising a CD123-binding domain and a CD3-binding domain to the patient, with the dosage being increased weekly for at least the first two or first three doses. For example, the composition can be delivered to the patient by IV infusion according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks: 12 μg. In some embodiments, the composition can be administered intravenously to the patient according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks: 18 μg. In some embodiments, the highest dosage administered to the patient is about 24 μg, about 36 μg, about 48 μg, about 60 μg, or about 100 μg. In some embodiments, the highest dose administered to a patient ranges from about 100 μg to about 130 μg.

[0038] In some embodiments, the composition is administered to the patient in a treatment cycle lasting about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or longer. In some embodiments, the composition may be administered to the patient for two or more treatment cycles, such as 2, 3, 4, 5, 6, 7, 8, or longer treatment cycles. In some embodiments, the treatment cycle lasts 4 weeks and may be repeated for up to 6 cycles. In some embodiments, the treatment cycle may be repeated for up to 36 cycles.

[0039] In some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: week 1 dose: 6 μg; and week 2 and subsequent weeks dose: 9 μg, and in some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: week 1 dose: 9 μg; and week 2 and subsequent weeks dose: 12 μg. In some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: week 1 dose: 12 μg; and week 2 and subsequent weeks dose: 18 μg.

[0040] In some embodiments, a method for treating a patient in need thereof comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 8, 15, and 22. In some embodiments, 6 μg is administered on day 1, 9 μg on day 8, 12 μg on day 15, and 12 μg on day 22. In some embodiments, 6 μg is administered on day 1, 9 μg on day 8, 12 μg on day 15, and 18 μg on day 22. In some embodiments, 6 μg is administered on day 1, 9 μg on day 8, 9 μg on day 15, and 9 μg on day 22. In some embodiments, 9 μg is administered on day 1, 12 μg on day 8, 12 μg on day 15, and 12 μg on day 22. In some embodiments, 12 μg is administered on day 1, 18 μg on day 8, 18 μg on day 15, and 18 μg on day 22. In some embodiments, patients treated according to the methods of the present disclosure exhibit a decrease in bone marrow blast percentage, and in some embodiments, patients exhibit a decrease in absolute blast count in the blood. In some embodiments, treatment results in a decrease in the patient's blast levels in the blood of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 45%, at least 50%, or more, compared to the patient's levels immediately before treatment.

[0041] In some embodiments, a method for treating a patient in need of treatment comprises administering to the patient a composition comprising a multispecific protein comprising a CD123-binding domain and a CD3-binding domain for a 28-day cycle. In some embodiments, the composition is administered to the patient once, twice, three times, or four times per week for the 28-day cycle. In some embodiments, the dosage is increased over the 28-day cycle. In some embodiments, the dosage is decreased over the 28-day cycle. In some embodiments, the dosage is increased every week for the 28-day cycle. In some embodiments, the dosage is decreased every week for the 28-day cycle.

[0042] In some embodiments, a method for treating a patient in need of treatment comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 8, 15, and 22 of a first 28-day cycle and on days 1, 8, 15, and 22 of at least one additional 28-day cycle. In some embodiments, the dose administered on day 22 of the first 28-day cycle is the same as the dose administered on days 1, 8, 15, and 22 of at least one additional 28-day cycle. In some embodiments, the patient is treated for two, three, four, five, six, seven, eight, or more additional 28-day cycles, with administration of the composition on days 1, 8, 15, and 22 of each cycle.

[0043] In some embodiments, methods for treating a patient in need of treatment comprise administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 2, 3, 4, 8, 11, 15, and 22 of an initial 28-day cycle. In some embodiments, methods for treating a patient in need of treatment comprise administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 2, 3, 4, 8, 11, 15, and 22 of an initial 28-day cycle, and thereafter on days 1, 8, 15, and 22 of at least one additional 28-day cycle. In some embodiments, the patient is treated for two, three, four, five, six, seven, eight, or more additional 28-day cycles, with administration of the composition occurring on days 1, 8, 15, and 22 of each cycle.

[0044] In some embodiments, methods for treating a patient in need of treatment comprise administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 2, 3, 4, 8, 11, 15, 18, 22, and 25 of an initial 28-day cycle. In some embodiments, methods for treating a patient in need of treatment comprise administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 2, 3, 4, 8, 11, 15, 18, 22, and 25 of an initial 28-day cycle, and thereafter on days 1, 8, 15, and 22 of at least one additional 28-day cycle. In some embodiments, the patient is treated for two, three, four, five, six, seven, eight, or more additional 28-day cycles, with administration of the composition occurring on days 1, 8, 15, and 22 of each cycle.

[0045] In some embodiments, a method for treating a patient in need thereof comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain during an initial 28-day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 9 μg of the multispecific protein is administered on day 2, 12 μg of the multispecific protein is administered on day 3, 18 μg of the multispecific protein is administered on day 4, 18 μg of the multispecific protein is administered on day 8, 18 μg of the multispecific protein is administered on day 11, 36 μg of the multispecific protein is administered on day 15, and 36 μg of the multispecific protein is administered on day 22 of the initial 28-day cycle. In some embodiments, the method further comprises administering to the patient the multispecific protein for at least one additional 28-day cycle, wherein 36 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

[0046] In some embodiments, a method for treating a patient in need thereof comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain during an initial 28-day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 12 μg of the multispecific protein is administered on day 2, 18 μg of the multispecific protein is administered on day 3, 24 μg of the multispecific protein is administered on day 4, 24 μg of the multispecific protein is administered on day 8, 24 μg of the multispecific protein is administered on day 11, 48 μg of the multispecific protein is administered on day 15, and 48 μg of the multispecific protein is administered on day 22 of the initial 28-day cycle. In some embodiments, the method further comprises administering the multispecific protein to the patient for at least one additional 28-day cycle, wherein 48 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

[0047] In some embodiments, a method for treating a patient in need thereof comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain during an initial 28-day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 12 μg of the multispecific protein is administered on day 2, 24 μg of the multispecific protein is administered on day 3, 36 μg of the multispecific protein is administered on day 4, 36 μg of the multispecific protein is administered on day 8, 36 μg of the multispecific protein is administered on day 11, 60 μg of the multispecific protein is administered on day 15, and 60 μg of the multispecific protein is administered on day 22 of the initial 28-day cycle. In some embodiments, the method further comprises administering to the patient the multispecific protein for at least one additional 28-day cycle, wherein 60 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

[0048] In some embodiments, a method for treating a patient in need thereof comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain during an initial 28-day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 12 μg of the multispecific protein is administered on day 2, 24 μg of the multispecific protein is administered on day 3, 36 μg of the multispecific protein is administered on day 4, 48 μg of the multispecific protein is administered on day 8, 48 μg of the multispecific protein is administered on day 11, 100 μg of the multispecific protein is administered on day 15, and 100 μg of the multispecific protein is administered on day 22 of the initial 28-day cycle. In some embodiments, the method further comprises administering the multispecific protein to the patient for at least one additional 28-day cycle, wherein 100 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

[0049] In some embodiments, the maximum dose administered to a patient in any one of the above-described schemes is increased by about 5% to about 40%, e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%. In these embodiments, this increased dose may be initially administered to a patient on the first day that the maximum dose was previously administered in the above-described scheme. [Brief explanation of the drawings]

[0050] [Figure 1A] 1A and 1B are schematic diagrams showing the structure of exemplary therapeutic proteins for use in the compositions and methods of the present disclosure. Figure 1A shows a homodimeric protein comprising two identical polypeptides, each comprising a CD3-binding domain and an Fc domain. Figure 1B shows a homodimeric protein comprising two identical polypeptides, each comprising a tumor-binding domain (e.g., a CD123-binding domain), an Fc domain, and a CD3-binding domain. An exemplary CD123 x CD3 bispecific therapeutic protein is referred to herein as TRI130. [Figure 1B] 1A and 1B are schematic diagrams showing the structure of exemplary therapeutic proteins for use in the compositions and methods of the present disclosure. Figure 1A shows a homodimeric protein comprising two identical polypeptides, each comprising a CD3-binding domain and an Fc domain. Figure 1B shows a homodimeric protein comprising two identical polypeptides, each comprising a tumor-binding domain (e.g., a CD123-binding domain), an Fc domain, and a CD3-binding domain. An exemplary CD123 x CD3 bispecific therapeutic protein is referred to herein as TRI130. [Figure 2]Schematic showing the design of a Phase 1 / 1b dose-escalation clinical trial in which TRI130 is administered to patients with relapsed or refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). [Figure 3A] Figure 1 shows the percentage of blasts in bone marrow aspirates plotted over time for patients from the Phase 1 / 1b study described in Figure 2 and Example 3. Data are graphed for patients in the cohort who received a maximum dose of 12 μg or greater. N = 14 patients evaluable for change from baseline. Cohorts 6a and 6b were studied with different escalating dosing schedules, as shown in Table 10. [Figure 3B] Figure 1 shows the percentage of blasts in bone marrow aspirates plotted over time for patients from the Phase 1 / 1b study described in Figure 2 and Example 3. Data are graphed for patients in the cohort who received a maximum dose of 12 μg or greater. N = 14 patients evaluable for change from baseline. Cohorts 6a and 6b were studied with different escalating dosing schedules, as shown in Table 10. [Figure 3C] Figure 1 shows the percentage of blasts in bone marrow aspirates plotted over time for patients from the Phase 1 / 1b study described in Figure 2 and Example 3. Data are graphed for patients in the cohort who received a maximum dose of 12 μg or greater. N = 14 patients evaluable for change from baseline. Cohorts 6a and 6b were studied with different escalating dosing schedules, as shown in Table 10. [Figure 3D] Figure 1 shows the percentage of blasts in bone marrow aspirates plotted over time for patients from the Phase 1 / 1b study described in Figure 2 and Example 3. Data are graphed for patients in the cohort who received a maximum dose of 12 μg or greater. N = 14 patients evaluable for change from baseline. Cohorts 6a and 6b were studied with different escalating dosing schedules, as shown in Table 10. [Figure 4A]Serum concentrations of interleukin-6 (IL-6, Figure 4A), interleukin-10 (IL-10, Figure 4B), interferon gamma (IFNγ, Figure 4C), and tumor necrosis factor alpha (TNFα, Figure 4D) in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) in the Phase 1 / 1b study described in Figure 2 and Example 3 are shown. Scheduled blood draws were performed at the time of administration of the first highest scheduled dose for each patient. Peak cytokine levels observed in unscheduled draws during IRR / CRS events are shown for comparison (four patients, N=6 events). [Figure 4B] Serum concentrations of interleukin-6 (IL-6, Figure 4A), interleukin-10 (IL-10, Figure 4B), interferon gamma (IFNγ, Figure 4C), and tumor necrosis factor alpha (TNFα, Figure 4D) in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) in the Phase 1 / 1b study described in Figure 2 and Example 3 are shown. Scheduled blood draws were performed at the time of administration of the first highest scheduled dose for each patient. Peak cytokine levels observed in unscheduled draws during IRR / CRS events are shown for comparison (four patients, N=6 events). [Figure 4C] Serum concentrations of interleukin-6 (IL-6, Figure 4A), interleukin-10 (IL-10, Figure 4B), interferon gamma (IFNγ, Figure 4C), and tumor necrosis factor alpha (TNFα, Figure 4D) in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) in the Phase 1 / 1b study described in Figure 2 and Example 3 are shown. Scheduled blood draws were performed at the time of administration of the first highest scheduled dose for each patient. Peak cytokine levels observed in unscheduled draws during IRR / CRS events are shown for comparison (four patients, N=6 events). [Figure 4D]Serum concentrations of interleukin-6 (IL-6, Figure 4A), interleukin-10 (IL-10, Figure 4B), interferon gamma (IFNγ, Figure 4C), and tumor necrosis factor alpha (TNFα, Figure 4D) in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) in the Phase 1 / 1b study described in Figure 2 and Example 3 are shown. Scheduled blood draws were performed at the time of administration of the first highest scheduled dose for each patient. Peak cytokine levels observed in unscheduled draws during IRR / CRS events are shown for comparison (four patients, N=6 events). DETAILED DESCRIPTION OF THE INVENTION

[0051] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, including but not limited to patent applications, papers, books, and articles, cited herein are hereby expressly incorporated by reference in their entirety for all purposes. In the event that one or more incorporated documents or portions of documents define a term that contradicts the definition of that term in this application, the definition set forth in this application shall prevail. However, any reference to references, papers, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0052] As used herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the stated range and fractions thereof, as appropriate (such as tenths and hundredths of integers), unless otherwise specified. As used herein, the terms "a" and "an" shall be understood to mean "one or more" of the listed components unless otherwise indicated. The use of a disjunctive conjunction (e.g., "or") shall be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably. Additionally, it should be understood that polypeptides containing various combinations of components (e.g., domains or regions) and substituents described herein are disclosed by the present application to the same extent as if each polypeptide were described individually. Thus, selection of specific components of individual polypeptides is within the scope of this disclosure.

[0053] definition The term "about" immediately preceding a numerical value means up to ±10% of the numerical value. For example, "about 40" means ±10% of 40 (i.e., 36-44), e.g., 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%, up to ±1%, less than ±1%, or other values ​​or ranges.

[0054] As used herein, the term "substantially" has its ordinary meaning as used in the art. For example, "substantially" can mean "significantly," "considerably," "largely," "mostly," or "essentially." In some embodiments, "substantially" can mean at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0055] The term "CD123" can refer to any isoform of CD123, also known as cluster of differentiation 123, interleukin-3 receptor alpha chain, and IL3RA. CD123 binds to the beta chain of the interleukin-3 receptor to form a receptor. CD123 is a type I transmembrane glycoprotein with an extracellular domain containing a predicted immunoglobulin-like domain and two FnIII domains. The CD123-binding domain of the present disclosure binds to the extracellular domain of CD123. CD123 is also known as the alpha chain of the human interleukin-3 (IL-3) receptor. CD123 is a type I transmembrane glycoprotein and a member of the cytokine receptor superfamily. The interleukin-3 receptor is a heterodimer formed by CD123 and the beta chain (CD131). IL-3 binds to CD123, and signaling is provided by CD131. IL-3 regulates the function and production of hematopoietic and immune cells and stimulates endothelial cell proliferation (Testa et al., Biomark Res. 2:4 (2014)).

[0056] CD123 is overexpressed in many hematological malignancies, including acute myeloid leukemia (AML), B-lymphoid leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), and a subset of hairy cell leukemia. While most AML patients respond well to initial therapy, the majority of AML patients are eventually diagnosed with relapsed or refractory disease (Ramos et al., J. Clin. Med. 4:665-695 (2015)). There is a need for CD123-targeting molecules with increased efficiency and efficacy, reduced adverse effects, and that can be used to treat disorders associated with CD123 dysregulation.

[0057] "CD3" is known in the art as a six-chain multiprotein complex (see, e.g., Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999), which is a subunit of the T cell receptor complex. In mammals, the CD3 subunit of the T cell receptor complex is a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows these chains to bind to positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif, or ITAM, known as an immunoreceptor tyrosine-based activation motif, while each CD3ζ chain has three ITAMs. ITAMs are thought to be important for the signaling capacity of the TCR complex. The CD3 used in this disclosure can be derived from a variety of animal species, including humans, monkeys, mice, rats, or other mammals.

[0058] "Cytokine release" or "cytokine storm" or "infusion reaction" refers to the release of cytokines from T cells. When cytokines are released into the circulation, systemic symptoms such as fever, nausea, chills, hypotension, tachycardia, asthenia, headache, rash, irritated throat, and difficulty breathing can occur. Some patients may experience severe, life-threatening reactions due to the massive release of cytokines. "Reduced" cytokine release refers to a reduction in the release of at least one cytokine (e.g., IFNγ, TNFα, IL-6, IL-2, IL-8, IL-10, IL-17, GM-CSF, IL-4, IL-12, IL-13, or IL-1β) following administration of a bispecific molecule disclosed herein compared to an OKT3 antibody (an antibody that binds to CD3) or another CD3-binding bispecific molecule. Reduced cytokine release can be measured using in vitro or in vivo assays.

[0059] As used herein, the term "step dosing" or "stepped dosing," or similar terms, refers to a dosing regimen in which a multispecific polypeptide described herein is administered to a patient on at least a first and a second dosing day, and the dose administered to the patient is held constant or increased between the first and second dosing days. For example, in some step dosing regimens, a patient may receive a first, second, third, and fourth dose, each administered on a different dosing day, with the second dose being higher than the first dose. The third dose may be higher than or the same as the second dose. The fourth dose may be higher than or the same as the third dose. In some embodiments, if a patient has an adverse reaction to a particular dose, subsequent doses may be reduced.

[0060] As used herein, the term "binding domain" or "binding region" refers to a domain, region, portion, or site of a protein, polypeptide, oligopeptide, peptide, antibody, or antibody-derived binding domain, receptor, or ligand capable of specifically recognizing and binding to a target molecule, such as an antigen, ligand, receptor, substrate, or inhibitor. Exemplary binding domains include antibody and antibody-like proteins or domains, antibody heavy and light chain variable regions, and single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab), receptor ectodomains, and ligands (e.g., cytokines, chemokines). In certain embodiments, a binding domain comprises or consists of an antigen-binding site (e.g., variable heavy and variable light chain sequences or three light chain complementarity-determining regions (CDRs) and three heavy chain CDRs from an antibody arranged in alternating framework regions (FRs) (e.g., human FRs, optionally containing one or more amino acid substitutions)). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, phage display library screening, and Biacore® interaction analysis.

[0061] A binding domain or protein containing a binding domain is one that is 5 M -1 or greater affinity or K a A binding domain "specifically binds" a target if it binds the target with an affinity (i.e., an equilibrium binding constant in the units of 1 / M of the specific binding interaction) but does not significantly bind other components present in the test sample. Binding domains can be classified as "high affinity" and "low affinity" binding domains. A "high affinity" binding domain has an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 1012 M -1 , or at least 10 13 M -1 K a A "low affinity" binding domain refers to a binding domain having at most 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 K a Alternatively, affinity refers to the equilibrium dissociation constant (K) of a particular binding interaction, with units M. d ) (e.g., 10 -5 M~10 -13 M, or about 500 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM). The affinity of binding domain polypeptides and single chain polypeptides according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., (1949) Ann. N.Y. Acad. Sci. 51:660, and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0062] As used herein, " conservative substitution " is clearly understood in the art as the substitution of one amino acid with another amino acid that has similar properties.Exemplary conservative substitutions [conservative substitutions] are well known in the art (see, for example, International Publication No. 97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p.8).In certain embodiments, conservative substitution comprises the substitution of leucine with serine.

[0063] As used herein, the term "derivative" refers to the modification of one or more amino acid residues of a peptide by chemical or biological means, such as, for example, glycosylation, alkylation, acylation, ester formation, or amide formation, with or without the use of an enzyme.

[0064] As used herein, a polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, a polypeptide or amino acid sequence derived from a particular sequence (sometimes referred to as the "starting" or "parent" or "parental" sequence) has an amino acid sequence that is essentially identical to the parent sequence or a portion thereof, where the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or at least 50-150 amino acids, or is otherwise identifiable to one of skill in the art as having its origin in the parent sequence. For example, a binding domain can be derived from an antibody, e.g., Fab, F(ab')2, Fab', scFv, single-domain antibody (sdAb), etc.

[0065] A polypeptide derived from another polypeptide may have one or more mutations or changes, such as one or more amino acid residues substituted with another amino acid residue or one or more amino acid insertions or deletions, compared to the parent polypeptide. In such embodiments, a polypeptide containing one or more mutations or changes from the parent polypeptide is referred to as a "variant." As used herein, the term "variant" or "variants" refers to a polynucleotide or polypeptide that has a sequence that differs from that of a reference polynucleotide or polypeptide but retains its essential properties. Typically, a variant polynucleotide or polypeptide sequence will be highly similar to, and in many regions identical to, the reference polynucleotide or polypeptide overall. For example, a variant polynucleotide or polypeptide may exhibit at least about 70%, at least about 80%, 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 compared to an active portion or full length of the reference polynucleotide or polypeptide. Polypeptides may contain amino acid sequences that are not naturally occurring. Such variations necessarily have less than 100% sequence identity or similarity with the parent polypeptide. In one embodiment, a variant has less than about 60% to 100% amino acid sequence identity or similarity with the amino acid sequence of the parent polypeptide. In other embodiments, a variant has less than about 75% to 100%, less than about 80% to 100%, less than about 85% to 100%, less than about 90% to 100%, or less than about 95% to 100% amino acid sequence identity or similarity with the amino acid sequence of the parent polypeptide.

[0066] As used herein, the term "sequence identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. If a position in one sequence is occupied by the same nucleic acid base or amino acid residue as the corresponding position in the comparison sequence, the sequences are said to be "identical" at that position. The percentage of sequence identity is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs, obtaining the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of sequence identity. The percentage of sequence identity is determined by comparing the two best-aligned sequences over the comparison window. The comparison window for polynucleotide sequences can be, for example, at least or more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleic acids in length. The comparison window for polypeptide sequences can be, for example, at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 300, or more amino acids in length.To optimally align sequences for comparison, a portion of the polynucleotide or polypeptide sequence in the comparison window can contain additions or deletions called gaps, while the reference sequence is kept constant.Optimal alignment is the alignment that generates the maximum possible number of "identical" positions between the reference sequence and the comparison sequence, even if there are gaps.The percentage "sequence identity" between two sequences can be determined using the program version "BLAST 2 Sequences," available from the National Center for Biotechnology Information as of September 1, 2004, which incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST 2 Sequences," the default parameters as of September 1, 2004, including word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expectation value (10), and any other desired parameters can be used, including, but not limited to, matrix options. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantial sequence identity" if the two sequences have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with each other.

[0067] As used herein, unless otherwise specified, amino acid residue positions in the variable region of an immunoglobulin molecule are numbered according to the IMGT numbering convention (Brochet, X, et al, Nucl. Acids Res. (2008) 36, W503-508), and amino acid residue positions in the constant region of an immunoglobulin molecule are numbered according to the EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). Other numbering conventions are known in the art (e.g., Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)).

[0068] As used herein, the term "dimer" refers to a biological entity consisting of two subunits bound to each other through one or more forms of intramolecular forces, including covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonds, and hydrophobic interactions), and that is stable under appropriate conditions (e.g., under physiological conditions, in aqueous solutions suitable for recombinant protein expression, purification, and / or storage, or under conditions for non-denaturing and / or non-reducing electrophoresis). As used herein, a "heterodimer" or "heterodimeric protein" refers to a dimer formed from two different polypeptides. Heterodimers do not include antibodies formed from four polypeptides (i.e., two light chains and two heavy chains). As used herein, a "homodimer" or "homodimeric protein" refers to a dimer formed from two identical polypeptides. All disclosures of polypeptides, including properties and activities (such as binding and RTCC), should be understood to include the dimeric form as well as other multimeric forms of the polypeptide.

[0069] When a polypeptide of the present disclosure is in a dimeric form (i.e., a dimeric protein), it contains two binding sites at the amino terminus and two binding sites at the carboxyl terminus. The binding domain is therefore considered to be bivalent (i.e., two binding moieties at each end) when the single-chain polypeptide is dimerized.

[0070] "Immunoglobulin constant region" or "constant region" is a term defined herein to refer to a peptide or polypeptide sequence corresponding to or derived from part or all of one or more constant domains of an immunoglobulin. In certain embodiments, the constant region comprises IgG CH2 and CH3 domains, e.g., IgG1 CH2 and CH3 domains. In certain embodiments, the constant region does not comprise a CH1 domain. In certain embodiments, the constant domains comprising the constant region are human. In some embodiments, the constant region of the fusion proteins of the present disclosure lacks or has minimal effector function, but retains the ability to bind to some Fc receptors, such as the neonatal Fc receptor (FcRn), and retains a relatively long in vivo half-life. For example, the constant region of the fusion proteins of the present disclosure does not result in or substantially reduces the induction of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement activation, and / or complement-dependent cytotoxicity (CDC). In other variations, fusion proteins of the present disclosure comprise a constant domain that retains one or more effector functions, such as one or both of ADCC and CDC. In certain embodiments, a binding domain of the present disclosure is fused to a human IgG1 constant region, where the IgG1 constant region has one or more of the following mutated amino acids: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamic acid at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (EU numbering). For example, any one or more of these amino acids can be changed to alanine. In a further embodiment, the IgG1 Fc domain has each of L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to alanine (i.e., L234A, L235A, G237A, E318A, K320A, and K322A, respectively), and optionally also has an N297A mutation (i.e., essentially eliminating glycosylation of the CH2 domain).

[0071] The term "light chain variable region" ("light chain variable domain" or "V L ") and a "heavy chain variable region" (also called a "heavy chain variable domain" or "V H ") refer to the variable binding regions from the antibody light and heavy chains, respectively. The variable binding regions are composed of separate, distinct subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). In one embodiment, the FRs are humanized. The term "CL" refers to an "immunoglobulin light chain constant region" or "light chain constant region," i.e., the constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or "heavy chain constant region," which can be further divided into CH1, CH2, and CH3 domains (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM) depending on the antibody isotype. "Fab" (Fragment Antigen Binding) is the portion of an antibody that binds to an antigen and comprises the variable region and CH1 domain of the heavy chain linked to the light chain via an interchain disulfide bond.

[0072] As used herein, the term "linker" generally refers to a short polypeptide sequence connecting two subdomains of a polypeptide. Non-limiting examples of linkers include flexible linkers containing glycine-serine repeats and linkers derived from (a) the interdomain region of a transmembrane protein (e.g., a type I transmembrane protein); or (b) an immunoglobulin hinge. In some embodiments, the linker provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. In certain embodiments, the linker consists of 5 to about 35 amino acids, e.g., about 15 to about 25 amino acids. As used herein, the phrase "linker between CH3 and CH1 or CL" refers to one or more amino acid residues (e.g., about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8-10, about 9-10, about 8-12, about 9-12, or about 10-12) between the C-terminus of the CH3 domain (e.g., wild-type CH3 or mutated CH3) and the N-terminus of the CH1 or CL domain (e.g., Cκ).

[0073] In some embodiments, the linker optionally comprises: (1) a single chain F V V in (scFv) H and V LThe term "Fc binding domain linker" may refer to (1) the polypeptide region between a first binding domain and a second binding domain in a multispecific polypeptide comprising two binding domains, or (2) the polypeptide region between a first binding domain and a second binding domain in a multispecific polypeptide comprising two binding domains. In the latter example, a linker connects two or more binding domains, and such a linker is referred to herein as an "Fc binding domain linker." In some embodiments, an Fc binding domain linker can connect or join two or more binding domains, resulting in a construct comprising the following structure: binding domain-Fc binding domain linker-binding domain. In some embodiments, the multispecific polypeptides described herein comprise, in order from amino terminus to carboxyl terminus, (i) a first binding domain, (ii) an Fc binding domain linker, and (iii) a second binding domain. In some embodiments, the multispecific polypeptides comprise, in order from amino terminus to carboxyl terminus, (i) a second binding domain, (ii) an Fc binding domain linker, and (iii) a first binding domain. In some embodiments, an Fc binding domain linker may join or link two or more binding domains by linking at least one binding domain to a non-binding domain polypeptide, such as an immunoglobulin Fc domain (i.e., a polypeptide comprising the structure: Ig hinge-Ig constant region). In such embodiments, the resulting construct may comprise the following structure: binding domain-Fc domain-Fc binding domain linker-binding domain. In some embodiments, the multispecific polypeptides described herein comprise, in order from amino terminus to carboxyl terminus, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) an Fc binding domain linker, and (v) a second binding domain. In some embodiments, the multispecific polypeptides comprise, in order from amino terminus to carboxyl terminus, (i) a second binding domain, (ii) an Fc binding domain linker, (iii) an immunoglobulin constant region, (iv) a hinge region, and (v) a first binding domain.The polypeptide region between the immunoglobulin constant region and the second binding domain in a multispecific polypeptide comprising two binding domains (e.g., an Fc binding domain linker) may also be referred to as a "carboxyl-terminal linker" or an "amino-terminal linker," depending on the orientation of the domains within the multispecific polypeptide. Non-limiting examples of linkers are provided in Table 1.

[0074] In some embodiments, "hinge" or "hinge region" refers to a polypeptide derived from an immunoglobulin hinge region and is located between the binding domain and the immunoglobulin constant region in a polypeptide described herein. A "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences inserted between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, the wild-type immunoglobulin hinge region sequence is human and may comprise a human IgG hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region).

[0075] "Modified immunoglobulin hinge region" or "variant immunoglobulin hinge region" refers to a hinge region that has one or more mutations, substitutions, insertions, or deletions compared to the corresponding parent wild-type immunoglobulin hinge region. In certain embodiments, the modified immunoglobulin hinge region is at least about 70% identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% identical) to a wild-type immunoglobulin hinge region. In certain embodiments, the modified immunoglobulin hinge region is a fragment of a wild-type immunoglobulin hinge region having a length of about 5 amino acids (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more amino acids) up to about 120 amino acids (e.g., about 10 to about 40 amino acids, or about 15 to about 30 amino acids, or about 15 to about 20 amino acids, or about 20 to about 25 amino acids). Typically, modified immunoglobulin hinge regions that are fragments of wild-type immunoglobulin hinge regions include the IgG core hinge region disclosed in U.S. Patent Application Publication Nos. 2013 / 0129723 and 2013 / 0095097 (e.g., a polypeptide comprising the sequence CXXC, where X is any amino acid (SEQ ID NO: 390)). Non-limiting examples of hinges are provided in Table 2.

[0076] As used herein, the term "humanization" refers to the process of using genetic engineering techniques to make antibodies or immunoglobulin-binding proteins and polypeptides from non-human species (e.g., mouse or rat) less immunogenic to humans, while still retaining the binding properties of the original antibody. In some embodiments, the binding domains (e.g., light and heavy chain variable regions, Fab, scFv) of antibodies or immunoglobulin-binding proteins and polypeptides are humanized. Non-human binding domains can be engineered using techniques such as "reshaping" (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 1991 9:266-271), "hyperchimerization" (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 Antibodies can be humanized using the technique known as CDR grafting (Jones et al., Nature 321:522 (1986)) and variations thereof, including CDR grafting (In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312). When derived from non-human sources, other regions of antibodies or immunoglobulin binding proteins and polypeptides, such as the hinge region and constant region domains, can also be humanized.

[0077] As used herein, an "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain" refers to an immunoglobulin domain of a polypeptide chain that selectively interacts with or binds to a different immunoglobulin domain of a second polypeptide chain, where the interaction of the different immunoglobulin heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second polypeptide chains (i.e., the formation of dimers between two different polypeptide chains, also referred to as "heterodimers"). An interaction between immunoglobulin heterodimerization domains "substantially contributes to or efficiently promotes" heterodimerization of the first and second polypeptide chains if there is a statistically significant reduction in dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimerization domain of the first polypeptide chain and / or the immunoglobulin heterodimerization domain of the second polypeptide chain. In certain embodiments, when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the first and second polypeptide chains form heterodimers with one another. Exemplary immunoglobulin heterodimerization domains include immunoglobulin CH1 domains, immunoglobulin CL domains (e.g., Cκ or Cλ isotypes), or derivatives thereof, including wild-type immunoglobulin CH1 and CL domains and engineered (or mutant) immunoglobulin CH1 and CL domains, as provided herein.

[0078] The terms "patient" and "subject" are used interchangeably. As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk for or suffering from a disease, disorder, or condition suitable for treatment or amelioration with a binding protein or multispecific polypeptide or composition thereof provided herein. "Patient" and "subject" are used interchangeably.

[0079] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not normally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that are approved by federal or state regulatory agencies or are listed in the United States Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans, are considered to be "pharmaceutically acceptable."

[0080] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used synonymously with gene, cDNA encoded by a gene, and mRNA. As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs, and derivatives, fragments, and homologs thereof.

[0081] The term "expression" refers to the biosynthesis of a product encoded by a nucleic acid. For example, in the case of a nucleic acid segment encoding a polypeptide of interest, expression involves transcription of the nucleic acid segment into mRNA and the translation of the mRNA into one or more polypeptides.

[0082] The terms "expression unit" and "expression cassette" are used interchangeably herein to refer to a nucleic acid segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell. An expression unit typically includes a transcriptional promoter, an open reading frame encoding the polypeptide of interest, and a transcriptional terminator, all in an operable configuration. In addition to a transcriptional promoter and terminator, an expression unit may further include other nucleic acid segments, such as, for example, an enhancer or polyadenylation signal.

[0083] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule that contains one or more expression units. In addition to one or more expression units, an expression vector may also contain additional nucleic acid segments, such as one or more origins of replication or one or more selectable markers. Expression vectors are usually derived from plasmid or viral DNA, or may contain elements of both.

[0084] As used herein, "polypeptide," "polypeptide chain," or "protein" refers to a contiguous arrangement of covalently linked amino acids. Polypeptides may form one or more intrachain disulfide bonds. With respect to the polypeptides described herein, reference to modifications or changes of amino acid residues corresponding to those designated by a SEQ ID NO: includes post-translational modifications of such residues. The term polypeptide protein also encompasses embodiments in which two polypeptide chains are linked together non-linearly, such as via an interchain disulfide bond. For example, a native immunoglobulin molecule consists of two heavy chain polypeptides and two light chain polypeptides.

[0085] As used herein, a "multispecific polypeptide" refers to a polypeptide comprising two or more binding domains, each capable of specifically binding to a target antigen. For example, a polypeptide described herein can comprise two, three, four, or more binding domains and can bind to two, three, four, or more target antigens. In some embodiments, a multispecific polypeptide is a bispecific polypeptide. As used herein, a "bispecific polypeptide" comprises two binding domains and can bind to two separate target antigens. In some embodiments, a bispecific polypeptide described herein comprises a first binding domain that specifically binds to a cell surface antigen expressed on a target cell. In some embodiments, a bispecific polypeptide described herein comprises a binding domain that specifically binds to a cell surface antigen expressed on an effector cell. The binding domains can be derived from an antibody (e.g., a variable heavy chain and / or a variable light chain, scFv), a ligand, or a receptor.

[0086] Multispecific polypeptides are disclosed, for example, in International Publication Nos. WO 2007 / 146968; WO 2010 / 040105; WO 2010 / 003108; WO 2016 / 094873; WO 2017 / 053469; U.S. Patent Application Publication No. 2006 / 0051844; and U.S. Patent No. 7,166,707; and U.S. Patent No. 8,409,577. These patents are incorporated herein by reference in their entireties. In certain embodiments, the multispecific polypeptides described herein are bispecific polypeptides and may have an scFv-Fc-scFv structure, also referred to herein as ADAPTIR™ polypeptides. The structure of a polypeptide comprising such a structure comprises, from N-terminus to C-terminus: a first scFv binding domain-an immunoglobulin (Ig) hinge region-an Ig constant region-a second scFv binding domain.

[0087] The protein or polypeptide may be an antibody or an antigen-binding fragment of an antibody. In some embodiments, the protein may be a recombinant multispecific protein. In other embodiments, the multispecific protein may be produced by chemical conjugation of two different monoclonal antibodies or by fusing two hybridoma cell lines to generate a hybrid hybridoma. Other multivalent formats that can be used include, for example, Kλ-bodies, dAbs, diabodies, TandAbs, nanobodies, Small Modular ImmunoPharmaceutials (SMIP™), DOCK-AND-LOCK® (DNL®), CrossMab Fab, CrossMab VH-VL, strand-exchange engineered domain body (SEEDbody), affibody, Fynomer, Kunitz domain, Albu-dab, two engineered Fv fragments with exchanged VHs (e.g., dual-affinity re-targeting molecule (DART)), scFv x Exemplary bispecific formats include scFv (e.g., BiTE), SVD-IG, Covx-body, peptibody, scFv-Ig, SVD-Ig, dAb-Ig, Knob-in-Hole, IgG1 antibody comprising matched mutation in the CH3 domain (e.g., duobody antibody), and triomab. Exemplary bispecific formats are discussed in Garber et al., Nature Reviews Drug Discovery 13:799-801 (2014), which is incorporated herein by reference in its entirety.Additional exemplary bispecific formats are discussed in Liu et al. Front. Immunol. 8:38 doi:10.2289 / fimmu.2017.00038 and Brinkmann and Kontermann, MABS 9:2,182-212 (2017), each of which is incorporated herein by reference in its entirety. In certain embodiments, the bispecific antibody may be an F(ab')2 fragment. The F(ab')2 fragment comprises two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds at the hinge region.

[0088] As will be understood by those skilled in the art, proteins and polypeptides are defined herein by the amino acid sequences of the individual polypeptide chains, which are indicated by the SEQ ID NOs referenced throughout this disclosure. For example, in some embodiments, the scFv-Fc-scFv proteins or polypeptides described herein consist of two scFv-Fc-scFv polypeptide chains linked by an interchain bond (e.g., an interchain disulfide bond) to form a dimeric scFv-Fc-scFv protein (e.g., a homodimeric or heterodimeric scFv-Fc-scFv protein). In such embodiments, the scFv-Fc-scFv protein is defined by the amino acid sequences of the individual scFv-Fc-scFv polypeptide chains. Polypeptides and proteins may also contain non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents can be added to proteins or polypeptides by the cell in which the protein is produced and vary depending on the cell type. Proteins and polypeptides are defined herein by their amino acid backbone structures; substituents such as carbohydrate groups are usually not specified but may be present nonetheless.

[0089] The term "light chain variable region" (or "light chain variable domain" or "VL" or "V" L ") and a "heavy chain variable region" (also called a "heavy chain variable domain" or "VH" or "V H") refer to the variable binding regions from antibody light and heavy chains, respectively. The variable binding regions are composed of separate, distinct subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs), usually comprising the following order from amino to carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In one embodiment, the FRs are humanized. The term "CL" refers to an "immunoglobulin light chain constant region" or "light chain constant region," i.e., the constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or "heavy chain constant region," which can be further divided into CH1, CH2, and CH3 domains (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM) depending on the antibody isotype. "Fab" (Fragment Antigen Binding) is the portion of an antibody that binds to an antigen and comprises the variable region and CH1 domain of a heavy chain linked to a light chain via an interchain disulfide bond.

[0090] The terms "amino-terminal" and "carboxyl-terminal" are used herein to indicate positions within a polypeptide. When the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to indicate proximity or relative position. For example, a particular sequence located carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl-terminus of the reference sequence, but is not necessarily at the carboxyl-terminus of the complete polypeptide.

[0091] As used herein, the terms "transformation," "gene transfer," and "transduction" refer to the introduction of a nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the introduction and incorporation of DNA, particularly recombinant DNA, into a cell. The nucleic acid to be transferred can be introduced into the cell via an expression vector.

[0092] As used herein, "antibody-dependent cellular cytotoxicity" and "ADCC" refer to a cell-mediated process in which nonspecific cytotoxic cells expressing FcγR (e.g., mononuclear cells such as natural killer (NK) cells and macrophages) recognize bound antibodies (or other proteins capable of binding FcγR) on target cells, subsequently causing lysis of the target cells. In principle, any effector cell bearing an activating FcγR can trigger ADCC mediation. The primary cells for mediating ADCC are NK cells, which express only FcγRIII, whereas monocytes can express FcγRI, FcγRII, and FcγRIII depending on their activation, localization, or differentiation state. For a review of FcγR expression on hematopoietic cells, see, e.g., Ravetch et al., 1991, Annu. Rev. Immunol., 9:457-92.

[0093] As used herein in reference to a polypeptide or protein, the term "having ADCC activity" means that a polypeptide or protein, e.g., one comprising an Fc domain (e.g., an immunoglobulin constant region having an immunoglobulin hinge region and CH2 and CH3 domains), such as from IgG (e.g., IgG1), can mediate antibody-dependent cellular cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g., FcγRIII) on a cytolytic immune effector cell (e.g., NK cell) expressing the Fc receptor. In some embodiments, a multispecific polypeptide or protein comprising an Fc domain may lack effector function (e.g., null ADCC activity) as a result of mutations in the CH2 and / or CH3 domains.

[0094] As used herein, "complement-dependent cytotoxicity" and "CDC" refer to the process by which components in normal serum ("complement"), together with antibodies or other C1q complement-binding proteins bound to a target antigen, result in the lysis of target cells expressing the target antigen. Complement consists of a group of serum proteins that act in a coordinated and sequenced manner to exert their effect.

[0095] As used herein, the terms "classical complement pathway" and "classical complement system" are synonymous and refer to a specific pathway for complement activation. The classical pathway requires an antigen-antibody complex for initiation and involves the sequential activation of nine major protein components, designated C1 through C9. At several steps in the activation process, the product is an enzyme that catalyzes the subsequent step. This cascade results in the amplification and activation of large amounts of complement with a relatively small initial signal.

[0096] As used herein in reference to a polypeptide or protein, the term "having CDC activity" means that a polypeptide or protein, e.g., one that includes an Fc domain (e.g., an immunoglobulin constant region having an immunoglobulin hinge region and CH2 and CH3 domains), such as from IgG (e.g., IgG1), can mediate complement-dependent cytotoxicity (CDC) through binding of the C1q complement protein and activation of the classical complement system. In some embodiments, a multispecific polypeptide or protein may lack effector function (e.g., null CDC activity) as a result of one or more mutations in the CH2 and / or CH3 domains.

[0097] As used herein, "enhanced effector cell activation" refers to the augmentation, prolongation, and / or potentiation of an effector cell response by a polypeptide or protein described herein. In some embodiments, enhanced effector cell activation refers to an increase in the cytotoxic activity of an effector cell. In some embodiments, enhanced effector cell activation refers to changes in cytokine production, cell proliferation, or cell surface molecule expression such that the ability of the effector cell to lyse a target cell is enhanced.

[0098] As used herein, the term "effector cell" refers to a cell of the immune system that can lyse or kill a target cell, such as a tumor cell. Herein, an effector cell may refer to a lymphocyte, such as a T cell, a natural killer (NK) cell, or an NKT cell, a monocyte, a macrophage, a dendritic cell, or a granulocyte. In some embodiments, the term effector cell refers to a T cell, an NK cell, or an NKT cell.

[0099] As used herein, the terms "treatment," "treating," or "ameliorating" refer to therapeutic or prophylactic / preventative treatment. Treatment is therapeutic if at least one symptom of the disease in the individual receiving treatment is improved, or if the treatment can slow the worsening of progressive disease in the individual, or prevent the onset of additional related diseases.

[0100] As used herein, the term "therapeutically effective amount (or therapeutically effective dosage)" or "effective amount (or effective dosage)" of a polypeptide or protein or composition thereof described herein refers to that amount of compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner or in terms of a statistically significant improvement in organ function. When referring to an individual active ingredient administered alone, a therapeutically effective amount refers to that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of the active ingredients that produce the therapeutic effect, whether administered sequentially or simultaneously (either in the same formulation or simultaneously in separate formulations).

[0101] Pharmaceutical Composition Described herein are stable pharmaceutical formulations of protein therapeutics, such as multispecific polypeptides, that prevent denaturation and / or prevent or substantially reduce aggregate formation, particularly upon freezing. In addition to the therapeutic protein, the pharmaceutical compositions described herein may further comprise one or more of a buffer, an excipient, and a surfactant. In some embodiments, the compositions comprise, consist of, or consist essentially of a buffer, an excipient, and a surfactant, wherein the multispecific protein is a dimer of two identical polypeptides, each polypeptide comprising, in amino-terminal to carboxyl-terminal order or carboxyl-terminal to amino-terminal order, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a second binding domain; and the buffer comprises or consists of succinate or a pharmaceutically acceptable salt or acid thereof.

[0102] In some embodiments, the composition comprises about 0.1 mg / ml to about 10 mg / ml of the multispecific protein. In some embodiments, the composition comprises about 1 mg / ml to about 5 mg / ml of the multispecific protein. In some embodiments, the composition comprises about 2 mg / ml of the multispecific protein. In some embodiments, the composition comprises about 2 mg / ml of the multispecific protein, about 5 mM succinate, about 6.5% weight / volume (w / v) sucrose, and about 0.02% w / v polysorbate 80.

[0103] In some embodiments, the composition substantially prevents degradation of the multispecific protein. In some embodiments, the composition delays or reduces degradation of the multispecific polypeptide compared to the same multispecific polypeptide stored in a histidine buffer under identical storage conditions. In some embodiments, the composition is substantially stable at 4°C for at least 1 year. In some embodiments, the composition substantially resists aggregate formation of the multispecific protein.

[0104] In some embodiments, the composition is capable of withstanding freeze-thaw conditions, hi some embodiments, the composition delays or reduces degradation of the multispecific polypeptide under freeze-thaw conditions compared to the multispecific polypeptide stored in a histidine buffer under the same freeze-thaw conditions.

[0105] In other embodiments, when formulated as disclosed herein, the CD123 x CD3-targeting multispecific polypeptide undergoes little or no degradation after lyophilization. For example, the CD123 x CD3-targeting multispecific polypeptide can be formulated in a succinate and sucrose formulation that exhibits reduced degradation after lyophilization compared to the same polypeptide formulated in a histidine buffer. Also provided herein are lyophilized anti-CD123 x anti-CD3 multispecific polypeptides, including, but not limited to, TRI130 and TRI129 formulated in about 5 mM succinate, about 6.5% weight / volume (w / v) sucrose, and about 0.02% w / v polysorbate 80. In some embodiments, the composition is lyophilized.

[0106] buffer solution As used herein, the term "buffer" or "buffering agent" refers to one or more components that, when added to an aqueous solution, can protect the solution against pH fluctuations upon addition of acid or alkali, or upon dilution with a solvent.

[0107] In some embodiments, the buffer comprises, consists of, or consists essentially of any pharmaceutically acceptable buffer. For example, the buffer may be potassium phosphate, acetic acid / sodium acetate, citric acid / sodium citrate, succinic acid / sodium succinate, tartaric acid / sodium tartrate, histidine / histidine HCl, glycine, Tris, glutamate, acetate, mixtures thereof, or pharmaceutically acceptable salts or acids thereof. In certain embodiments, the buffer comprises, consists of, or consists essentially of succinate or a pharmaceutically acceptable salt or acid thereof.

[0108] In some embodiments, the concentration of the buffer in the composition is about 1 mM to about 500 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 1 to about 10 mM, about 5 mM to about 50 mM, about 5 mM to about 20 mM, or about 5 mM to about 10 mM. In some embodiments, the composition comprises about 1 mM to about 10 mM of a succinate ester, or a pharmaceutically acceptable salt or acid thereof. In some embodiments, the composition comprises about 5 mM of a succinate ester, or a pharmaceutically acceptable salt or acid thereof.

[0109] In some embodiments, the pH of the composition is 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, or 11.5. In some embodiments, the pH of the composition is about 3.0 to about 6.0. In some embodiments, the pH of the composition is about 4.0 to about 5.5. In some embodiments, the pH of the composition is about 4.8.

[0110] excipients As referred to herein, excipients are pharmacologically inactive substances that are formulated with the active pharmaceutical ingredient of the composition. Excipients may aid in lubrication, flowability, disintegration, or taste, and may impart certain antimicrobial functions.

[0111] Representative excipients that can be used in the compositions disclosed herein include pharmaceutical binders, diluents, release-retarding excipients, lubricants, glidants, gas-generating agents, coating systems, solvents, and colorants. Suitable excipients include those listed as excipients in Tables 3-5 of Handbook of Pharmaceutical Excipients, Third Edition, Edited by A.H. Kibbe, American Pharmaceutical Association and Pharmaceutical Press (2000), and E.C. Cole et al., Advanced Drug Delivery Reviews 60 (2008), 747-756. For example, the excipient can be selected from the group consisting of polypropylene glycol; polyethylene glycol, polyoxyethylene castor oil derivatives, polyoxyethylene glycerol oxystearate, saturated polyglycolized glycerides, polyethylene polypropylene glycol, vitamin E, and vitamin E TPGS (d-α-tocopheryl polyethylene glycol polyethylene glycol 1000 succinate).

[0112] In some embodiments, the composition comprises about 1% to about 20%, about 1% to about 10%, about 5% to about 15%, or about 10% weight / volume (w / v) of the excipient. In some embodiments, the composition comprises about 1% to about 12%, e.g., about 6.5%, of the excipient.

[0113] In some embodiments, the excipient comprises, consists of, or consists essentially of a sugar. In some embodiments, the composition comprises about 1% to about 12% w / v sugar. In some embodiments, the composition comprises about 4% to about 8% w / v sugar. In some embodiments, the composition comprises about 6.5% w / v sugar. In some embodiments, the sugar is sucrose.

[0114] surfactant As described herein, a "surfactant" is a surface-active molecule that contains both a hydrophobic portion (eg, an alkyl chain) and a hydrophilic portion (eg, carboxyl and carboxylate groups).

[0115] Suitable surfactants for use in the compositions described herein include, but are not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and derivatives; Triton; sodium lauryl sulfate; sulfate); sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic, PF68, etc.). In certain embodiments, the surfactant comprises or consists of polysorbate 80.

[0116] In some embodiments, the composition comprises about 0.001% to about 1%, about 0.01% to about 0.5%, or about 0.01% to about 0.1% w / v surfactant, hi some embodiments, the composition comprises about 0.02% w / v surfactant.

[0117] In some embodiments, the composition comprises about 0.001% to about 1% w / v, about 0.01% to about 0.5% w / v, or about 0.01% to about 0.1% w / v of polysorbate 80. In some embodiments, the composition comprises about 0.02% w / v of polysorbate 80.

[0118] Therapeutic Proteins The compositions described herein can be used in conjunction with many different protein therapeutics described herein.

[0119] Binding domain In some embodiments, the therapeutic protein comprises a binding domain. The binding domain can provide specific binding to at least one cell surface molecule (e.g., a cell surface receptor). The binding domain can be an antibody or fragment thereof, or in the form of a fusion protein in any of a variety of formats (e.g., the fusion protein can be in the form of a bispecific or multispecific molecule). In other embodiments, the binding domain can comprise, for example, a specific cytokine or molecule that targets the binding domain polypeptide to, for example, a specific cell type, a toxin, an additional cellular receptor, or an antibody.

[0120] In some embodiments, the binding domains described herein are derived from antibodies and include variable heavy chains (V H ) and variable light chain (V L ) for example, V H and V L The binding domains and variable chains can be arranged in any order that still retains some degree of binding to the target. In some embodiments, the binding domain comprises: (i) an immunoglobulin heavy chain variable region (V) comprising HCDR1, HCDR2, and HCDR3; H and (ii) an immunoglobulin light chain variable region (V) comprising LCDR1, LCDR2, and LCDR3. L ) is included.

[0121] In some embodiments, the polypeptides and proteins described herein comprise a binding domain that is an scFv. In such embodiments, the binding domain may also be referred to as an scFv domain. In some embodiments, the binding domain comprises a V domain specific for a target of interest. H and V L In certain embodiments, the V is a single chain Fv fragment (scFv) comprising the V region. H and V L The regions are human or humanized. In some variations, the binding domains are V and VL linked by peptide linkers. L and V H It is a single-chain Fv (scFv) containing the region.

[0122] In certain embodiments, the binding domain of the polypeptides described herein comprises (i) an immunoglobulin light chain variable region (V) comprising CDRs LCDR1, LCDR2, and LCDR3. L ), and (ii) an immunoglobulin heavy chain variable region (V) comprising CDRs HCDR1, HCDR2, and HCDR3. H In some embodiments, the amino acid sequences provided for the polypeptide constructs do not include a human immunoglobulin leader sequence. The CDR sequences and amino acid substitution positions shown are those defined using the IMGT criteria (Brochet et al, Nucl. Acids Res. (2008) 36, W503-508).

[0123] In certain embodiments, binding domain V of the present disclosure L and / or V H The region is the parent V L and / or V H V of the area L and / or V H derived from known monoclonal antibodies (e.g., 1618 / 1619 described in WO 2016 / 185016) L and / or V HWhen compared to the sequence, the V may optionally contain about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of the above changes. The insertions, deletions, or substitutions may be at or near the amino or carboxyl termini, or both, of the region, provided that each CDR contains zero changes or a maximum of one, two, or three changes. L and / or V H In some embodiments, a binding domain comprising a modified VL and / or VH region can still specifically bind to its target with similar or higher affinity than the parent binding domain.

[0124] V L and V H The use of peptide linkers for linking regions is well known in the art, and numerous papers exist in this particular field. In some embodiments, the peptide linker is a 15-mer and consists of three repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 128) ((Gly4Ser)3) (SEQ ID NO: 59). Other linkers have been used, and phage display technology, as well as selective infective phage technology, have been used to diversify and select appropriate linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., Protein Eng. 11, 405-410, 1998). In certain embodiments, V L and V H The region has the formula (Gly4Ser) n(where n=1-5) (SEQ ID NO: 129). For example, in some embodiments, the linker comprises (Gly4Ser)4 (SEQ ID NO: 61). Other suitable linkers can be obtained by optimizing simple linkers by random mutagenesis. In some embodiments, the V of the scFv described herein H In some embodiments, the V region of the scFvs described herein may be located N-terminal to the linker sequence. L The region may be located C-terminal to the linker sequence.

[0125] In some embodiments, the binding domain may bind to a tumor antigen such as CD123, PSMA, CD19, CD33, 5T4, or HER2. In some embodiments, the binding site may be a CD3 binding domain. In some embodiments, the binding domain may bind to 4-1-BB. In some embodiments, the binding domain may bind to OX40. In some embodiments, the combined multispecific protein binds to both 4-1-BB and OX40.

[0126] hinge In addition to the binding domain, the therapeutic polypeptide may further comprise a hinge region. In some embodiments, the hinge is a modified immunoglobulin hinge in which one or more cysteine ​​residues of the wild-type immunoglobulin hinge region are substituted with one or more amino acid residues (e.g., serine or alanine). Exemplary modified immunoglobulin hinges, carboxyl-terminal linkers, and amino-terminal linkers include an immunoglobulin human IgG1 hinge region in which one, two, or three cysteine ​​residues found in the wild-type human IgG1 hinge are substituted with one, two, or three different amino acid residues (e.g., serine or alanine). The modified immunoglobulin hinge may additionally have a proline substituted with another amino acid residue (e.g., serine or alanine). For example, the modified human IgG1 hinge described above may additionally have a proline substituted with another amino acid residue located carboxyl-terminal to the three cysteines of the wild-type human IgG1 hinge region. In one embodiment, the prolines in the core hinge region are not substituted. In certain embodiments, the hinge, carboxyl-terminal linker, or amino-terminal linker polypeptide comprises or is a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a wild-type immunoglobulin hinge region, such as a wild-type human IgG1 hinge, a wild-type human IgG2 hinge, or a wild-type human IgG4 hinge. Immunoglobulin constant domains Therapeutic proteins may also comprise an immunoglobulin constant (Fc) domain (also referred to herein as a constant region, Fc domain, Fc region, etc.). In some embodiments, the constant region comprises an IgG CH2 and CH3 domain, e.g., an IgG1 CH2 and CH3 domain. In some embodiments, the constant region does not comprise a CH1 domain. In some embodiments, the immunoglobulin constant region is a human Fc domain. In some embodiments, the immunoglobulin constant region comprises one, two, three, or more amino acid substitutions compared to a wild-type immunoglobulin constant region to reduce or prevent binding to FcγR1, FcγRIIa, FcγRIIb, FcγRIIa, and FcγRIIIb. In some embodiments, the constant domains comprising the constant region are human or derived from human sequences. In some embodiments, the Fc domain comprises one or more mutations in the Fc region to reduce or prevent complement fixation and interaction with Fcγ receptors. In some embodiments, the immunoglobulin constant region comprises one, two, three, or more amino acid substitutions compared to the wild-type immunoglobulin constant region to reduce or prevent Fc-mediated T cell activation. In some embodiments, the immunoglobulin constant region comprises one, two, three, or more amino acid substitutions compared to the wild-type immunoglobulin constant region to prevent or reduce CDC activity. In some embodiments, the immunoglobulin constant region comprises one, two, three, or more amino acid substitutions compared to the wild-type immunoglobulin constant region to prevent or reduce ADCC activity.

[0127] In some embodiments, the Fc region comprises one or more mutations in the CH2 domain at positions 234, 235, 237, and 322 according to the EU numbering system. In some embodiments, the Fc domain comprises mutations in the CH2 domain at positions 234, 235, 237, 318, 320, and 322 according to the EU numbering system. In some embodiments, the Fc domain comprises CH2 domain mutations L234A, L235A, G237A, and K322A according to the EU numbering system. In some embodiments, the Fc domain comprises CH2 domain mutations L234A, L235A, G237A, E318A, K320A, and K322A according to the EU numbering system. In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising substitutions E233P, L234A, L235A, G237A, and K322A and a deletion of G236 according to the EU numbering system. In some embodiments, the Fc domain is derived from human IgG1. In some embodiments, two or more mutations in the IgG1 Fc domain prevent or substantially reduce signaling via Fc-mediated cross-linking.

[0128] In some embodiments, the immunoglobulin constant region comprises the amino acid sequence of any one of SEQ ID NOs: 32-35, or a variant thereof. The inclusion of an immunoglobulin constant region delays clearance of the polypeptides and proteins of the present disclosure from circulation after administration to a subject. Mutations or other changes in the immunoglobulin constant region further allow for relatively easy modulation of polypeptide effector functions (e.g., ADCC, ADCP, CDC, complement fixation, and Fc receptor binding), which can be increased or decreased depending on the disease being treated, as known in the art and described herein. In certain embodiments, the polypeptides and proteins described herein comprise an immunoglobulin constant region capable of mediating one or more of these effector functions. In other embodiments, one or more of these effector functions are reduced or absent in the immunoglobulin constant regions of the polypeptides or proteins described herein compared to the corresponding wild-type immunoglobulin constant region.

[0129] The constant regions present in the polypeptides and proteins of the present disclosure may comprise or be derived from part or all of a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof. For example, an immunoglobulin constant region may comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, two CH4 domains, and portions of a CH2 domain and a CH3 domain. In certain embodiments, the polypeptides or proteins described herein do not comprise a CH1 domain.

[0130] The polypeptides or proteins described herein may comprise wild-type or modified immunoglobulin CH2 domains from particular immunoglobulin classes or subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD) and from various species (including human, mouse, rat, and other mammals). In certain embodiments, the CH2 domain of a polypeptide or protein described herein is a wild-type human immunoglobulin CH2 domain, such as the wild-type CH2 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD set forth in SEQ ID NOS: 115, 199-201, and 195-197, respectively, of U.S. Patent Application Publication No. 2013 / 0129723 (the sequences of which are incorporated herein by reference). In a specific embodiment, the CH2 domain is a wild-type human IgG1 CH2 domain as set forth in SEQ ID NO: 115 of US Patent Application Publication No. 2013 / 0129723 (the sequence is incorporated herein by reference).

[0131] In certain embodiments, a modified CH2 region in a polypeptide or protein of the disclosure comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild-type immunoglobulin CH2 region, such as the CH2 region of wild-type human IgG1, IgG2, or IgG4, or murine IgG2a (e.g., IGHG2c).

[0132] The modified immunoglobulin CH2 region in the polypeptides or proteins of the present disclosure can be derived from the CH2 regions of various immunoglobulin isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, and various species (including human, mouse, rat, and other mammals). In certain embodiments, the modified immunoglobulin CH2 region in the fusion proteins of the present disclosure can be derived from the CH2 region of human IgG1, IgG2, or IgG4, or mouse IgG2a (e.g., IGHG2c), the sequences of which are set forth in SEQ ID NOs: 115, 199, 201, and 320 of U.S. Patent Application Publication No. 2013 / 0129723 (the sequences are incorporated herein by reference). In certain embodiments, the modified CH2 domain of the polypeptides or proteins described herein is a modified human IgG1 CH2 domain having mutations known in the art to enhance or decrease immunological activity (i.e., effector function), such as ADCC, ADCP, CDC, complement binding, Fc receptor binding, or any combination thereof.

[0133] In certain embodiments, the CH2 domain of a polypeptide or protein described herein is a modified immunoglobulin CH2 region (e.g., a modified human IgG1 CH2 domain) that contains one or more amino acid deletions or substitutions. In some embodiments, the CH2 domain contains an amino acid substitution of asparagine at position 297 (e.g., an asparagine-to-alanine substitution). Such an amino acid substitution reduces or eliminates glycosylation at this position and abrogates efficient Fc binding to FcγR and C1q. The sequence of a modified human IgG1 CH2 domain with an Asn-to-Ala substitution at position 297 is set forth in SEQ ID NO: 324 of U.S. Patent Application Publication No. 2013 / 0129723 (the sequence is incorporated herein by reference). In some embodiments, the modified CH2 domain contains at least one substitution or deletion at positions 234-238. For example, the immunoglobulin CH2 region can include substitutions at positions 234, 235, 236, 237, or 238; positions 234 and 235; positions 234 and 236; positions 234 and 237; positions 234 and 238; positions 234-236; positions 234, 235 and 237; positions 234, 236 and 238; positions 234, 235, 237, and 238; substitutions at positions 236-238; or any other combination of substitutions of 2, 3, 4, or 5 amino acids at positions 234-238. In some embodiments, the modified CH2 region includes one or more (e.g., 2, 3, 4, or 5) amino acid deletions at positions 234-238, e.g., at one of positions 236 or 237, while the other positions are substituted. In certain embodiments, the amino acid residues at one or more of positions 234-238 are substituted with one or more alanine residues. In further embodiments, only one amino acid residue at positions 234-238 is deleted, while one or more of the remaining amino acids at positions 234-238 may be substituted with another amino acid (e.g., alanine or serine).

[0134] In some embodiments, the mutation reduces or eliminates ADCC activity or Fc receptor binding ability of the polypeptide comprising the modified CH2 domain.

[0135] In certain embodiments, the CH2 domain of a polypeptide or protein described herein is a modified immunoglobulin CH2 region (e.g., a modified human IgG1 CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331. For example, the immunoglobulin CH2 region can comprise substitutions at positions 253, 310, 318, 320, 322, or 331, positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of substitutions of two, three, four, five, or six amino acids at positions 253, 310, 318, 320, 322, or 331. In such embodiments, the mutations reduce or eliminate CDC activity of the polypeptide comprising the modified CH2 domain.

[0136] In certain embodiments, in addition to the amino acid substitution at position 297, the modified CH2 region of a polypeptide or protein described herein (e.g., a modified human IgG1 CH2 domain) can further comprise one or more (e.g., two, three, four, or five) additional substitutions at positions 234-238. For example, the immunoglobulin CH2 region can include substitutions at positions 234 and 297, positions 234, 235, and 297, positions 234, 236, and 297, positions 234-235 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any other combination of substitutions of two, three, four, or five amino acids at positions 234-238 in addition to position 297. Additionally or alternatively, the modified CH2 region may comprise one or more (e.g., two, three, four, or five) amino acid deletions at positions 234-238, such as position 236 or position 237. The additional mutations reduce or eliminate ADCC activity or Fc receptor binding ability of a polypeptide comprising the modified CH2 domain. In certain embodiments, an amino acid residue at one or more of positions 234-238 is substituted with one or more alanine residues. In further embodiments, only one amino acid residue at positions 234-238 may be deleted, while one or more of the remaining amino acids at positions 234-238 may be substituted with another amino acid (e.g., alanine or serine).

[0137] In certain embodiments, in addition to one or more (e.g., two, three, four, or five) amino acid substitutions at positions 234-238, a mutated CH2 region of a polypeptide or protein described herein (e.g., an engineered human IgG1 CH2 domain) in a fusion protein of the disclosure may contain one or more (e.g., two, three, four, five, or six) additional amino acid substitutions (e.g., substitutions with alanine) at one or more positions involved in complement fixation (e.g., positions I253, H310, E318, K320, K322, or P331). Examples of mutated immunoglobulin CH2 regions include human IgG1, IgG2, IgG4, and mouse IgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320, and 322. An exemplary mutant immunoglobulin CH2 region is the murine IGHG2c CH2 region with alanine substitutions at L234, L235, G237, E318, K320, and K322.

[0138] In still further embodiments, in addition to the amino acid substitution at position 297 and additional deletions or substitutions at positions 234-238, a modified CH2 region of a polypeptide or protein described herein (e.g., a modified human IgG1 CH2 domain) can further comprise one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331. For example, an immunoglobulin CH2 region can comprise (1) a substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more (e.g., two, three, four, five, or six) amino acid substitutions at positions I253, H310, E318, K320, K322, and P331, e.g., one, two, or three substitutions at positions E318, K320, and K322. The amino acid at the above positions may be substituted with alanine or serine.

[0139] In certain embodiments, the substituted CH2 region of a polypeptide or protein described herein comprises (i) an amino acid substitution of an asparagine at position 297 and one amino acid substitution at positions 234, 235, 236, or 237; (ii) an amino acid substitution of an asparagine at position 297 and amino acid substitutions at two positions between 234 and 237; (iii) an amino acid substitution of an asparagine at position 297 and amino acid substitutions at three positions between 234 and 237; (iv) an amino acid substitution of an asparagine at position 297, amino acid substitutions at positions 234, 235, and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three positions between 234 and 237 and amino acid substitutions at positions 318, 320, and 322; or (vi) amino acid substitutions at three positions between 234 and 237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320, and 322.

[0140] Exemplary modified immunoglobulin CH2 regions with an amino acid substitution of asparagine at position 297 include a human IgG1 CH2 region with alanine substitutions at L234, L235, G237, and N297 and a deletion at G236 (SEQ ID NO: 325 of U.S. Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference); a human IgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO: 326 of U.S. Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference); a human IgG4 CH2 region with alanine substitutions at F234, L235, G237, and N297 and a deletion at G236 (SEQ ID NO: 327 of U.S. Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference); a human IgG4 CH2 region (SEQ ID NO: 322 of US Patent Application Publication No. 2013 / 0129723, the sequence of which is incorporated herein by reference), a human IgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NO: 343 of US Patent Application Publication No. 2013 / 0129723, the sequence of which is incorporated herein by reference), a human IgG4 CH2 region with alanine substitutions at L235 and N297 (SEQ ID NO: 344 of US Patent Application Publication No. 2013 / 0129723, the sequence of which is incorporated herein by reference), a human IgG4 CH2 region with alanine substitutions at G236 and N297 (SEQ ID NO: 345 of US Patent Application Publication No. 2013 / 0129723, the sequence of which is incorporated herein by reference), and a human IgG4 CH2 region with alanine substitutions at G237 and N297 The CH2 region (SEQ ID NO: 346 of U.S. Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference) can be used in the polypeptides of the present disclosure.

[0141] In certain embodiments, in addition to the above amino acid substitutions, the modified CH2 region of a polypeptide or protein described herein (e.g., a modified human IgG1 CH2 domain) may contain one or more additional amino acid substitutions at one or more positions other than those described above. Such amino acid substitutions may be conservative or non-conservative amino acid substitutions. For example, in certain embodiments, in a modified IgG2 CH2 region, P233 can be changed to E233 (see, e.g., SEQ ID NO: 326 in U.S. Patent Application Publication No. 2013 / 0129723, the sequence of which is incorporated herein by reference). Additionally, or alternatively, in certain embodiments, the modified CH2 region may contain one or more amino acid insertions, deletions, or both. The insertion, deletion, or substitution may occur anywhere in the immunoglobulin CH2 region, such as at the N- or C-terminus of the wild-type immunoglobulin CH2 region resulting from the hinge-mediated connection of the CH2 region to another region (e.g., a binding domain or an immunoglobulin heterodimerization domain).

[0142] In certain embodiments, the modified CH2 domain of a polypeptide or protein described herein is a human IgG1 CH2 domain (SEQ ID NO: 595 of US Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference) with alanine substitutions at positions 235, 318, 320, and 322 (i.e., a human IgG1 CH2 domain with L235A, E318A, K320A, and K322A substitutions), optionally with an N297 mutation (e.g., to alanine). In certain embodiments, the modified CH2 domain is a human IgG1 CH2 domain (SEQ ID NO: 596 of U.S. Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference) with alanine substitutions at positions 234, 235, 237, 318, 320, and 322 (i.e., a human IgG1 CH2 domain with L234A, L235A, G237A, E318A, K320A, and K322A substitutions), optionally with an N297 mutation (e.g., to alanine).

[0143] In some embodiments, the immunoglobulin constant region of a polypeptide or protein described herein comprises a human IgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A according to the EU numbering system.

[0144] The CH3 domains that can form the immunoglobulin constant regions of the polypeptides or proteins described herein can be wild-type immunoglobulin CH3 domains from a particular immunoglobulin class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM) of various species (including humans, mice, rats, and other mammals), or modified immunoglobulin CH3 domains thereof. In certain embodiments, the CH3 domains of the polypeptides described herein are wild-type human immunoglobulin CH3 domains, such as the wild-type CH3 domains of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, IgE, or IgM set forth in SEQ ID NOS: 116, 208-210, 204-207, and 212, respectively, of U.S. Patent Application Publication No. 2013 / 0129723 (the sequences of which are incorporated herein by reference). In a specific embodiment, the CH3 domain is a wild-type human IgG1 CH3 domain as set forth in SEQ ID NO: 116 of US Patent Application Publication No. 2013 / 0129723 (the sequence is incorporated herein by reference).

[0145] In certain embodiments, the CH3 domain of a polypeptide described herein is a modified human immunoglobulin CH3 domain, such as a modified CH3 domain based on or derived from the wild-type CH3 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, IgE, or IgM. For example, the modified CH3 domain can be a human IgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions numbered according to EU numbering). Mutations at such positions can be involved in complement fixation. In certain other embodiments, the modified CH3 domain of a polypeptide described herein can be a human IgG1 CH3 domain, except with one or two amino acid substitutions at positions F405 or Y407. Amino acids at such positions are involved in interactions with other CH3 domains. In certain other embodiments, the modified CH3 domain of a polypeptide described herein can be a modified human IgG1 CH3 domain with its last lysine deleted. The sequence of this modified CH3 domain is shown in SEQ ID NO: 761 of US Patent Application Publication No. 2013 / 0129723 (the sequence is incorporated herein by reference).

[0146] In certain embodiments, the polypeptides or proteins described herein comprise a CH3 domain containing a so-called "knobs-into-holes" mutation (see Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al., Protein Engineering 9:617-21, 1966). More specifically, mutations can be introduced into each of the CH3 domains of each polypeptide chain to allow these two CH3 domains to pair with each other due to the steric complementarity required for CH3 / CH3 bonding. For example, the CH3 domain in one single-chain polypeptide of a polypeptide heterodimer may contain a T366W mutation (a "knob" mutation, which replaces a small amino acid with a larger one), and the CH3 domain in the other single-chain polypeptide of the polypeptide heterodimer may contain a Y407A mutation (a "hole" mutation, which replaces a large amino acid with a small one). Other exemplary knob-into-hole mutations include: (1) a T366Y mutation in one CH3 domain and a Y407T mutation in the other CH3 domain; and (2) a T366W mutation in one CH3 domain and T366S, L368A, and Y407V mutations in the other CH3 domain.

[0147] The CH4 domain capable of forming the immunoglobulin constant region of the polypeptides or proteins described herein can be a wild-type immunoglobulin CH4 domain or a modified immunoglobulin CH4 domain derived from an IgE or IgM molecule. In certain embodiments, the CH4 domain of a polypeptide described herein is a wild-type human immunoglobulin CH4 domain, such as the wild-type CH4 domains of human IgE and IgM set forth in SEQ ID NOs: 213 and 214, respectively, of U.S. Patent Application Publication No. 2013 / 0129723 (the sequences are incorporated herein by reference). In certain embodiments, the CH4 domain of a polypeptide described herein is a modified human immunoglobulin CH4 domain, such as a modified CH4 domain based on or derived from the CH4 domain of human IgE or IgM, which has mutations that increase or decrease immunological activity known to bind to the IgE or IgM Fc region.

[0148] In certain embodiments, the immunoglobulin constant region of a polypeptide or protein described herein comprises a combination of CH2, CH3, or CH4 domains (i.e., two or more constant region domains selected from CH2, CH3, and CH4). For example, an immunoglobulin constant region may comprise a CH2 and a CH3 domain or a CH3 and a CH4 domain. In certain other embodiments, an immunoglobulin constant region may comprise two CH3 domains and no CH2 or CH4 domains (i.e., two or more CH3 domains only). The multiple constant region domains forming the immunoglobulin constant region of a polypeptide described herein can be based on or derived from the same immunoglobulin molecule, or immunoglobulin molecules of the same class or subclass. In certain embodiments, the immunoglobulin constant region is an IgG CH2-CH3 (e.g., IgG1 CH2-CH3, IgG2 CH2-CH3, and IgG4 CH2-CH3), and may be human (e.g., human IgG1, IgG2, and IgG4) CH2-CH3. For example, in certain embodiments, the immunoglobulin constant region of a polypeptide described herein comprises (1) wild-type human IgG1 CH2 and CH3 domains, (2) a human IgG1 CH2 with an N297A mutation (i.e., CH2(N297A)) and a wild-type human IgG1 CH3, or (3) a human IgG1 CH2(N297A) and a modified human IgG1 CH3 in which the last lysine has been deleted. Alternatively, the multiple constant region domains of a polypeptide or protein described herein can be based on or derived from different immunoglobulin molecules, or immunoglobulin molecules of different classes or subclasses. For example, in certain embodiments, the immunoglobulin constant region comprises both a human IgM CH3 domain and a human IgG1 CH3 domain. The multiple constant region domains forming the immunoglobulin constant region of a polypeptide described herein can be linked directly together or can be linked to each other via one or more (e.g., about 2-10) amino acids.

[0149] Exemplary immunoglobulin constant regions that can be used in the polypeptides or proteins described herein are set forth in SEQ ID NOs: 305-309, 321, 323, 341, 342, and 762 of U.S. Patent Application Publication No. 2013 / 0129723 (the sequences are incorporated herein by reference). Further exemplary immunoglobulin constant regions that can be used in the polypeptides or proteins described herein are provided in the table below. [Table 1] JPEG2026021465000003.jpg164159

[0150] In certain embodiments, the immunoglobulin constant regions of each polypeptide chain of a homodimeric or heterodimeric protein described herein are identical to one another. In certain other embodiments, the immunoglobulin constant region of one polypeptide chain of a heterodimer is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer. For example, one immunoglobulin constant region of a heterodimeric protein may contain a CH3 domain with a "knob" mutation, while the other immunoglobulin constant region of the heterodimeric protein may contain a CH3 domain with a "hole" mutation.

[0151] Fc-binding domain linker In some embodiments, the polypeptide may further comprise an Fc binding domain linker. In some embodiments, the Fc binding domain linker can be used to link an immunoglobulin constant region to a C-terminal binding domain (e.g., a CD3 binding domain). In some embodiments, the Fc binding domain linker can be used as a hinge domain and / or incorporated into an scFv. In some embodiments, the Fc binding domain linker is a Gly4Ser linker (SEQ ID NO: 128). In some embodiments, the Fc binding domain linker is a 20-mer and consists of four repeats of the Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 128) amino acid sequence ((Gly4Ser)4) (SEQ ID NO: 61). In some embodiments, the Fc binding domain linker comprises an amino acid sequence selected from any one of SEQ ID NOs: 50-70. Other linkers have been used, and phage display technology, as well as selective infective phage technology, have been used to diversify and select appropriate linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., Protein Eng. 11, 405-410, 1998). In certain embodiments, V L and V H The region has the formula (Gly4Ser) n (where n=1-5) (SEQ ID NO: 129). Other suitable linkers can be obtained by optimizing simple linkers by random mutagenesis. In some embodiments, the bispecific molecule does not comprise a hinge region or a constant region.

[0152] In certain embodiments, the Fc binding domain linker is a flexible linker sequence comprising a glycine-serine (e.g., Gly4Ser, SEQ ID NO: 128) repeat. In certain embodiments, the linker comprises three Gly4Ser repeats (SEQ ID NO: 59) followed by a proline residue. In certain embodiments, the proline residue is followed by an amino acid selected from the group consisting of glycine, arginine, and serine. In some embodiments, the Fc binding domain linker comprises or consists of a sequence selected from SEQ ID NOs: 50-70.

[0153] Some exemplary hinge, Fc binding domain linker sequences suitable for use according to the present disclosure are set forth in Table 2. Additional exemplary hinge and linker regions are set forth in SEQ ID NOs: 241-244, 601, 78, 763-791, 228, 379-434, 618-749 of U.S. Patent Application Publication No. 2013 / 0129723 (the foregoing sequences are incorporated herein by reference). [Table 2] JPEG2026021465000005.jpg227159

[0154] In addition to the domains described above, therapeutic polypeptides may further comprise immunoglobulin dimerization / heterodimerization domains, junction amino acids, tags, additional binding domains, etc. In some embodiments, the polypeptides and proteins described herein are conjugated to a drug or toxic moiety.

[0155] Bispecific / multispecific proteins In some embodiments, the therapeutic protein can be a bispecific or multispecific protein. Non-limiting examples of bispecific molecules include scFv-Fc-scFv molecules, scFv-Ig molecules, and scFv-scFv molecules. In some embodiments, the bispecific molecules described herein comprise or consist of a first binding domain, scFv, linked to a second binding domain, scFv, and do not include other sequences such as an immunoglobulin constant region. In some embodiments, the therapeutic protein can be a bispecific or multispecific protein comprising, in amino-terminal to carboxyl-terminal or carboxyl-terminal to amino-terminal order, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) (optionally) an Fc binding domain linker, and (v) a second binding domain.

[0156] In some embodiments, the multispecific protein can comprise, from N- to C-terminus, a CD3-binding domain, a hinge region, an immunoglobulin constant region, and a tumor antigen-binding domain, which can bind to, for example, CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0157] In some embodiments, the multispecific protein can comprise, from N- to C-terminus, a tumor antigen-binding domain, a hinge region, an immunoglobulin constant region, and a CD3-binding domain. The tumor antigen-binding domain can bind to, for example, CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0158] In some embodiments, the multispecific protein can comprise, from N- to C-terminus, a 4-1-BB binding domain, a hinge region, an immunoglobulin constant region, and a tumor antigen binding domain, which can bind to, for example, CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0159] In some embodiments, the multispecific protein can comprise, from N- to C-terminus, a tumor antigen-binding domain, a hinge region, an immunoglobulin constant region, and a 4-1-BB-binding domain. The tumor antigen-binding domain can bind to, for example, CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0160] Homodimers / heterodimers In some embodiments, a Therapeutic protein can be a homodimer or a heterodimer. In some embodiments, a Therapeutic protein is a dimer of two identical polypeptides, each comprising, in amino-terminal to carboxyl-terminal order or in carboxyl-terminal to amino-terminal order, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) (optionally) an Fc-binding domain linker, and (v) a second binding domain. In some embodiments, a bispecific or multispecific protein is a dimer of two identical polypeptides, each comprising, in amino-terminal to carboxyl-terminal order or in carboxyl-terminal to amino-terminal order, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) (optionally) an Fc-binding domain linker, and (v) a second binding domain. In other embodiments, a bispecific protein described herein is a diabody.

[0161] In certain embodiments, the hinge present in a polypeptide that forms a heterodimer with another polypeptide chain can be an immunoglobulin hinge, such as a wild-type immunoglobulin hinge region or a modified immunoglobulin hinge region thereof. In certain other embodiments, the hinge of one polypeptide chain of a heterodimeric protein is identical to the corresponding hinge of the other polypeptide chain of the heterodimer. In certain other embodiments, the hinge of one chain differs from the hinge of the other chain (in their length or sequence). Different hinges in different chains allow for differential engineering of the binding affinities of the binding domains to which the hinges are linked, thereby allowing the heterodimer to selectively bind to the target of one binding domain over the target of the other binding domain.

[0162] In other embodiments, the polypeptides and proteins described herein contain a heterodimerization domain capable of heterodimerizing with a different heterodimerization domain in a second, non-identical polypeptide chain. In certain variations, the second polypeptide chain for heterodimerization contains a second binding domain. Thus, in certain embodiments of the present disclosure, two non-identical polypeptide chains, one containing a first binding domain and the second optionally containing a second binding domain, dimerize to form a heterodimeric binding protein. When it is desired to form a heterodimer from two non-identical polypeptide chains in which one or both polypeptide chains contain a binding domain, a dimerization / heterodimerization domain can be used. In certain embodiments, one polypeptide chain member of certain heterodimers described herein does not contain a binding domain. Examples of types of heterodimers include those described in U.S. Patent Application Publication Nos. 2013 / 0095097 and 2013 / 0129723, and WO 2016 / 094873.

[0163] In certain embodiments, the first and second polypeptide chains dimerize through the inclusion of an "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain." As used herein, "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain" refers to an immunoglobulin domain of a first polypeptide chain that selectively interacts with or binds to a different immunoglobulin domain of a second polypeptide chain, where the interaction of the different immunoglobulin domains substantially contributes to or efficiently promotes heterodimerization of the first and second polypeptide chains (i.e., the formation of a dimer between two different polypeptide chains, also referred to as a "heterodimer"). The immunoglobulin heterodimerization domains in the polypeptide chains of a heterodimer are different from each other and can therefore be differentially engineered to promote heterodimerization of both chains and minimize homodimerization of either chain. The immunoglobulin heterodimerization domains provided herein enable efficient heterodimerization between different polypeptides and facilitate purification of the resulting heterodimeric protein.

[0164] As provided herein, immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different polypeptide chains according to the present disclosure include wild-type and modified immunoglobulin CH1 and CL domains, e.g., human CH1 and CL domains. In certain embodiments, the immunoglobulin heterodimerization domain is a wild-type CH1 domain, such as a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain, as set forth in SEQ ID NOs: 114, 186-192, and 194 of U.S. Patent Application Publication No. 2013 / 0129723, or SEQ ID NO: 114 of U.S. Patent Application Publication No. 2013 / 0129723, respectively (the above sequences are incorporated herein by reference). In a further embodiment, a cysteine ​​residue in the wild-type CH1 domain (e.g., human CH1) that is involved in forming a disulfide bond with the wild-type immunoglobulin CL domain (e.g., human CL) is deleted or substituted in the modified immunoglobulin CH1 domain, such that a disulfide bond is not formed between the modified CH1 domain and the wild-type CL domain.

[0165] The polypeptides and proteins described herein may be produced using scaffolds such as those generally disclosed in U.S. Patent Application Publication Nos. 2013 / 0129723 and 2013 / 0095097. These patent application publications are incorporated herein by reference in their entireties. The polypeptides described herein may comprise two non-identical polypeptide chains, each comprising an immunoglobulin heterodimerization domain. The interacting immunoglobulin heterodimerization domains are different. In one embodiment, the immunoglobulin heterodimerization domain comprises a CH1 domain or a derivative thereof. In another embodiment, the immunoglobulin heterodimerization domain comprises a CL domain or a derivative thereof. In one embodiment, the CL domain is a Cκ or Cλ isotype or a derivative thereof.

[0166] Exemplary Protein Therapeutics: Anti-CD123 x Anti-CD3 Polypeptides and Dimers Thereof Exemplary protein therapeutics can bind both CD123-expressing cells and the T cell receptor complex on T cells to induce target-dependent T cell cytotoxicity, activation, and proliferation.

[0167] Thus, in certain embodiments, a therapeutic protein used in connection with the methods and compositions described herein is a bispecific single-chain molecule comprising a CD123-binding domain and a CD3-binding domain. In some embodiments, the CD123- and / or CD3-binding domain is derived from an antibody and comprises a variable heavy chain (VH) and a variable light chain (VL). For example, the CD123- and / or CD3-binding domain can be an scFv comprising a VH and a VL. These binding domains and variable chains can be arranged in any order that still retains some degree of binding to the target. For example, the variable domains may be (VH CD123)-(VL CD123)-(VH CD3)-(VL CD3), (VL CD123)-(VH CD123)-(VH CD3)-(VL CD3), (VH CD123)-(VL CD123)-(VL CD3)-(VH CD3), (VL CD123)-(VH CD123)-(VL CD3)-(VH CD3), (VH CD3)-(VL CD3)-(VH CD123)-(VL CD123), (VL CD3)-(VH CD3)-(VL CD123)-(VH CD123), (VH CD3)-(VL CD3)-(VL CD123)-(VH CD123), or (VL CD3)-(VH The VH and VL regions in the CD3-binding domain may be arranged in the order VH-VL or VL-VH. The pair of VH and VL regions in the CD3-binding domain may be in the format of a single-chain antibody (scFv). The VH and VL regions are arranged in the order VH-VL or VL-VH. In some embodiments, an scFv may bind to CD123 more efficiently than an antibody comprising the same VH and VL region sequences in the same orientation. In certain embodiments, an scFv may bind to CD123 more efficiently in the VL-VH orientation than in the VH-VL orientation. The VH region may be arranged N-terminal to the linker sequence. The VL region may be arranged C-terminal to the linker sequence. The domain arrangement in the CD3-binding domain of a bispecific single-chain molecule may be VH-VL, with the CD3-binding domain being arranged C-terminal to the CD123-binding domain. A bispecific molecule may comprise a CD3-binding scFv linked to a CD123-binding scFv. These scFvs are linked by a short peptide.In some embodiments, the bispecific single chain molecule does not comprise a hinge region or a constant region (see, e.g., U.S. Patent Application Publication Nos. 2013 / 0295121, WO 2010 / 037836, 2004 / 106381, and 2011 / 121110; each of which is incorporated by reference in its entirety).

[0168] The CD123-bispecific binding construct may comprise one or more of the sequences shown in Table 3, Table 4, and / or Table 5. [Table 3] JPEG2026021465000007.jpg221159JPEG2026021465000008.jpg220159JPEG2026021465000009.jpg22015 9JPEG2026021465000010.jpg229159JPEG2026021465000011.jpg224159JPEG2026021465000012.jpg22215 9JPEG2026021465000013.jpg226159JPEG2026021465000014.jpg222159JPEG2026021465000015.jpg22315 9JPEG2026021465000016.jpg224159JPEG2026021465000017.jpg221159JPEG2026021465000018.jpg55159 [Table 4] [Table 5] JPEG2026021465000021.jpg78159

[0169] In certain embodiments, the CD123-binding domain comprises (i) an immunoglobulin light chain variable region (VL) comprising CDRs LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) comprising CDRs HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 144, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 146, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 148. In certain embodiments, the CD123-binding domain comprises (i) an immunoglobulin light chain variable region (VL) comprising CDRs LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) comprising CDRs HCDR1, HCDR2, and HCDR3. In some such embodiments, (i) LCDR1 has the amino acid sequence set forth in SEQ ID NO: 138 or a sequence that differs from SEQ ID NO: 138 by at least one amino acid substitution; (ii) LCDR2 has the amino acid sequence set forth in SEQ ID NO: 140 or a sequence that differs from SEQ ID NO: 140 by at least one amino acid substitution; (iii) LCDR3 has the amino acid sequence set forth in SEQ ID NO: 142 or a sequence that differs from SEQ ID NO: 142 by at least one amino acid substitution; (iv) HCDR1 has the amino acid sequence set forth in SEQ ID NO: 144 or a sequence that differs from SEQ ID NO: 144 by at least one amino acid substitution; (v) HCDR2 has the amino acid sequence set forth in SEQ ID NO: 146 or a sequence that differs from SEQ ID NO: 146 by at least one amino acid substitution; and (vi) HCDR3 has the amino acid sequence set forth in SEQ ID NO: 148 or a sequence that differs from SEQ ID NO: 148 by at least one amino acid substitution. The above amino acid substitutions may be conservative or non-conservative amino acid substitutions. In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 differ from the recited sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.In certain embodiments, the CDRs of the disclosure comprise about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions) compared to the CDR sequences of known monoclonal antibodies, or a combination of the above changes. For example, the present disclosure includes recombinant polypeptides comprising: (i) an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 138 or a sequence that differs from SEQ ID NO: 138 by one or two amino acid substitutions; (ii) an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 140 or a sequence that differs from SEQ ID NO: 140 by one or two amino acid substitutions; (iii) an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 142 or a sequence that differs from SEQ ID NO: 142 by one or two amino acid substitutions; (iv) an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 144 or a sequence that differs from SEQ ID NO: 144 by one or two amino acid substitutions; (v) an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 146 or a sequence that differs from SEQ ID NO: 146 by one or two amino acid substitutions; and (vi) an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 148 or a sequence that differs from SEQ ID NO: 148 by one or two amino acid substitutions. The amino acid substitutions may be conservative or non-conservative amino acid substitutions.

[0170] In a related embodiment, the recombinant polypeptide of the present disclosure comprises a light chain variable region (V L ) (e.g., SEQ ID NO: 134), or the heavy chain variable region (V H), or both (e.g., SEQ ID NO: 136). In one embodiment, the CD123 binding domain of the recombinant polypeptide is an scFv comprising, in a VH-VL orientation, a variable heavy chain comprising SEQ ID NO: 136 and a variable light chain comprising SEQ ID NO: 134. In another embodiment, the CD123 binding domain of the recombinant polypeptide is an scFv comprising, in a VL-VH orientation, a variable light chain comprising SEQ ID NO: 134 and a variable heavy chain comprising SEQ ID NO: 136. For example, in certain embodiments, a polypeptide of the present disclosure comprises the amino acid sequence of SEQ ID NO: 337. The present disclosure includes recombinant polypeptides that are at least about 80%, at least about 85%, at least about 88%, 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%, at least about 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO:337.

[0171] In certain embodiments, the CD123 binding domain comprises (i) an immunoglobulin light chain variable region (V) comprising CDRs LCDR1, LCDR2, and LCDR3. L ), and (ii) an immunoglobulin heavy chain variable region (V) comprising CDRs HCDR1, HCDR2, and HCDR3. HIn some such embodiments, (i) LCDR1 has the amino acid sequence set forth in SEQ ID NO: 154 or a sequence that differs from SEQ ID NO: 154 by at least one amino acid substitution; (ii) LCDR2 has the amino acid sequence set forth in SEQ ID NO: 156 or a sequence that differs from SEQ ID NO: 156 by at least one amino acid substitution; (iii) LCDR3 has the amino acid sequence set forth in SEQ ID NO: 158 or a sequence that differs from SEQ ID NO: 158 by at least one amino acid substitution; (iv) HCDR1 has the amino acid sequence set forth in SEQ ID NO: 160 or a sequence that differs from SEQ ID NO: 160 by at least one amino acid substitution; (v) HCDR2 has the amino acid sequence set forth in SEQ ID NO: 162 or a sequence that differs from SEQ ID NO: 162 by at least one amino acid substitution; and (vi) HCDR3 has the amino acid sequence set forth in SEQ ID NO: 164 or a sequence that differs from SEQ ID NO: 164 by at least one amino acid substitution. The amino acid substitutions may be conservative or non-conservative amino acid substitutions. In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 differ from the recited sequences by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the CDRs of the disclosure comprise about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of the above changes, compared to the CDR sequences of known monoclonal antibodies.

[0172] In a related embodiment, the CD123 binding domain comprises a light chain variable region (V L ) (e.g., SEQ ID NO: 17), or the heavy chain variable region (V H), (e.g., SEQ ID NO: 16), or both.

[0173] In certain embodiments, the CD123 binding domain is a humanized immunoglobulin V L and / or V H Contains the immunoglobulin V region L and V H Techniques for humanizing regions are known in the art and are discussed, for example, in U.S. Patent Application Publication No. 2006 / 0153837. In certain embodiments, the CD123 binding domain is a human immunoglobulin V L and / or V H Includes the area.

[0174] Essentially, humanization by CDR grafting involves replacing only the CDRs of a non-human antibody onto a human variable region framework and a human constant region. In theory, this should substantially reduce or eliminate immunogenicity (except in the presence of allotypic or idiotypic differences). However, it has also been reported that some framework residues of the original antibody must also be preserved (Reichmann et al., Nature, 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10, 029 (1989)).

[0175] Framework residues that need to be conserved can be identified by computer modeling. Alternatively, essential framework residues may be identified by comparison with known antigen-binding site structures (Padlan, Molec. Immunol., 31(3):169-217 (1994), incorporated herein by reference).

[0176] Residues that may affect antigen binding are divided into several groups. The first group includes residues adjacent to the surface of the antigenic site, which may therefore make direct contact with the antigen. These residues include amino-terminal residues and residues adjacent to the CDRs. The second group includes residues that may contact the CDR or another peptide chain in the antibody, thereby altering the structure or relative alignment of the CDR. The third group includes amino acids with buried side chains that may affect the structural integrity of the variable domain. Residues in these groups are usually found in the same positions (Padlan, 1994, supra), although their identified positions may differ depending on the numbering system (see Kabat et al., "Sequences of proteins of immunological interest," 5th ed., Pub. No. 91-3242, USDept. Health & Human Services, NIH, Bethesda, Md., 1991).

[0177] Knowledge in the art about humanized antibodies is applicable to polypeptides according to the present disclosure, even though they are not antibodies.

[0178] In some embodiments, the anti-CD123 scFv comprises an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12; and an LCDR1 comprising SEQ ID NO: 13, an LCDR2 comprising SEQ ID NO: 14, and an LCDR3 comprising SEQ ID NO: 15. In some embodiments, the anti-CD123 scFv comprises a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 136, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 134. In some embodiments, the anti-CD123 scFv comprises a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16. In some embodiments, the anti-CD123 scFv comprises a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17. In some embodiments, the tumor antigen binding domain is an anti-CD123 scFv, and the scFv comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:18.

[0179] In some embodiments, the present disclosure relates to a CD123 binding domain, wherein (i) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 134, and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 136.

[0180] In further embodiments, each CDR contains no more than one, two, or three substitutions, insertions, or deletions compared to those from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g., CD123).

[0181] In certain embodiments, the CD123 binding domain does not inhibit IL-3 binding to CD123.

[0182] In certain embodiments, a CD123-binding molecule or protein may comprise a cell-binding domain for recruiting T cells to target cells expressing CD123. In certain embodiments, the CD123-binding proteins described herein may comprise (i) a binding domain that specifically binds to the TCR complex or a component thereof (e.g., TCRα, TCRβ, CD3γ, CD3δ, and CD3ε), and (ii) another binding domain that specifically binds to CD123. The CD123-binding protein may utilize essentially any binding domain that binds T cells, for example, an antibody-derived binding domain. Exemplary anti-CD3 antibodies from which the CD3-binding domain can be derived include the CRIS-7 monoclonal antibody (Reinherz, EL et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986); SEQ ID NO: 341 (QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITR) and SEQ ID NO: 342 (QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWGQGTLVTVSA)). L and V HThe amino acid sequences are shown in SEQ ID NO: 343 (diqmtqspsslsasvgdrvtitcsasssvsymnwyqqkpgkapkrliydtsklasgvpsrfsgsgsgtdftltisslqpedfatyycqqwssnpptfgggtkveik) and SEQ ID NO: 344 (qvqlvqsgaevkkpgasvkvsckasgytfisytmhwvrqapgqglewmgyinprsgythynqklkdkatltadksastaymelsslrsedtavyycarsayydydgfaywgqgtlvtvss)), respectively. L and V H Amino acid sequences include BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691); OKT3 monoclonal antibody (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and its derivatives such as OKT3 ala-ala (also called OKT3 AA-FL or OKT3 FL), a humanized Fc variant with alanine substitutions at positions 234 and 235 (Herold et al. (2003) J. Clin. Invest. 11:409); visilizumab (Carpenter et al. (2002) Blood 99:2712), G19-4 monoclonal antibody (Ledbetter et al., 1986, J. Immunol. 136:3945), 145-2C11 monoclonal antibody (Hirsch ... and 145-2C11 monoclonal antibody (Hirsch et al. (2002) Blood 99:2712). al. (1988) J. Immunol. 140:3766) and I2C monoclonal antibody (see, e.g., U.S. Patent Application Publication Nos. 2011 / 0293619 and 20120244162). For example, the CD3 binding domain may comprise a CD3 binding domain disclosed in US Patent Application Publication No. 2012 / 0244162, which comprises a CD3 binding domain comprising a VL region selected from SEQ ID NOs: 17, 21, 35, 39, 53, 57, 71, 75, 89, 83, 107, 111, 125, 129, 143, 147, 161, 165, 179 and 183 of US Patent Application Publication No. 2012 / 0244162 and / or a VH region selected from SEQ ID NOs: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 and 181 of US Patent Application Publication No. 2012 / 0244162. In some embodiments, the CD3 binding domain comprises an amino acid sequence selected from SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, and 187 of U.S. Patent Application Publication No. 2012 / 0244162. In some embodiments, the CD3 binding domain is one described in, or derived from, WO 2004 / 106380, WO 2005 / 040220A1, U.S. Patent Application Publication No. 2014 / 0099318. An exemplary anti-TCR antibody is the BMA031 monoclonal antibody (Borst et al. (1990) Human Immunology 29:175-188). The CD3 binding domain may be derived from any of the antibodies or sequences described in WO 2013 / 158856, which is incorporated herein by reference in its entirety.

[0183] In some embodiments, the second binding domain of a CD123-binding polypeptide described herein comprises (i) an immunoglobulin light chain variable region comprising LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein (a) LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 348, 349, and 350, respectively, and HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 345, 346, and 347, respectively; or (b) LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 354, 355, and 356, respectively, and HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 351, 352, and 353, respectively. In some embodiments, the second binding domain of a CD123-binding polypeptide described herein comprises (i) an immunoglobulin light chain variable region comprising LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein (a) LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 182, 183, and 184, respectively, and HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 182, 183, and 184, respectively. , having the amino acid sequences set forth in SEQ ID NOs: 351, 352 and 353, and HCDR1, HCDR2 and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 357, 359 and 359, respectively; or (b) LCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 359, 367 and 368, respectively, and HCDR1, HCDR2 and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 363, 364 and 365, respectively.In some embodiments, the second binding domain of a CD123-binding polypeptide described herein comprises (i) an immunoglobulin light chain variable region comprising LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein (a) LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 372, 373, and 374, respectively, or (b) LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 378, 379, and 380, respectively, and HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 375, 376, and 377, respectively. In some embodiments, the second binding domain comprising the CDR sequences detailed in this paragraph is humanized.

[0184] In some embodiments of a CD123 binding protein comprising two binding domains that specifically bind CD3ε, the second binding domain competes with CRIS-7, HuM291, or I2C monoclonal antibody for binding to CD3ε. In some embodiments, the CD3 binding domain comprises an immunoglobulin light chain variable region (V) derived from CRIS-7, HuM291, or I2C monoclonal antibody. L ) and immunoglobulin heavy chain variable region (V H ) (e.g., V of the second binding domain L and V H (The CDRs of the light and heavy chains of the monoclonal antibody may be humanized variable regions, respectively.) The second binding domain may comprise the light chain variable region, the heavy chain variable region, or both, of the DRA222, TSC455, or TSC456 CD3 binding domain. The amino acid sequences of DRA222, TSC455, and TSC456 are provided in Table 4. The DRA222 binding domain is also described in WO 2013 / 158856. TSC455 is also known as TSC394 F87Y. TSC455 is also known as TSC394 E86D F87Y or TSC394 DY.

[0185] In some embodiments, the second binding domain specifically binds CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 93% identical, at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 384; or an amino acid sequence that is at least 94% identical, at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 385; and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 82% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 383.

[0186] In some embodiments, the second binding domain is a CD3 binding domain comprising an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HCDR3 comprising SEQ ID NO: 21; and an LCDR1 comprising SEQ ID NO: 22, an LCDR2 comprising SEQ ID NO: 23, and an LCDR3 comprising SEQ ID NO: 24. In some embodiments, the CD3 antigen binding domain is an anti-CD3 scFv comprising a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 384. In some embodiments, the CD3 binding domain comprises a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 25. In some embodiments, the CD3 binding domain comprises a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 26. In some embodiments, the CD3 binding domain is an anti-CD3 scFv that comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:27.

[0187] In some embodiments, CD123-binding polypeptides or proteins that further comprise a CD3-binding domain may have low levels of high molecular weight aggregates generated during recombinant expression of the polypeptide or protein. CD123-binding polypeptides or proteins that further comprise a CD3-binding domain may exhibit relatively long stability in human serum, depending on the CD3-binding domain present in the polypeptide or protein.

[0188] In certain variations, the CD3 binding domain comprises one or more CD3 binding sequences (e.g., CDRs or variable regions) disclosed in U.S. Patent Application Publication Nos. 2013 / 0129730, 2011 / 0293619, U.S. Patent No. 7,635,472, WO 2010 / 037836, WO 2004 / 106381, or WO 2011 / 121110. In some embodiments, the CD3 binding domain comprises one or more mutations shown in Table 6. [Table 6]

[0189] In various embodiments, the CD3 binding domain comprises one or more of the sequences shown in Table 7. [Table 7]

[0190] In some embodiments, the therapeutic protein comprises, in order from amino terminus to carboxyl terminus, a first binding domain, a hinge region, an immunoglobulin constant region, and a second binding domain. In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the first binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, and HCDR3 comprises SEQ ID NO: 12. In some embodiments, LCDR1 comprises SEQ ID NO: 13, LCDR2 comprises SEQ ID NO: 14, and LCDR3 comprises SEQ ID NO: 15. In some embodiments, HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, and HCDR3 comprises SEQ ID NO: 12; and LCDR1 comprises SEQ ID NO: 13, LCDR2 comprises SEQ ID NO: 14, and LCDR3 comprises SEQ ID NO: 15. In some embodiments, the first binding domain comprises a sequence at least 95% identical to SEQ ID NO: 18. In some embodiments, the second binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 comprises SEQ ID NO: 19, HCDR2 comprises SEQ ID NO: 20, and HCDR3 comprises SEQ ID NO: 21. In some embodiments, LCDR1 comprises SEQ ID NO: 22, LCDR2 comprises SEQ ID NO: 23, and LCDR3 comprises SEQ ID NO: 24. In some embodiments, HCDR1 comprises SEQ ID NO: 19, HCDR2 comprises SEQ ID NO: 20, HCDR3 comprises SEQ ID NO: 21; and LCDR1 comprises SEQ ID NO: 22, LCDR2 comprises SEQ ID NO: 23, and LCDR3 comprises SEQ ID NO: 24. In some embodiments, the second binding domain comprises a sequence at least 95% or 100% identical to SEQ ID NO: 27. In some embodiments, the therapeutic protein comprises the sequence of SEQ ID NO: 31.

[0191] The structural format of the multispecific anti-CD123 and anti-CD3 molecules disclosed herein induces potent tumor cell lysis but reduced cytokine release compared to multispecific anti-CD123 and anti-CD3 molecules in other structural formats. Without being bound by any theory, the polypeptide structural format disclosed herein (e.g., in order from amino terminus to carboxyl terminus: (a) a binding domain that is a CD123-binding domain; (b) a hinge region; (c) an immunoglobulin constant region; and (d) a second binding domain that is a human or humanized binding domain that specifically binds T cells, CD3, CD3ε, or a T cell receptor (TCR) complex) induces a moderate level of T cell receptor (TCR) stimulation compared to other T cell engagers. It has been widely demonstrated that the strength or magnitude of the TCR signal regulates the outcome of T cell activation. TCR stimulation triggers multiple cellular events, including the initiation of effector functions (e.g., cytolysis), as well as cytokine secretion and cell division (Corse, Gottschalk, and Allison. J Immunol 2011, 186:5039-5045). These separate cellular events can proceed with different kinetics and reach different maximum levels, depending on the strength of the TCR stimulation and additional factors. The multispecific structural formats disclosed herein are potent enough to cause tumor cell lysis and induce multiple T cell divisions over multiple days, yet moderate enough to limit the amount of cytokine secretion.

[0192] In some embodiments, a multispecific polypeptide comprising a CD123 binding domain and a CD3 binding domain, when bound to the CD3 protein on T cells, induces reduced cytokine release from the T cells compared to an OKT3 antibody control. In some embodiments, a multispecific polypeptide comprising a CD123 binding domain and a CD3 binding domain reduces cytokine release from the T cells compared to a multispecific polypeptide comprising a CD3 binding domain derived from OKT3 or I2C. In some embodiments, a multispecific polypeptide comprising a CD123 binding domain (e.g., a CD123 binding domain comprising an amino acid sequence at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 312 and / or SEQ ID NO: 337) and a CD3 binding domain in an scFv-Fc-scFv format induces reduced cytokine release in non-human primates or humans compared to a bispecific polypeptide comprising a CD123 binding domain and an I2C-derived CD3 binding domain in a bispecific T cell engager (scFv-scFv) format or a dual affinity retargeting format.

[0193] Also provided herein are pharmaceutical compositions comprising the therapeutic proteins described herein. In some embodiments, the compositions comprise 1-20 mg / ml, 2.5-12 mg / ml, or 5-10 mg / ml of therapeutic protein. In some embodiments, the compositions comprise about 2.5 mg / ml to about 12 mg / ml, or about 5 mg / ml to about 10 mg / ml of therapeutic protein. In some embodiments, the compositions comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 mg / ml of therapeutic protein. In some embodiments, the compositions comprise about 5 mg / ml of therapeutic protein.

[0194] How to use The present disclosure provides methods for treating a subject for a disease or disorder, the methods comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure.

[0195] In some embodiments, the disease or disorder is cancer. The cancer may be selected from, for example, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, B-cell acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML).

[0196] In some embodiments, the disease or disorder can be an inflammatory disease or disorder. In some embodiments, the inflammatory disease or disorder can be an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is irritable bowel syndrome, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), psoriasis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, asthma, multiple sclerosis, dermatomyositis, polymyositis, pernicious anemia, primary biliary cirrhosis, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid syndrome (aPL), or autoimmune hepatitis. In some embodiments, the inflammatory disease or disorder is selected from type 1 diabetes, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, pemphigus vulgaris, Sjogren's syndrome, temporal arteritis, autoimmune hemolytic anemia, bullous pemphigoid, vasculitis, celiac disease, endometriosis, hidradenitis suppurativa, interstitial cystitis, morphea, scleroderma, narcolepsy, neuromyotonia, leukoplakia, autoimmune inner ear disease, and myasthenia gravis. In some embodiments, the inflammatory disease or disorder is psoriasis.

[0197] In some embodiments, the inflammatory disease or disorder can be a "neuroimmune disease," such as neuropathic pain, osteoarthritis, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease.

[0198] In some embodiments, the inflammatory disease or disorder can be an adverse transplant-related event, i.e., transplant rejection, allograft disease, or graft-versus-host disease.

[0199] In some embodiments, for the therapeutic methods and uses described herein, the proteins or polypeptides described herein are delivered in a manner compatible with conventional methods associated with the management of the disease or disorder for which treatment is indicated. In the present disclosure herein, a therapeutically effective amount of the protein or polypeptide is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.

[0200] Subjects for administration of the proteins of the present disclosure include patients at risk of developing a particular disorder as well as patients who have been shown to have such a disorder. Typically, the subject has been diagnosed with a disorder for which treatment is required. Furthermore, the subject may be monitored for any changes in the disorder during the course of treatment (e.g., an increase or decrease in clinical symptoms of the disorder). In some variations, the subject does not suffer from another disorder requiring treatment.

[0201] In preventive applications, pharmaceutical compositions or drugs comprising the proteins of the present disclosure are administered to patients susceptible to or at risk of a particular disorder in an amount sufficient to eliminate or reduce the risk of the disorder or delay its onset. In therapeutic applications, compositions or drugs comprising the proteins of the present disclosure are administered to patients suspected of or already suffering from such a disorder in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. An amount appropriate to achieve this is referred to as a therapeutically effective dose or amount. In both preventive and therapeutic regimens, agents are usually administered in several dosages until a sufficient response (e.g., suppression of inappropriate angiogenic activity) is achieved. Typically, the response is monitored, and if the desired response begins to wane, repeat dosages are administered.

[0202] To identify patients for treatment with the methods of the present disclosure, recognized screening methods can be used to determine risk factors associated with a particular disorder or to determine the status of a pre-existing disorder identified in a subject. Such methods can include, for example, determining whether an individual has relatives diagnosed with a particular disorder. Screening methods can also include routine workup to determine familial status for a particular disorder, for example, one known to have a hereditary component. For example, various cancers are also known to have a specific hereditary component. Hereditary components of cancer include, for example, mutations in multiple transforming genes (e.g., Ras, Raf, EGFR, cMet, etc.), specific HLA and killer cell inhibitory receptor (KIR) molecules, or mechanisms by which cancer cells can directly or indirectly regulate the immune suppression of cells such as NK cells and T cells (see, for example, Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann, Clin. Exp. Immunol. 149:1-8, 2007). For this purpose, nucleotide probes can be routinely used to identify individuals who carry genetic markers associated with specific disorders of interest.In addition, a variety of immunological methods are known in the art that are useful for identifying markers of specific disorders.For example, various ELISA immunoassay methods are available and known in the art, using monoclonal antibody probes to detect antigens associated with specific tumors.Screening can be carried out as indicated by known patient symptomatology, age factors, related risk factors, etc.These methods allow clinicians to routinely select patients who need the methods described herein for treatment.

[0203] For administration, the pharmaceutical compositions of the present disclosure may comprise (i) a therapeutic protein / polypeptide; and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition may comprise (i) a therapeutic protein / peptide, (ii) a buffer, (iii) an excipient, and (iv) a surfactant.

[0204] Pharmaceutical compositions comprising the polypeptides or proteins described herein may be formulated into a dosage form selected from the group consisting of oral unit dosage forms, intravenous unit dosage forms, intranasal unit dosage forms, suppository unit dosage forms, intradermal unit dosage forms, intramuscular unit dosage forms, intraperitoneal unit dosage forms, subcutaneous unit dosage forms, epidural unit dosage forms, sublingual unit dosage forms, and intracerebral unit dosage forms. The oral unit dosage form may be selected from the group consisting of tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained release formulations, aerosols, and sprays.

[0205] Pharmaceutical compositions comprising polypeptide or protein described herein can be administered to subjects in therapeutically effective amounts.In the method of the present disclosure, polypeptide or protein described herein can be administered to subjects by various administration methods, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal and oral administration routes.For the purpose of prevention and treatment, antagonist can be administered to subjects by single bolus delivery, by long-term continuous delivery, or by repeated administration protocol (for example, hourly, daily, weekly or monthly).

[0206] In this regard, the determination of an effective dosage is typically based on animal model studies followed by human clinical trials, and is further guided by determining an effective dosage and administration protocol that significantly reduces the incidence or severity of the target disorder in the model subject. The effective amount of the composition of the present disclosure will vary depending on many factors, including the means of administration, the target site, the patient's physiological condition, whether the patient is human or animal, other drugs administered, whether the treatment is prophylactic or therapeutic, and the specific activity of the composition itself and its ability to elicit the desired response in an individual. Typically, the patient is a human, although for some diseases, the patient may be a non-human mammal. Typically, the administration regimen is adjusted to provide an optimal therapeutic response, i.e., to optimize safety and efficacy.

[0207] Also provided herein is the use of a composition of the present disclosure in the manufacture of a medicament for the treatment of cancer. For example, the composition of the present disclosure can be used for the treatment of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Also provided is a method comprising administering to a patient a composition comprising a multispecific polypeptide comprising a CD123-binding domain and a CD3-binding domain at a weekly dose of about 0.3, about 1, about 3, about 6, about 9, about 12, about 18, about 20, about 24, about 30, about 36, about 50, about 48, about 60, about 75, or about 100 μg via IV infusion. Typically, patients are treated once or twice weekly for 4 to 6 weeks. Patients may receive the same dose each week, or the dose may be increased, for example, weekly.

[0208] In some embodiments, the dosage is increased weekly, with the initial dosage being less than the amount that the patient would be expected to tolerate. This type of titration regimen reduces the risk of the patient developing infusion-related reactions or cytokine release syndrome. In some embodiments, a multispecific protein comprising a CD123-binding domain and a CD3-binding domain (e.g., TRI130 or TRI129) can be administered intravenously to a patient, with at least the first two or three doses being increased weekly. For example, the composition of the present disclosure can be administered by IV infusion according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks: 12 μg. In some embodiments, a patient can be administered the composition of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks: 18 μg. In some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: week 1 dose: 6 μg; and week 2 and subsequent weeks dose: 9 μg, and in some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: week 1 dose: 9 μg; and week 2 and subsequent weeks dose: 12 μg. In other embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: week 1 dose: 12 μg; and week 2 and subsequent weeks dose: 18 μg.

[0209] In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks dose: 12 μg. In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks dose: 18 μg. In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 12 μg; and Week 4 and subsequent weeks dose: 12 μg. In some embodiments, patients may receive a composition of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; and Week 4 and subsequent weeks dose: 24 μg. In some embodiments, patients may receive a composition of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; and Week 4 and subsequent weeks dose: 36 μg. In some embodiments, patients may receive a composition of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; and Week 4 and subsequent weeks dose: 48 μg. In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; and Week 4 and subsequent weeks dose: 60 μg.

[0210] In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following treatment schedule: Day 1: 6 μg; Day 2: 9 μg; Day 3: 12 μg; Day 4: 18 μg; Day 8: 18 μg; Day 11: 18 μg; Day 15: 36 μg; Day 22: 36 μg; followed by a weekly dose of 36 μg.

[0211] In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following treatment schedule: Day 1: 6 μg; Day 2: 12 μg; Day 3: 18 μg; Day 4: 24 μg; Day 8: 24 μg; Day 11: 24 μg; Day 15: 48 μg; Day 22: 48 μg; followed by a weekly dose of 48 μg.

[0212] In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following treatment schedule: Day 1: 6 μg; Day 2: 12 μg; Day 3: 24 μg; Day 4: 36 μg; Day 8: 36 μg; Day 11: 36 μg; Day 15: 60 μg; Day 22: 60 μg; followed by weekly doses of 60 μg.

[0213] In some embodiments, patients may be administered the compositions of the present disclosure intravenously according to the following treatment schedule: Day 1: 6 μg; Day 2: 12 μg; Day 3: 24 μg; Day 4: 36 μg; Day 8: 48 μg; Day 11: 48 μg; Day 15: 100 μg; Day 22: 100 μg; followed by weekly doses of 100 μg.

[0214] In some embodiments, a method for treating a patient in need thereof comprises administering to the patient a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain on days 1, 8, 15, and 22. In some embodiments, 6 μg is administered on day 1, 9 μg on day 8, 12 μg on day 15, and 12 μg on day 22. In some embodiments, 6 μg is administered on day 1, 9 μg on day 8, 12 μg on day 15, and 18 μg on day 22. In some embodiments, 6 μg is administered on day 1, 9 μg on day 8, 9 μg on day 15, and 9 μg on day 22. In some embodiments, 9 μg is administered on day 1, 12 μg on day 8, 12 μg on day 15, and 12 μg on day 22. In some embodiments, 12 μg is administered on day 1, 18 μg is administered on day 8, 18 μg is administered on day 15, and 18 μg is administered on day 22.

[0215] In some embodiments, patients treated according to the methods of the present disclosure exhibit a decrease in bone marrow blast percentage, and in some embodiments, patients exhibit a decrease in absolute blast counts in the blood. In some embodiments, treatment results in a decrease in the patient's blast levels of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the patient's levels immediately before treatment.

[0216] In some embodiments, patients treated according to the methods of the present disclosure exhibit a complete remission (CR). As used herein, a complete remission is defined as a reduction of bone marrow blasts by up to 5%, absence of circulating blasts and blasts with Auer rods, absence of extramedullary disease, and an absolute neutrophil count (ANC) of ≥ 1.0 x 10 9 / L (1,000 / μL) and PLT ≥ 100x10 9 / L (100,000 / μL). In some embodiments, patients treated according to the methods of the present disclosure have minimal residual disease (CR). MRD ) without the CR. As used herein, CR MRD refers to a CR that is negative for genetic markers by quantitative reverse transcription polymerase chain reaction (RT-qPCR) or negative by multiparameter flow cytometry. In some embodiments, patients treated according to the methods of the present disclosure achieve complete remission (CR) with inadequate recovery of hematopoietic function. i ) indicates CR. i Residual neutropenia (ANC < 1.0x10 9 / L [1,000 / μL]) or thrombocytopenia (PLT < 100x10 9 Includes all criteria for CR listed above except for CR (100,000 / μL [100,000 / μL]).

[0217] In some embodiments, patients treated according to the methods of the present disclosure exhibit a morphologically leukemia-free state (MLFS). As used herein, MLFS refers to bone marrow blasts <5% (i.e., the bone marrow should not be simply "aplastic"; at least 200 cells should be counted or cellularity should be at least 10%); the absence of blasts with Auer rods; and the absence of extramedullary disease. Hematological recovery is not required.

[0218] In some embodiments, patients treated according to the methods of the present disclosure exhibit a partial response (PR). As used herein, PR includes all hematological criteria of a CR described above plus a reduction in bone marrow blast percentage to 5-25% and at least a 50% reduction in pretreatment bone marrow blast percentage.

[0219] In some embodiments, patients treated according to the methods of the present disclosure exhibit stable disease (SD) and CR. MRD ,CR,CR i , PR, and MLFS, but without progressive disease (i.e., an increase in the percentage of bone marrow blasts in the blood and / or an increase in absolute blast count).

[0220] Patients treated with a CD123 x CD3-targeting multispecific polypeptide (e.g., TRI130 or TRI129) at the same weekly dose or on an escalating treatment regimen may also have their infusion time (i.e., length of infusion) adjusted to further reduce the likelihood of infusion reactions or cytokine release syndrome. To reduce the risk of adverse events, the first dose is administered IV to the patient over several hours, e.g., about 20-24 hours. In some embodiments, the first dose of the composition is administered over a period of about 20-24 hours, the second dose over a period of about 8 hours, the third dose over a period of about 6 hours, and the fourth and subsequent doses over a period of about 4 hours. In some embodiments, the first dose of the composition is administered over a period of about 20-24 hours, the second dose over a period of about 8 hours, the third dose over a period of about 6 hours, and the fourth and subsequent doses over a period of about 4 hours, with the first, second, third, and fourth doses being the same. The composition can also be administered to a subject by continuous IV infusion, for example, for up to 72 hours.

[0221] Patients treated with a CD123 x CD3 targeting multispecific polypeptide (e.g., TRI130 or TRI129) may also be treated with one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered simultaneously or at about the same time as the CD123 x CD3 targeting multispecific polypeptide. In some embodiments, the one or more additional therapeutic agents are administered prior to administration of the multispecific polypeptide (i.e., as a "premedication"), such as about 1-3 hours prior to administration. In some embodiments, the one or more additional therapeutic agents are administered after administration of the one or more multispecific polypeptides.

[0222] In some embodiments, the one or more additional therapeutic agents are diphenhydramine, acetaminophen, and / or dexamethasone. In some embodiments, the one or more additional therapeutic agents may be administered intravenously or orally. In some embodiments, dexamethasone may be administered at a dosage of about 10 to about 20 mg. In some embodiments, methylprednisolone may be administered at a dosage of about 1 mg / kg. In some embodiments, acetaminophen may be administered at a dosage of about 650 or about 1,000 mg. In some embodiments, acetaminophen is administered three times daily for one day, with the first dose administered 1 to 3 hours before administration of the CD123 x CD3-targeting multispecific polypeptide. In some embodiments, the one or more additional therapeutic agents may include an antihistamine, such as diphenhydramine. Diphenhydramine may be administered at a dosage of about 50 mg. In some embodiments, the one or more additional therapeutic agents may include allopurinol. In some embodiments, allopurinol is administered at least two days prior to administration of the CD123 x CD3 targeting multispecific polypeptide. In some embodiments, the one or more additional therapeutic agents may include tocilizumab.

[0223] In some embodiments, a method for treating a disorder characterized by overexpression of CD123 in a patient in need of treatment comprises administering to the patient an effective amount of a pharmaceutical composition comprising a recombinant polypeptide comprising a CD123-binding domain and a CD3-binding domain (e.g., TRI130 or TRI129) at any dosage or dosing regimen described herein. In some embodiments, a method for treating a disorder characterized by overexpression of CD123 in a patient in need of treatment comprises administering to the patient an effective amount of a pharmaceutical composition comprising a recombinant polypeptide comprising a CD123-binding domain and a CD3-binding domain (e.g., TRI130 or TRI129) at any dosage or dosing regimen described herein, wherein administration of the pharmaceutical composition induces reduced cytokine levels in the subject compared to administration of (a) a dual affinity retargeting antibody comprising the CD123-binding domain and the CD3-binding domain of the recombinant polypeptide; or (b) a bispecific T cell engager molecule comprising the CD123-binding domain and the CD3-binding domain of the recombinant polypeptide. In some embodiments, the disorder is cancer, e.g., AML or MDS. In some embodiments, the subject was previously treated with a different CD123 binding molecule, and the subject experienced an adverse event after the previous treatment. In some embodiments, the adverse event was excessive cytokine release. In some embodiments, the cytokine levels were levels of IFNγ, TNFα, IL-6, IL-2, IL-8, IL-10, IL-17, GM-CSF, IL-4, IL-12, IL-13, or IL-1β, or any combination thereof. In some embodiments, the cytokine levels were levels of IFNγ, IL-2, TNFα, and IL-10. In some embodiments, the cytokine levels are measured in an in vitro activated T cell assay.

[0224] Pharmaceutical compositions comprising the proteins and polypeptides described herein can be supplied as kits containing a container containing the pharmaceutical composition described herein. The pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted before injection. Such kits may further include written information regarding the indications and usage of the pharmaceutical composition. In some embodiments, the kit includes the pharmaceutical composition and an IV stabilization solution (0.1 M succinate buffer and 0.08% w / v polysorbate 80, pH 6.0, or a similar solution designed to prevent or reduce the potential for adhesion of the multispecific polypeptide to plastic tubing and bags).

[0225] The present disclosure will be further clarified by the following examples, which are merely illustrative of the disclosure and are not intended to be limiting in any way.

[0226] Example The present invention will be described in further detail by reference to the following examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the present invention should not be construed as being limited to the following examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following examples, make and utilize the compounds of the present invention and practice the claimed methods. The following examples, therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0227] Example 1: Anti-CD123 x Anti-CD3 Bispecific Protein Formulation It is important that therapeutic protein drugs maintain their formulation quality during long-term storage at distribution centers, transportation, and storage and administration sites. After production and formulation, proteins can be exposed to a variety of temperatures, including frozen (-80°C, -20°C), refrigerated (4°C), and room temperature (25°C). Conditions that affect the stability of protein drugs include pH, protein concentration, and the concentrations of salts and excipients that may be included in the formulation.

[0228] To evaluate a 5 mM succinate, 6.5% sucrose, 0.02% w / v polysorbate 80 (abbreviated as SSuT), pH 4.8 formulation for anti-CD123 x anti-CD3 bispecific (anti-CD123-hinge region-CH2-CH3-anti-CD3), Chinese hamster ovary (CHO) cells were stably transfected with DNA plasmids encoding TRI-129 or TRI-130 along with a selectable marker. After transfection, these cells were grown under selective pressure to ensure stable integration of the bispecific protein-encoding genes into the CHO cell genome and the death of cells that did not contain the selectable marker.

[0229] TRI-129 and TRI-130 CHO pools were initially grown in shake flasks and then transferred to 10 L bioreactors and cultured in the designated animal component-free medium. After approximately two weeks of culture, the cell culture supernatant was clarified using a combination of depth filtration and sterile filtration, followed by purification using a combination of affinity and mixed-mode column chromatography. The two-step purified protein was diafiltered into succinate buffer using a tangential flow filtration (TFF) device, followed by the addition of appropriate volumes of concentrated stock sucrose solution and polysorbate 80 to obtain the designated composition. TRI-129 and TRI-130 ADAPTIR proteins were evaluated in both the 2 mg / mL and 10 mg / mL preferred formulation buffers. Stability in SSuT was compared to protein in Dulbecco's phosphate-buffered saline (dPBS).

[0230] In one experiment, TRI-129 and TRI-130 were formulated in SSut or PBS, and samples were stored at 4°C and 25°C. Sample purity was measured using an analytical size exclusion chromatography (SEC) assay at the beginning of the study and after 9 days of storage. The change in % purity of the formulation after 9 days is reported in Table 8 below. Negative values ​​for samples in dPBS indicate a decreasing purity of the sample, indicating a redistribution of the formulation peak area from the main peak to aggregates / higher molecular weight species (HMW). After 9 days, both TRI-129 and TRI-130 in SSuT showed much smaller purity changes compared to samples in dPBS, indicating that this formulation has significant stabilizing properties. [Table 8]

[0231] During production and / or storage, it is common for protein therapeutics to be subjected to one or more freeze-thaw cycles from -80°C or -20°C to 4°C or room temperature. It is important that the selected bulk active pharmaceutical ingredient and formulation stabilize the protein and prevent dramatic changes in drug quality. TRI-129 and TRI-130 formulated in SSuT were investigated for their ability to resist freeze aggregation. Freeze aggregation is the formation of aggregates resulting from freezing and thawing a protein solution. The stability of TRI-129 and TRI-130 was investigated at 2 and 10 mg / mL. After assessing sample purity by analytical SEC, 200 μL was placed at -80°C or -20°C for several hours to allow sufficient time for the sample to freeze completely. The sample was removed from the freezer and allowed to thaw completely at room temperature. The sample was subjected to a total of three freeze-thaw cycles at -80°C or -20°C and then retested by SEC to measure the change in %MP. As shown in Table 9 below, both TRI-129 and TRI-130 showed minimal change in %MP at either -80° C. or -20° C. The higher protein concentration sample at 10 mg / mL showed a slightly greater change in %MP than the sample at 2 mg / mL. [Table 9]

[0232] Example 2: Determination of the No Observed Adverse Effect Level (NOAEL) and the Estimated Minimum Effective Level (MABEL) for TRI130 No observed adverse effect level (NOAEL) A 28-day repeat-dose toxicity study, including a 5-week recovery period, was conducted in nonhuman primates (NHPs). Animals in four study groups received weekly doses of vehicle or 0.5, 2.5, or 10 mg / kg TRI130. Measured parameters included safety pharmacology, laboratory evaluation, and necropsy with complete histopathology. There were no TRI130-related clinical adverse findings, changes in animal or organ weights, or abnormal macroscopic or microscopic findings in histopathology. Minimal cytokines were detected after dosing and attenuated after the second dose compared with the first. The expected pharmacodynamic effects of the anti-CD3 binding domain of the molecule were observed, accompanied by transient T cell redistribution. The elimination half-life was approximately 73 hours at the high dose. The no-observed-adverse-effect level (NOAEL) was 10 mg / kg in NHPs, which corresponds to a human equivalent dose (HED) of approximately 3.2 mg / kg.

[0233] Estimated Minimum Pharmacological Effective Level (MABEL) TRI130 recruits T cells to lyse tumor cells expressing the target antigen CD123, with maximal activity occurring at very low levels of TCR occupancy (data not shown). To calculate the estimated minimum effective dose (MABEL), an in vitro activity assay was used instead of in vitro receptor occupancy.

[0234] The MABEL was determined using the effective concentration required to elicit 10% activity (EC10) (Muller et al., 2009). While the potency of TRI130 in these T cell activation assays may vary depending on the degree of T cell activation and the effector-to-target cell ratio (E:T), activation assays are more sensitive for calculating the MABEL than redirected T cell cytotoxicity (RTCC) assays. Both RTCC and T cell activation assays were evaluated as potential assays for predicting the MABEL relative to the starting clinical dose. The RTCC assay, using purified T cells with the CD123+KG1a tumor cell line at a 10:1 E:T ratio, had a mean EC10 of 5.8 pM evaluated from five donors (ranging from 4.4 to 6.7 pM across donors evaluated). In contrast, the T cell activation assay was a more sensitive assay for estimating the MABEL. To measure in vitro-induced T cell activation, T cells were isolated from peripheral blood mononuclear cells and incubated with TRI130 in the presence of CD123+ tumor cells (MOLM-13). The upregulation of CD69 and CD25 on T cells was monitored for 20 hours using multicolor flow cytometry after gating on viable CD4+ and CD8+ T cells. These assays assessed activation of both CD4 and CD8 T cells from three donors. The average EC10 calculations for these assays were 1.2 pM (range 0.7–1.6 pM) for CD4 T cells and 1.3 pM (range 0.9–2.0 pM) for CD8 T cells. This assay was a more conservative estimate of MABEL and was used to estimate the starting dose for patients. For TRI130, 0.7 pM (0.113 ng / ml) was the most conservative approach for MABEL based on the CD4+ T cell responses from donors.

[0235] Elimination and volume estimates for Group 4 in the single-dose NHP study were determined using a WinNonlin (v6.4) precompiled two-compartment model for intravenous (IV) administration. Allometric scaling was used to predict human elimination and volume parameter estimates that could be used to simulate dosing strategies that could result in a Cmax below the EC10 (MABEL) value of 0.7 pM, the lowest EC10 measured from an individual donor in an activation assay. Using this modeling, a dose of approximately 0.005 μg / kg would have a Cmax below the MABEL concentration of 0.113 ng / mL (0.7 pM).

[0236] A flat dose or fixed dose, rather than weight-based dosing, will be utilized in this study. Several studies have shown that fixed dosing compared to weight-based dosing performed similarly across many similar monoclonal antibodies, and that the PK variability introduced by either dosing regimen was modest compared to the variability typically observed in pharmacodynamics, efficacy, and safety (Wang et al., 2009). Assuming a 60 kg patient and a MABEL dose of 0.005 μg / kg, the starting dose in the study is 0.3 μg.

[0237] Example 3: Administration of TRI130, a Formulated Anti-CD123 x Anti-CD3 Therapeutic Candidate, to Patients In an ongoing Phase 1 / 1b, open-label, dose-escalation study of patients with relapsed or refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), patients are administered formulated TRI130 (5 mM succinate, 6.5% sucrose, 0.02% w / v polysorbate 80, pH 4.8). The study is conducted in two parts. The first part is a Phase 1, open-label, dose-escalation study to determine the recommended dose for Phase 1b. Phase 1b is an open-label proliferation study to evaluate the clinical activity and safety of the drug at the recommended dose. The study design is outlined in Figure 2. Endpoints include safety, immunogenicity, pharmacokinetics, pharmacodynamics, and clinical activity.

[0238] Table 10 is the dosing schedule showing the amount administered weekly (IV) to Cohorts 1-10 (dose escalation cohorts). In both parts of the study, patients will receive the drug intravenously weekly for six 28-day cycles unless there is disease progression, intolerable toxicity, or early withdrawal of consent. If patients respond, there is the option for longer-term treatment. [Table 10]

[0239] The drug is supplied in a single-use vial containing 2 mg of formulation in 1 mL of liquid at a concentration of 2 mg / mL. The formulation is mixed with an IV stabilizing solution to prevent adhesion of the formulation to the IV bag and IV tubing set. The stabilizing solution is supplied sterile in a 10 mL vial consisting of 0.1 M succinate buffer and 0.08% w / v polysorbate 80, pH 6.0, and is refrigerated (2-8°C).

[0240] For Cohorts 1-4, dosing is administered by IV infusion over approximately 20-24 hours during the first dose (Cycle 1, Day 1), 8 hours (± 1 hour) during the second dose (Cycle 1, Day 8), 6 hours (± 1 hour) during the third dose (Cycle 1, Day 15), and 4 hours (± 1 hour) during all subsequent doses (Cycle 1, Day 22 and beyond). For Cohorts 5 and beyond, dosing is administered by IV infusion in hourly escalations over 20-24 hours after the first dose. The same dose is administered over 8 hours (± 30 minutes) for the second dose, 6 hours (± 30 minutes) for the third dose, and 4 hours (± 30 minutes) for the fourth and all subsequent doses.

[0241] If necessary, all dose infusions may be delayed and / or interrupted to extend the administration time for up to 72 hours to manage or prevent any adverse events, particularly infusion-related reactions (IRR) or cytokine release syndrome (CRS). If the infusion is extended beyond 60 hours, patients should be observed for 12 hours after the completion of the infusion. Table 10 discloses a stepped dosing schedule (starting with Cohort 5) that has the potential to reduce the likelihood of IRR and / or CRS.

[0242] The frequency of patient dosing is weekly for up to 6 months. The weekly dosing schedule was selected based on cynomolgus monkey toxicology studies with escalating TRI130 doses (data not shown). The half-life of TRI130 after a single dose for individual animals receiving 0.25-1 mg / kg ranged from approximately 25-113 hours, with longer half-life estimates associated with animals in the higher dose group (1 mg / kg).

[0243] To mitigate infusion-related reactions (IRR) and cytokine release syndrome (CRS), patients receive post-premedication: diphenhydramine, acetaminophen, and dexamethasone. All premedications are administered 1-3 hours before the infusion begins. The dose of any premedication may be reduced if necessary due to complications, in the opinion of the investigator. Dexamethasone is optional after cycle 2, day 15, unless the patient experiences an IRR or CRS with the previous dose. The doses of the three premedications are: 1. Dexamethasone 10-20 mg IV or methylprednisolone 1 mg / kg IV, or equivalent; dosage at the investigator's discretion based on patient comorbidities. 2. Acetaminophen 650 or 1,000 mg, or equivalent, administered orally (PO) three times daily for one day (650 mg or 1,000 mg dose at the investigator's discretion), with the first dose administered 1-3 hours before the study drug infusion; and 3. Antihistamine: Diphenhydramine 50 mg PO or IV, or equivalent. If the investigator chooses to administer allopurinol for tumor lysis prophylaxis, it must be initiated at least 2 days before the start of study drug.

[0244] Phase 1 - dose escalation study Treatment was initiated at the estimated minimum effective dose (MABEL) in patient cohorts. Enrolled patients had either: 1) relapsed or refractory AML and were ineligible for or refused intensive chemotherapy or allogeneic stem cell transplantation; or 2) relapsed or refractory MD, with more than 5% blasts in the bone marrow or any circulating blasts in the peripheral blood, and had failed a previous hypomethylating agent (HMA); failure was defined as intolerance to the HMA, lack of response (no CR for at least six cycles), or IWG-defined progression during or after treatment with an HMA. The demographics of the 32 patients enrolled in the ongoing Phase 1 dose-escalation study (through Cohort 7) are shown in Table 11 below. For patients enrolled through Cohort 7, the median age was 67 years, and 79% of patients had AML. The average number of doses administered per patient was 8.5, and the average treatment duration was 54 days. [Table 11] JPEG2026021465000028.jpg21159

[0245] Treatment-related adverse events in 32 patients enrolled in the ongoing Phase 1 dose-escalation study are shown in Table 12 below. 34% of patients experienced one or more IRR / CRS events (grade ≥ 3, reported in 16%). The most common symptoms were dyspnea, fever, hypotension, hypoxia, tachycardia, and chills / chills. Notably, IRR / CRS was the only treatment-related serious adverse event occurring in more than two patients. Three of the 11 patients who experienced IRR / CRS events received tocilizumab to treat them. [Table 12]

[0246] The percentage of blasts in the patients' bone marrow aspirates was monitored over time. As shown in Figures 3A-3D, a decrease in bone marrow blasts was observed in several patients receiving the highest dose of 12 μg or higher. Two patients had a decrease in bone marrow blasts of 29% to 0% (Cohort 6b, Figure 3C) and 33% to 4% (Cohort 6a, Figure 3B), respectively. Absolute neutrophil and platelet counts met the criteria for complete remission. Both patients, as well as the one blast-reduced patient in Cohort 7, remain on study.

[0247] Serum cytokines were assessed at scheduled time points before and after administration of each patient's maximum dose, and also at intervals during infusion-related reaction or cytokine release syndrome events. As shown in Figures 4A-4D, cytokines were not assessed between scheduled collections. Increased cytokines, particularly IL-6, were observed during IRR / CRS adverse events. Notably, in this small dataset, no correlation was observed between maximum cytokine concentrations and dose level or event grade.

[0248] Thus, in this preliminary study, administration of TRI130 at a dose of 24 μg was tolerated with a manageable safety profile. As noted above, two patients had complete responses (CRs). Cytokines were not significantly elevated unless IRR / CRS adverse events were concurrently present. Preliminary data did not suggest evidence of treatment-induced antidrug antibodies (ADAs).

[0249] Phase 1 - Additional Dose Escalation Cohorts Following completion of the dose-limiting toxicity (DLT) observation period in Cohort 7, patients will be enrolled in four additional sequential cohorts (Cohorts A, B, C, and D) while Cohorts 8 and above are treated. These cohorts will be run sequentially and separate from Cohorts 8–10, achieving more rapid dose escalation.

[0250] Patients in Cohorts A, B, C, and D receive continuous IV dosing (20-24 hours / day) over the first 4 days of Cycle 1, then twice weekly during Week 2, followed by weekly dosing for Cycle 1 and the remaining cycles. Table 14 shows the dosing schedules for Cohorts A, B, C, and D. Week 1 of dosing for Cohort A utilizes the dosing tested in Cohort 6a (6 μg on Day 1, 9 μg on Day 2, 12 μg on Day 3, and 18 μg on Day 4). During Week 2, the 18 μg dose is administered on Days 8 and 11. In Week 3, the dose is increased to 36 μg and maintained at this level. The benefit of escalating the daily dose during Week 1 is that C max The dose is gradually increased, which may reduce the likelihood of IRR / CRS. Active treatment with tocilizumab is administered for symptomatic treatment of grade ≥2 and for unresponsive IRR or CRS within 2 hours of the middle administration period. [Table 13]

[0251] For Cohorts A-D, dosing will be by IV infusion over 20-24 hours with hourly dose escalation. The same dose will be administered over 8 hours (±30 minutes) for the second dose, over 6 hours (±30 minutes) for the third dose, and over 4 hours (±30 minutes) for the fourth and all subsequent doses.

[0252] Premedication will be administered prior to Cycle 1, Day 1; Cycle 1, Day 8; Cycle 1, Day 11; Cycle 1, Day 15; and Cycle 1, Day 22. For all subsequent doses, dexamethasone is optional, but acetaminophen and diphenhydramine are required.

[0253] Phase 1b - proliferation The recommended dosing regimen will be further investigated in two expansion cohorts of the same type of patient: 1) Cohort 1 consists of 24 patients with relapsed or refractory AML who are ineligible for intensive chemotherapy or allogeneic stem cell transplantation, and 2) Cohort 2 consists of 24 patients with relapsed or refractory MD who have more than 5% blasts in the bone marrow or any circulating blasts in the peripheral blood and who have failed a previous HMA; failure is defined as intolerance to the HMA, lack of response (no CR for at least 6 cycles), or IWG-defined progression during or after treatment with the HMA. Serum samples will be collected for serial PK assessment of drug levels.

Claims

1. 1. A composition comprising a multispecific protein, a buffer, an excipient, and a surfactant, (a) the multispecific protein is a dimer of two identical polypeptides, each polypeptide arranged in amino-terminal to carboxyl-terminal order or in carboxyl-terminal to amino-terminal order: (i) a first binding domain; (ii) the hinge region; (iii) an immunoglobulin constant region, and (iv) a second binding domain; and (b) the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof; composition.

2. 10. The composition of claim 1, comprising about 1 mM to about 10 mM of a succinate ester or a pharmaceutically acceptable salt or acid thereof.

3. 3. The composition of claim 2, comprising about 5 mM succinate or a pharmaceutically acceptable salt or acid thereof.

4. The composition of any one of claims 1 to 3, wherein the excipient comprises or consists of a sugar.

5. The composition of claim 4 , wherein the sugar is sucrose.

6. 6. The composition of claim 4 or 5, comprising about 1% weight / volume (w / v) to about 12% w / v of the sugar.

7. The composition of claim 6, comprising about 6.5% (w / v) of the sugar.

8. The composition of any one of claims 1 to 7, wherein the surfactant comprises or consists of polysorbate 80.

9. 9. The composition of claim 8, comprising about 0.02% w / v polysorbate 80.

10. 10. The composition of any one of claims 1 to 9, comprising about 0.1 mg / ml to about 10 mg / ml of the multispecific protein.

11. 11. The composition of claim 10, comprising about 1 mg / ml to about 5 mg / ml of the multispecific protein.

12. 12. The composition of claim 11, comprising about 2 mg / ml of the multispecific protein.

13. 13. The composition of any one of claims 1 to 12, comprising about 5 mM succinate, about 6.5 weight / volume (w / v) % sucrose, and about 0.02 w / v % polysorbate 80.

14. The composition of any one of claims 1 to 13, having a pH of about 4.0 to about 5.

5.

15. 15. The composition of claim 14 having a pH of about 4.

8.

16. The composition of any one of claims 1 to 15, wherein the immunoglobulin constant region is a human Fc domain.

17. 17. The composition of any one of claims 1 to 16, wherein the immunoglobulin constant region comprises immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD.

18. The composition of any one of claims 1 to 17, wherein the first binding domain is a CD3-binding domain and the second binding domain is a tumor antigen-binding domain.

19. 19. The composition of claim 18, wherein the polypeptide comprises, from N-terminus to C-terminus, the CD3 binding domain, the hinge region, the immunoglobulin constant region, and the tumor antigen binding domain.

20. The composition of any one of claims 1 to 17, wherein the first domain is a tumor antigen-binding domain and the second binding domain is a CD3 antigen-binding domain.

21. 21. The composition of claim 20, wherein the polypeptide comprises, from N-terminus to C-terminus, the tumor antigen binding domain, the hinge region, the immunoglobulin constant region, and the CD3 binding domain.

22. The composition of any one of claims 1 to 17, wherein the first binding domain is a 4-1-BB binding domain and the second binding domain is a tumor antigen binding domain.

23. The composition of claim 22, wherein the polypeptide comprises, from N-terminus to C-terminus, the 4-1-BB binding domain, the hinge region, the immunoglobulin constant region, and the tumor antigen binding domain.

24. The composition of any one of claims 1 to 17, wherein the first binding domain is a tumor antigen domain and the second binding domain is a 4-1-BB binding domain.

25. The composition of claim 24, wherein the polypeptide comprises, from N-terminus to C-terminus, the tumor antigen-binding domain, the hinge region, the immunoglobulin constant region, and the 4-1-BB binding domain.

26. The composition of any one of claims 18 to 25, wherein the tumor antigen-binding domain binds to CD123, PSMA, CD19, CD33, or HER2.

27. The composition of any one of claims 1 to 17, wherein the first binding domain or the second binding domain is a 4-1-BB binding domain.

28. The composition of any one of claims 1 to 17, wherein the first binding domain or the second binding domain is an OX40 binding domain.

29. The composition of any one of claims 1 to 17, wherein the first binding domain is a 4-1-BB binding domain and the second binding domain is an OX40 binding domain.

30. The composition of any one of claims 1 to 17, wherein the first binding domain is an OX40 binding domain and the second binding domain is a 4-1-BB binding domain.

31. The composition of claim 29 or 30, wherein the 4-1-BB binding domain is an scFv and the OX40 binding domain is an scFv.

32. The composition of any one of claims 1 to 17, wherein the first binding domain is an OX40 domain and the second binding domain is a tumor antigen-binding domain.

33. The composition of any one of claims 1 to 17, wherein the first binding domain is a tumor antigen-binding domain and the second binding domain is an OX40-binding domain.

34. At least one of the first binding domain and the second binding domain comprises: (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3; The composition of any one of claims 1 to 33, comprising:

35. 35. The composition of any one of claims 1 to 34, wherein at least one of the first binding domain and the second binding domain is a single chain variable fragment (scFv).

36. 36. The composition of claim 35, wherein the light chain variable region of the scFv is carboxy-terminal to the heavy chain variable region of the scFv.

37. 36. The composition of claim 35, wherein the light chain variable region of the scFv is amino terminal to the heavy chain variable region of the scFv.

38. The composition of any one of claims 35 to 37, wherein the scFv comprises a linker polypeptide.

39. 39. The composition of claim 38, wherein the linker polypeptide is between the light chain variable region and the heavy chain variable region of the scFv.

40. The linker polypeptide is Gly ４ 40. The composition of claim 38 or 39, comprising Ser (SEQ ID NO: 128).

41. The linker polypeptide has the formula (Gly ４ Ser) ｎ 41. The composition of claim 40, comprising:

42. the tumor antigen-binding domain is HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 11, and HCDR3 comprising SEQ ID NO: 12; and LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 14, and LCDR3 comprising SEQ ID NO: 15; The composition of any one of claims 18 to 26, which is an anti-CD123 scFv comprising:

43. 27. The composition of any one of claims 18 to 26, wherein the tumor antigen-binding domain is an anti-CD123 scFv comprising a VH comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 136, and a VL comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:

134.

44. 27. The composition of any one of claims 18 to 26, wherein the tumor antigen-binding domain is an anti-CD123 scFv, and the scFv comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:

27.

45. the CD3 binding domain is HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 20, and HCDR3 comprising SEQ ID NO: 21; and LCDR1 comprising SEQ ID NO: 22, LCDR2 comprising SEQ ID NO: 23, and LCDR3 comprising SEQ ID NO: 24; The composition of any one of claims 18 to 21, which is an anti-CD3 scFv comprising:

46. The composition of any one of claims 18 to 21, wherein the CD3 antigen-binding domain is an anti-CD3 scFv comprising a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO:

384.

47. The composition of any one of claims 18 to 21, wherein the CD3 binding domain is an anti-CD3 scFv comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:

27.

48. 48. The composition of any one of claims 18-21 or 45-47, wherein each polypeptide comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:

31.

49. 49. The composition of any one of claims 1 to 48, wherein the immunoglobulin constant region comprises one, two, three or more amino acid substitutions compared to a wild-type immunoglobulin constant region to reduce or prevent binding to FcγR1, FcγRIIa, FcγRIIb, FcγRIIa, and FcγRIIIb.

50. 49. The composition of any one of claims 1-48, wherein the immunoglobulin constant region comprises one, two, three or more amino acid substitutions compared to a wild-type immunoglobulin constant region to reduce or prevent Fc-mediated T cell activation.

51. 49. The composition of any one of claims 1-48, wherein the immunoglobulin constant region comprises one, two, three or more amino acid substitutions compared to a wild-type immunoglobulin constant region to prevent or reduce CDC activity.

52. 49. The composition of any one of claims 1-48, wherein the immunoglobulin constant region comprises one, two, three or more amino acid substitutions compared to a wild-type immunoglobulin constant region to prevent or reduce ADCC activity.

53. 53. The composition of any one of claims 1 to 52, wherein the immunoglobulin constant region comprises a human IgG1 CH2 domain having one, two, three or more amino acid substitutions compared to a wild-type human IgG1 CH2 domain.

54. 54. The composition of any one of claims 1 to 53, wherein the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A according to the EU numbering system.

55. 55. The composition of claim 54, wherein the immunoglobulin constant region comprises a human IgGl CH2 domain comprising the substitutions L234A, L235A, G237A, E318A, K320A and K322A according to the EU numbering system.

56. 54. The composition of any one of claims 1 to 53, wherein the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions E233P, L234A, L235A, G237A, and K322A, and a deletion of G236, according to the EU numbering system.

57. 57. The composition of any one of claims 1 to 56, wherein the hinge region is derived from an immunoglobulin hinge region.

58. 58. The composition of any one of claims 1 to 57, wherein each polypeptide comprises an Fc binding domain linker between the immunoglobulin constant region and the second binding domain.

59. the Fc binding domain linker is Gly ４ 59. The composition of claim 58, comprising the sequence Ser (SEQ ID NO: 128).

60. The Fc binding domain linker has the formula (Gly ４ Ser) ｎ 60. The composition of claim 59, comprising:

61. The composition of any one of claims 1 to 60, which is for intravenous or subcutaneous administration.

62. 62. The composition of any one of claims 1 to 61, which substantially prevents degradation of the multispecific protein.

63. 63. The composition of any one of claims 1-62, which delays or reduces degradation of the multispecific polypeptide compared to the same multispecific polypeptide stored in a histidine buffer under identical storage conditions.

64. 64. The composition of any one of claims 1 to 63, which is substantially stable at 4°C for at least 1 year.

65. 65. The composition of any one of claims 1 to 64, which is substantially resistant to the formation of aggregates of the multispecific protein.

66. 66. The composition of any one of claims 1 to 65, which forms fewer aggregates than the same multispecific polypeptide stored in a histidine buffer under identical storage conditions.

67. A composition comprising a fusion protein, a buffer, an excipient, and a surfactant, (a) the fusion protein is a dimer of two identical polypeptides, each polypeptide consisting, in order from amino terminus to carboxyl terminus: (i) a first binding domain that specifically binds to CD123; (ii) the hinge region; (iii) an immunoglobulin constant region, and (iv) a second binding domain that specifically binds to CD3; Including, (b) the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof; composition.

68. 68. The composition of claim 67, comprising about 5 mM succinate, about 6.5 weight / volume (w / v)% sucrose, and about 0.02 w / v% polysorbate 80.

69. 69. The composition of claim 67 or 68, comprising about 0.1 mg / ml to about 10 mg / ml of the multispecific protein.

70. 70. The composition of claim 69, comprising about 1 mg / ml to about 5 mg / ml of the multispecific protein.

71. 71. The composition of claim 70, comprising about 2 mg / ml of the multispecific protein.

72. 72. The composition of any one of claims 67 to 71, having a pH of about 4.0 to about 5.

5.

73. 73. The composition of claim 72, having a pH of about 4.

8.

74. The composition of any one of claims 67 to 73, wherein the immunoglobulin constant region is a human Fc domain.

75. 75. The composition of any one of claims 67-74, wherein the immunoglobulin constant region comprises immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD.

76. 76. The composition of any one of claims 1 to 75, wherein the CH2 domain is a human IgG1 CH2 domain having one, two, three or more amino acid substitutions compared to a wild-type human IgG1 CH2 domain.

77. 77. The composition of any one of claims 67 to 76, wherein the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A according to the EU numbering system.

78. 78. The composition of any one of claims 67 to 77, wherein the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, E318A, K320A and K322A according to the EU numbering system.

79. 77. The composition of any one of claims 67 to 76, wherein the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the substitutions E233P, L234A, L235A, G237A, and K322A and deletion of G236 according to the EU numbering system.

80. 80. The composition of any one of claims 67 to 79, wherein the hinge region is derived from an immunoglobulin hinge region.

81. 81. The composition of any one of claims 67 to 80, wherein each polypeptide comprises an Fc binding domain linker between the immunoglobulin constant region and the second binding domain.

82. the Fc binding domain linker is Gly ４ 82. The composition of claim 81, comprising the sequence Ser (SEQ ID NO: 128).

83. The Fc binding domain linker has the formula (Gly ４ Ser) ｎ 83. The composition of claim 82, comprising:

84. The composition of any one of claims 67 to 83, which is for intravenous or subcutaneous administration.

85. At least one of the first binding domain and the second binding domain comprises: (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3; The composition of any one of claims 67 to 84, comprising:

86. 86. The composition of any one of claims 67 to 85, wherein at least one of the first binding domain and the second binding domain is a single chain variable fragment (scFv).

87. 87. The composition of claim 86, wherein the light chain variable region of the scFv is carboxy-terminal to the heavy chain variable region of the scFv.

88. 87. The composition of claim 86, wherein the light chain variable region of the scFv is amino terminal to the heavy chain variable region of the scFv.

89. The composition of any one of claims 86 to 88, wherein the scFv comprises a linker polypeptide.

90. 90. The composition of claim 89, wherein the linker polypeptide is between the light chain variable region and the heavy chain variable region of the scFv.

91. The linker polypeptide is Gly ４ 91. The composition of claim 89 or 90, comprising a Ser (SEQ ID NO: 128) linker.

92. The linker polypeptide has the formula (Gly ４ Ser) ｎ 92. The composition of claim 91, comprising:

93. the first binding domain comprises: HCDR1 comprising the sequence of SEQ ID NO: 10, HCDR2 comprising the sequence of SEQ ID NO: 11, and HCDR3 comprising the sequence of SEQ ID NO: 12; and LCDR1 comprising the sequence of SEQ ID NO: 13, LCDR2 comprising the sequence of SEQ ID NO: 14, and LCDR3 comprising the sequence of SEQ ID NO: 15; The composition of any one of claims 67 to 92, which is an anti-CD123 scFv comprising:

94. 93. The composition of any one of claims 67 to 92, wherein the first binding domain is an anti-CD123 scFv comprising a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 136, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO:

134.

95. 93. The composition of any one of claims 67 to 92, wherein the first binding domain is an anti-CD123 scFv comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:

18.

96. the second binding domain comprises: HCDR1 comprising the sequence of SEQ ID NO: 19, HCDR2 comprising the sequence of SEQ ID NO: 20, and HCDR3 comprising the sequence of SEQ ID NO: 21; and LCDR1 comprising the sequence of SEQ ID NO: 22, LCDR2 comprising the sequence of SEQ ID NO: 23, and LCDR3 comprising the sequence of SEQ ID NO: 24; The composition of any one of claims 67 to 95, which is an anti-CD3 scFv comprising:

97. 96. The composition of any one of claims 67 to 95, wherein the second binding domain is an anti-CD3 scFv comprising a VH comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO:

384.

98. 96. The composition of any one of claims 67 to 95, wherein the second binding domain is an anti-CD3 scFv comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:

27.

99. 96. The composition of any one of claims 67 to 95, wherein each polypeptide comprises the sequence of SEQ ID NO:31, or a sequence that is at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:

31.

100. the first binding domain is an anti-CD123 scFv comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15; and the second binding domain is an anti-CD3 scFv comprising an immunoglobulin heavy chain variable region (VH) comprising HCDR1 of SEQ ID NO: 19, HCDR2 of SEQ ID NO: 20, and HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 23, and LCDR3 of SEQ ID NO: 24; The composition according to any one of claims 67 to 95.

101. 101. The composition of any one of claims 67 to 100, which substantially prevents degradation of the multispecific protein.

102. 102. The composition of any one of claims 67-101, which delays or reduces degradation of the multispecific polypeptide compared to the same multispecific polypeptide stored in a histidine buffer under identical storage conditions.

103. 103. The composition of any one of claims 67 to 102, which is substantially stable at 4°C for at least one year.

104. 104. The composition of any one of claims 67 to 103, which is substantially resistant to the formation of aggregates of the multispecific protein.

105. 105. The composition of any one of claims 67 to 104, which forms fewer aggregates than the same multispecific polypeptide stored in a histidine buffer under identical storage conditions.

106. A composition comprising a fusion protein, a buffer, an excipient, and a surfactant, (a) the fusion protein (i) a first binding domain that specifically binds to CD123, a first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15; and (ii) a second binding domain that specifically binds to CD3, the second binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO:

24. Including, (b) the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof; composition.

107. 1. A composition comprising a fusion protein, about 5 mM succinate, about 6.5% weight / volume (w / v) sucrose, and about 0.02% w / v polysorbate 80, wherein the fusion protein is selected from the group consisting of Fab, Fab', F(ab')2, scFv, disulfide-linked Fv, scFv x scFv (BiTE), scFv-Fc (SMIP), scFv-Fc-scFv, quadroma, Kλ-body, dAb, diabody, nanobody, DOCK-AND-LOCK® (DNL®), CrossMab Fab, CrossMab VH-VL, strand-exchange engineered domain body, and the like. body (SEEDbody), affibody, Fynomer, Kunitz domain, Albu-dab, two engineered Fv fragments with exchanged VHs (e.g., dual-affinity re-targeting molecule (D.A.R.T.)), SVD-IG, Covx-body, peptibody, SVD-Ig, dAb-Ig, Knob-in-Hole, IgG1 antibody comprising matched mutations in the CH3 domain A composition comprising a mAb (e.g., a duobody antibody) and a triomab.

108. A composition comprising a fusion protein, about 5 mM succinate, about 6.5 weight / volume (w / v) % sucrose, and about 0.02 w / v % polysorbate 80, (a) the fusion protein (i) a first binding domain that specifically binds to CD123, a first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15; and (ii) a second binding domain that specifically binds to CD3, a second binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO:

24. A composition comprising:

109. A composition comprising a fusion protein, a buffer, an excipient, and a surfactant, (a) the fusion protein (i) a first binding domain that specifically binds to CD123, a first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising SEQ ID NO: 136; and an immunoglobulin light chain variable region (VL) comprising SEQ ID NO: 134; and (ii) a second binding domain that specifically binds to CD3, A second binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising SEQ ID NO: 383 or 387; and an immunoglobulin light chain variable region (VL) comprising SEQ ID NO:

384. and (b) the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof; composition.

110. A composition comprising a fusion protein, a buffer, an excipient, and a surfactant, (a) the fusion protein (i) a first binding domain that specifically binds to CD123, the first binding domain comprising SEQ ID NO: 18; and (ii) a second binding domain that specifically binds to CD3, the second binding domain comprising SEQ ID NO: 27; (b) the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof; composition.

111. A composition comprising a fusion protein, a buffer, an excipient, and a surfactant, (a) the fusion protein is a dimer of two identical polypeptides, each polypeptide arranged in amino-terminal to carboxyl-terminal order or in carboxyl-terminal to amino-terminal order; (i) a first binding domain that specifically binds to CD123, a first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15; (ii) a hinge region of SEQ ID NO: 47; (iii) an immunoglobulin constant region of SEQ ID NO: 131; (iv) an Fc-binding domain linker of SEQ ID NO: 132, and (v) a second binding domain that specifically binds to CD3, a second binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO:

24. Including, (b) the buffer comprises or consists of a succinate ester or a pharmaceutically acceptable salt or acid thereof; composition.

112. A composition comprising a fusion protein, about 5 mM succinate, about 6.5 weight / volume (w / v) % sucrose, and about 0.02 w / v % polysorbate 80, The fusion protein is a dimer of two identical polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus: (i) a first binding domain that specifically binds to CD123, a first binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 10, an HCDR2 of SEQ ID NO: 11, and an HCDR3 of SEQ ID NO: 12; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 13, an LCDR2 of SEQ ID NO: 14, and an LCDR3 of SEQ ID NO: 15; (ii) a hinge region of SEQ ID NO: 47; (iii) an immunoglobulin constant region of SEQ ID NO: 131; (iv) an Fc-binding domain linker of SEQ ID NO: 132, and (v) a second binding domain that specifically binds to CD3, a second binding domain comprising an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 of SEQ ID NO: 19, an HCDR2 of SEQ ID NO: 20, and an HCDR3 of SEQ ID NO: 21; and an immunoglobulin light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and an LCDR3 of SEQ ID NO: 24; A composition comprising:

113. A composition comprising a fusion protein, about 5 mM succinate, about 6.5 weight / volume (w / v) % sucrose, and about 0.02 w / v % polysorbate 80, the fusion protein comprises or consists of SEQ ID NO: 31, about 2 mg / ml of the fusion protein; having a pH of about 4.8; composition.

114. 114. A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1 to 113.

115. The autoimmune disease is irritable bowel syndrome, inflammatory bowel disease, psoriasis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, asthma, multiple sclerosis, dermatomyositis, polymyositis, pernicious anemia, primary biliary cirrhosis, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid syndrome (aPL), autoimmune hepatitis, type 1 diabetes, Goodpasture's syndrome, gray's syndrome, The method of claim 114, wherein the condition is Busu disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, pemphigus vulgaris, Sjogren's syndrome, temporal arteritis, autoimmune hemolytic anemia, bullous pemphigoid, vasculitis, celiac disease, endometriosis, hidradenitis suppurativa, interstitial cystitis, morphea, scleroderma, narcolepsy, neuromyotonia, leukoplakia, autoimmune inner ear disease, or myasthenia gravis.

116. 114. A method for treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the composition of any one of claims 1 to 113.

117. 117. The method of claim 116, wherein the cancer is selected from a carcinoma or a sarcoma.

118. 117. The method of claim 116, wherein the cancer is selected from melanoma, kidney cancer, pancreatic cancer, lung cancer, bowel cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, or blood cancer.

119. 119. The method of claim 118, wherein the hematological cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, B-cell acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML).

120. 114. Use of a composition according to any one of claims 1 to 113 for treating an autoimmune disease in a subject.

121. 114. Use of a composition according to any one of claims 1 to 113 in the manufacture of a medicament for treating an autoimmune disease.

122. 114. Use of a composition according to any one of claims 1 to 113 for treating cancer in a subject.

123. 114. Use of a composition according to any one of claims 1 to 113 in the manufacture of a medicament for treating cancer.

124. A method of treating cancer in a patient comprising administering a composition comprising a multispecific protein comprising a CD123 binding domain and a CD3 binding domain to a subject by IV infusion at a dose of 0.3, 1, 3, 6, 9, 12, 18, 20, 24, 30, 36, 48, 50, 60, 75, or 100 μg.

125. 112. A method of treating cancer in a subject comprising administering to a patient by IV infusion the composition of any one of claims 42-48, 93-100, and 108-111 at a dose of 0.3, 1, 3, 6, 9, 12, 18, 20, 24, 30, 36, 48, 50, 60, 75, or 100 μg.

126. 126. The method of claim 124 or 125, wherein the first dose of the composition is administered to the patient by IV infusion over a period of 20 to 24 hours.

127. 127. The method of claim 126, wherein a second dose of the composition is administered to the patient by IV infusion over an 8-hour period, the second dose being the same as the first dose.

128. 128. The method of claim 127, wherein a third dose of the composition is administered to the patient by IV infusion over a period of six hours, the third dose being the same as the first and second doses.

129. 129. The method of any one of claims 124-128, wherein the fourth and subsequent doses of the composition are administered to the patient by IV infusion over a period of about 20 to about 4 hours.

130. 126. The method of claim 124 or 125, wherein the composition is administered to the patient by continuous IV infusion for up to 72 hours.

131. 131. The method of any one of claims 124-130, wherein the composition is administered on days 1, 8, 15, and 22.

132. 132. The method of claim 131, wherein 6 μg is administered on day 1, 9 μg is administered on day 8, 12 μg is administered on day 15, and 12 μg is administered on day 22.

133. 132. The method of claim 131, wherein 6 μg is administered on day 1, 9 μg is administered on day 8, 12 μg is administered on day 15, and 18 μg is administered on day 22.

134. 132. The method of claim 131, wherein 6 μg is administered on day 1, 9 μg is administered on day 8, 9 μg is administered on day 15, and 9 μg is administered on day 22.

135. 132. The method of claim 131, wherein 9 μg is administered on day 1, 12 μg is administered on day 8, 12 μg is administered on day 15, and 12 μg is administered on day 22.

136. 132. The method of claim 131, wherein 12 μg is administered on day 1, 18 μg is administered on day 8, 18 μg is administered on day 15, and 18 μg is administered on day 22.

137. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; Week 4 dose and subsequent weekly doses: 12 μg.

138. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 9 μg; Week 3 dose: 12 μg; Week 4 dose and subsequent weekly doses: 18 μg.

139. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 12 μg; Week 4 dose and subsequent weekly doses: 12 μg.

140. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; Week 4 dose and subsequent weekly doses: 24 μg.

141. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; Week 4 dose and subsequent weekly doses: 36 μg.

142. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; Week 4 dose and subsequent weekly doses: 48 μg.

143. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; Week 2 dose: 12 μg; Week 3 dose: 18 μg; Week 4 dose and subsequent weekly doses: 60 μg.

144. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 6 μg; and Week 2 dose and subsequent weekly doses: 9 μg.

145. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 9 μg; and Week 2 dose and subsequent weekly doses: 12 μg.

146. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV according to the following weekly treatment schedule: Week 1 dose: 12 μg; and Week 2 dose and subsequent weekly doses: 18 μg.

147. 131. The method of any one of claims 124-130, wherein the composition is administered to a patient during an initial 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 9 μg of the multispecific protein is administered on day 2, 12 μg of the multispecific protein is administered on day 3, 18 μg of the multispecific protein is administered on day 4, 18 μg of the multispecific protein is administered on day 8, 18 μg of the multispecific protein is administered on day 11, 36 μg of the multispecific protein is administered on day 15, and 36 μg of the multispecific protein is administered on day 22 of the initial 28 day cycle.

148. 148. The method of claim 147, further comprising administering the multispecific protein to the patient for at least one additional 28-day cycle, wherein 36 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

149. 131. The method of any one of claims 124-130, wherein the composition is administered to a patient during an initial 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 12 μg of the multispecific protein is administered on day 2, 18 μg of the multispecific protein is administered on day 3, 24 μg of the multispecific protein is administered on day 4, 24 μg of the multispecific protein is administered on day 8, 24 μg of the multispecific protein is administered on day 11, 48 μg of the multispecific protein is administered on day 15, and 48 μg of the multispecific protein is administered on day 22 of the initial 28 day cycle.

150. 150. The method of claim 149, further comprising administering the multispecific protein to the patient for at least one additional 28-day cycle, wherein 48 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

151. 131. The method of any one of claims 124-130, wherein the composition is administered to a patient during an initial 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 12 μg of the multispecific protein is administered on day 2, 24 μg of the multispecific protein is administered on day 3, 36 μg of the multispecific protein is administered on day 4, 36 μg of the multispecific protein is administered on day 8, 36 μg of the multispecific protein is administered on day 11, 60 μg of the multispecific protein is administered on day 15, and 60 μg of the multispecific protein is administered on day 22 of the initial 28 day cycle.

152. 152. The method of claim 151, further comprising administering the multispecific protein to the patient for at least one additional 28-day cycle, wherein 60 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

153. 131. The method of any one of claims 124-130, wherein the composition is administered to a patient during an initial 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 1, 12 μg of the multispecific protein is administered on day 2, 24 μg of the multispecific protein is administered on day 3, 36 μg of the multispecific protein is administered on day 4, 48 μg of the multispecific protein is administered on day 8, 48 μg of the multispecific protein is administered on day 11, 100 μg of the multispecific protein is administered on day 15, and 100 μg of the multispecific protein is administered on day 22 of the initial 28 day cycle.

154. 154. The method of claim 153, further comprising administering the multispecific protein to the patient for at least one additional 28-day cycle, wherein 100 μg of the multispecific protein is administered on days 1, 8, 15, and 22, respectively, of the at least one additional 28-day cycle.

155. 131. The method of any one of claims 124-130, wherein the composition is administered to the patient by IV and the dosage is increased weekly for the first several weeks.

156. 131. The method of any one of claims 124-130, wherein the patient is administered the composition once, twice, three times, or four times each week.

157. 157. The method of any one of claims 124-156, wherein the composition is administered to the patient with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

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