Dosing regimens for protein therapeutics

Abstract

Provided herein is a method for treating cancer, comprising administering to a subject in need of cancer treatment: i) a multispecific protein comprising a CD123 binding domain and a CD3 binding domain; and ii) a second anti-cancer agent. The anti-cancer agent used in the methods described herein can be, for example, a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is venetoclax, azacitidine, decitabine, daunorubicin, cytarabine, idarubicin, mitoxantrone, or etoposide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 253,714, filed October 8, 2021, and U.S. Provisional Patent Application No. 63 / 191,488, filed May 21, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] Description of electronically submitted text files A computer-readable copy of the text file, i.e., sequence listing, submitted electronically herewith (file name: APVO_069_02WO_SeqList_ST25.txt, date of recording: May 20, 2022, file size: approximately 105,091 bytes) is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to bispecific or multispecific protein therapeutics for treating cancer. More specifically, the present disclosure relates to bispecific or multispecific proteins comprising a CD123 binding domain and a CD3 binding domain. The present disclosure also relates to clinical methods including administration regimens of the protein therapeutics, alone or in combination with one or more additional anti-cancer agents, to subjects in need thereof. [Background technology]

[0004] One of the most important and frequent side effects associated with the use of bispecific T cell binding antibodies is neurotoxicity. Neurotoxicity can be severe, life-threatening, or fatal. Another potential complication associated with treatment using such antibodies is a systemic inflammatory syndrome known as cytokine release syndrome (CRS). Thus, dosing strategies are needed to reduce the risks associated with cytokine release and other toxic effects in patients receiving bispecific and multispecific therapies that act through T cell engagement (i.e., T cell engagers). This class of therapy includes bispecific therapies targeting CD123 and CD3. mAb14045 (Xencor), a CD123×CD3 bispecific antibody molecule evaluated in patients with relapsed or refractory acute myeloid leukemia and other CD123-expressing hematological malignancies, received a partial clinical trial suspension by the FDA in 2019 due to two patient deaths in a Phase I clinical trial, including one death due to CRS.

[0005] Administration strategies designed to reduce the potential for severe effects of cytokine release, including CRS, may not be therapeutically effective. Thus, there remains a need for methods to deliver therapeutically effective amounts of T cell engagers (e.g., CD123xCD3 therapeutics) to patients in a manner that reduces the risk of toxicity, including cytokine toxicity. Summary of the Invention

[0006] Described herein are methods of treating cancer, including administration strategies. The methods may include administering a multispecific protein, such as a multispecific protein comprising a CD123 binding domain and a CD3 binding domain, to a subject in need thereof. In some embodiments, the multispecific protein is administered in combination with a second anti-cancer agent, such as a chemotherapeutic agent. The administration strategies described herein may provide therapeutic benefit while minimizing the potential for neurotoxicity and / or cytokine release syndrome.

[0007] Thus, in some embodiments, a method for treating cancer is provided herein, the method comprising administering to a subject in need thereof i) a multispecific protein comprising a CD123 binding domain and a CD3 binding domain, and ii) a second anti-cancer agent. In some embodiments, the method further comprises administering to the subject a third anti-cancer agent. The anti-cancer agent used in the methods described herein can be, for example, a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is venetoclax, azacitidine, decitabine, daunorubicin, cytarabine, idarubicin, mitoxantrone, or etoposide.

[0008] In some embodiments, provided herein is a method for treating cancer, the method comprising administering to a subject in need thereof a multispecific protein comprising a CD123 binding domain and a CD3 binding domain.

[0009] In some embodiments, the multispecific protein comprises a dimer of two identical polypeptides, each polypeptide comprising, from amino-terminus to carboxyl-terminus or carboxyl-terminus to amino-terminus, (i) a CD123 binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a CD3 binding domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD123 binding domain, a hinge region, an immunoglobulin constant region, and a CD3 binding domain. In some embodiments, at least one of the CD123 and CD3 binding domains 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, the CD123 binding domain is an scFv comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HDCR3 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 CD123 binding domain is an 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 CD123 binding domain is an scFv, and the scFv comprises a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27. In some embodiments, the CD3 binding domain is an scFv comprising an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HDCR3 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 binding domain is an 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 scFv, and the scFv comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27. In some embodiments, each polypeptide comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:31.

[0010] In some embodiments, the multispecific protein is administered to the subject by IV infusion. In some embodiments, the multispecific protein is administered to the 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. In some embodiments, the multispecific protein is administered once or twice a week. In some embodiments, the second anticancer agent is administered to the subject orally or by IV infusion.

[0011] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle. In some embodiments, cytarabine is administered intravenously on days 1-5 of the first 28 day cycle and days 1-5 of at least one additional 28 day cycle. In some embodiments, the dose of cytarabine is about 1 g / m 2 In some embodiments, mitoxantrone, etoposide, and cytarabine are administered intravenously on days 1-6 of a first 28-day cycle and at least one additional 28-day cycle. In some embodiments, the dose of mitoxantrone is about 6 mg / m 2 / day and the dose of etoposide is approximately 80 mg / m 2 / day, and the dose of cytarabine is approximately 1 g / m 2 / day.

[0012] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 15 and 12 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle. In some embodiments, venetoclax is administered orally on days 1-21 of the first 28 day cycle and days 1-21 of at least one additional 28 day cycle. In some embodiments, the dose of venetoclax is about 100 to about 400 mg / day. In some embodiments, azacitidine is administered intravenously on days 1-7 of the first 28 day cycle and at least one additional 28 day cycle. In some embodiments, the dose of azacitidine is about 75 mg / m 2 It is.

[0013] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22. In some embodiments, cytarabine is administered by intravenous infusion on days 1-7 of the first 28 day cycle and days 1-7 of at least one additional 28 day cycle. In some embodiments, the dose of cytarabine is about 100 to about 200 mg / m 2In some embodiments, idarubicin is administered by intravenous infusion on days 1-3 of a first 28-day cycle and days 1-3 of at least one additional 28-day cycle. In some embodiments, the dose of idarubicin is about 12 mg / m 2 It is.

[0014] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle. In some embodiments, azacitidine is administered orally on days 1-14 of the first 28 day cycle and at least one additional 28 day cycle. In some embodiments, the dose of azacitidine is about 300 mg / day.

[0015] In some embodiments, the cancer is carcinoma or sarcoma.In some embodiments, the cancer is melanoma, kidney cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, or blood cancer.In some embodiments, the cancer is 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). [Brief description of the drawings]

[0016] [Figure 1A]

[0023] Figure 1A is a schematic diagram showing the structure of an exemplary therapeutic protein for use with 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] 1A-1C are schematic diagrams showing the structure of exemplary therapeutic proteins for use with the compositions and methods of the present disclosure. FIG. 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×CD3 bispecific therapeutic protein is referred to herein as TRI130. [Diagram 2] FIG. 1 is a schematic showing the design of a Phase 1 / 1b dose-escalation clinical study 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 aspirate plotted over time for patients in the Phase 1 / 1b study described in Figure 2 and Example 2. Data are graphed for patients in the cohort receiving the highest dose of > 12 μg. N = 14 patients evaluable for change from baseline. Different stepwise dosing regimens were tested in Co 6a and 6b as shown in Table 10. [Figure 3B] Figure 1 shows the percentage of blasts in bone marrow aspirate plotted over time for patients in the Phase 1 / 1b study described in Figure 2 and Example 2. Data are graphed for patients in the cohort receiving the highest dose of > 12 μg. N = 14 patients evaluable for change from baseline. Different stepwise dosing regimens were tested in Co 6a and 6b as shown in Table 10. [Figure 3C] Figure 1 shows the percentage of blasts in bone marrow aspirate plotted over time for patients in the Phase 1 / 1b study described in Figure 2 and Example 2. Data are graphed for patients in the cohort receiving the highest dose of > 12 μg. N = 14 patients evaluable for change from baseline. Different stepwise dosing regimens were tested in Co 6a and 6b as shown in Table 10. [Figure 3D]Figure 1 shows the percentage of blasts in bone marrow aspirate plotted over time for patients in the Phase 1 / 1b study described in Figure 2 and Example 2. Data are graphed for patients in the cohort receiving the highest dose of > 12 μg. N = 14 patients evaluable for change from baseline. Different stepwise dosing regimens were tested in Co 6a and 6b as shown in Table 10. [Figure 4A] Serum concentrations of interleukin-6 (IL-6, FIG. 4A), interleukin-10 (IL-10, FIG. 4B), interferon-gamma (IFN-γ, FIG. 4C), and tumor necrosis factor-alpha (TNF-α, FIG. 4D) are shown in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) from the Phase 1 / 1b study described in FIG. 2 and Example 2. Scheduled blood draws were collected at the first administration of each patient's highest scheduled dose. Peak cytokine levels observed during unscheduled collections during IRR / CRS events are shown for comparison (N=6 events in 4 patients). [Figure 4B] Serum concentrations of interleukin-6 (IL-6, FIG. 4A), interleukin-10 (IL-10, FIG. 4B), interferon-gamma (IFN-γ, FIG. 4C), and tumor necrosis factor-alpha (TNF-α, FIG. 4D) are shown in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) from the Phase 1 / 1b study described in FIG. 2 and Example 2. Scheduled blood draws were collected at the first administration of each patient's highest scheduled dose. Peak cytokine levels observed during unscheduled collections during IRR / CRS events are shown for comparison (N=6 events in 4 patients). [Figure 4C]Serum concentrations of interleukin-6 (IL-6, FIG. 4A), interleukin-10 (IL-10, FIG. 4B), interferon-gamma (IFN-γ, FIG. 4C), and tumor necrosis factor-alpha (TNF-α, FIG. 4D) are shown in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) from the Phase 1 / 1b study described in FIG. 2 and Example 2. Scheduled blood draws were collected at the first administration of each patient's highest scheduled dose. Peak cytokine levels observed during unscheduled collections during IRR / CRS events are shown for comparison (N=6 events in 4 patients). [Figure 4D] Serum concentrations of interleukin-6 (IL-6, FIG. 4A), interleukin-10 (IL-10, FIG. 4B), interferon-gamma (IFN-γ, FIG. 4C), and tumor necrosis factor-alpha (TNF-α, FIG. 4D) are shown in patient samples from scheduled blood draws (pre-dose, approximately 15-30 minutes post-dose, and approximately 20-26 hours post-dose; N=26) from the Phase 1 / 1b study described in FIG. 2 and Example 2. Scheduled blood draws were collected at the first administration of each patient's highest scheduled dose. Peak cytokine levels observed during unscheduled collections during IRR / CRS events are shown for comparison (N=6 events in 4 patients). [Figure 5A] 5A-5E are schematic diagrams showing the dosing regimens used in Cohort 1-Cohort 5 of the expansion study described in Example 3. It will be understood by one of skill in the art that the dosing regimens shown in Figures 5A-5E may be used to administer any of the multispecific proteins disclosed herein. [Figure 5B] 5A-5E are schematic diagrams showing the dosing regimens used in Cohort 1-Cohort 5 of the expansion study described in Example 3. It will be understood by one of skill in the art that the dosing regimens shown in Figures 5A-5E may be used to administer any of the multispecific proteins disclosed herein. [Figure 5C]5A-5E are schematic diagrams showing the dosing regimens used in Cohort 1-Cohort 5 of the expansion study described in Example 3. It will be understood by one of skill in the art that the dosing regimens shown in Figures 5A-5E may be used to administer any of the multispecific proteins disclosed herein. [Figure 5D] 5A-5E are schematic diagrams showing the dosing regimens used in Cohort 1-Cohort 5 of the expansion study described in Example 3. It will be understood by one of skill in the art that the dosing regimens shown in Figures 5A-5E may be used to administer any of the multispecific proteins disclosed herein. [Figure 5E] 5A-5E are schematic diagrams showing the dosing regimens used in Cohort 1-Cohort 5 of the expansion study described in Example 3. It will be understood by one of skill in the art that the dosing regimens shown in Figures 5A-5E may be used to administer any of the multispecific proteins disclosed herein. [Figure 6A] Figure 1 shows pharmacokinetic (PK) data. TRI130 concentrations are shown for patients from cohort 6a of the dose escalation study at various study time points. The horizontal hashed line represents the lower limit of quantification (LLOQ) of the assay. All other measurements were within the limit of detection but not in the quantifiable range. The dashed vertical line represents the end of treatment (EOT) for each patient. [Figure 6B] FIG. 1 shows anti-drug antibody (ADA) response values ​​and titers in patients from cohort 6a of the dose escalation study at various study time points. Dashed vertical lines represent the EOT of each patient. Open symbols indicate follow-up samples. [Figure 7A] Figure 1 shows PK data. TRI130 concentrations are shown for various patients from cohort 6b of the dose escalation study at various study time points. The dashed vertical lines represent the end of treatment (EOT) for each patient. [Figure 7B] Figure 1 shows ADA response values ​​and titers in various patients from cohort 6a of the dose escalation study at various study time points. The dashed vertical lines represent the EOT of each patient. Open symbols indicate follow-up samples. [Figure 8A] FIG. 1 shows PK simulation of TRI130 concentration over time in cohort A compared to cohort 7. [Figure 8B] FIG. 13 shows PK simulation of TRI130 concentration over time in Cohort B compared to Cohort 7. [Figure 8C] FIG. 13 shows PK simulation of TRI130 concentration over time in Cohort C compared to Cohort 7. [Figure 8D] FIG. 13 shows PK simulation of TRI130 concentration over time in Cohort D compared to Cohort 7. [Figure 8E] The traces of these simulations are overlaid. [Figure 9A] FIG. 1 shows the estimated maximum concentration (Cmax), time to maximum (Tmax), minimum concentration (Cmin) and area under the curve (AUC) for different cohorts. [Figure 9B] FIG. 1 shows Cmax vs. Cmin at Cmax, AUC:Cmax, and the ratio of highest Cmax:Cmin in cycle 1 for various cohorts. [Figure 10] Figure 15 shows depletion of putative circulating AML-LSC cells in VENAZA-resistant relapsed AML patients receiving TRI130 monotherapy. The numbers in the upper left corner of each graph in the first and second columns of the graph represent the total number of LSCs, the majority of which co-expressed both CD123 and CD33. Virtually all CD34+CD38- cells were CD123+ and CD33+, consistent with AML. The size of this CD123+CD33+CD34+CD38- AML LSC population, indicated by the arrow in the third column of panel A, was significantly reduced by TRI130 monotherapy. See also Figure 15. [Figure 11]Figure 15 shows circulating AML-LSC cells in VENAZA-resistant relapsed AML patients receiving TRI130 monotherapy. Virtually all CD34+CD38- cells were CD123+ and CD33+, consistent with AML. The size of the CD123+CD33+CD34+CD38- AML LSC population (indicated by the arrow in the third column of the graph) did not change significantly during TRI130 monotherapy. See also Figure 15. [Figure 12] Figure 13 shows the onset and duration of stable (SD), PR, CR, clearance of peripheral blasts, and development of PD, each indicated by a specific symbol. Arrows indicate patients alive. See also Figures 13A-B. [Figure 13A]Table 13A shows patient characteristics of AML (FIG. 13A) and MDS (FIG. 13B) patients enrolled in the study described in Example 4. C: cycle number, D: days in a particular cycle; AZA: Azacitidine; MDS: MDS-rf: MDS-related features; abn: abnormal; AZA: azacytidine; MDS: myelodysplastic syndrome; myelodysplastic syndrome; PD: progressive disease; IM-1: intermediate-1, IM-2: intermediate-2; SD: unchanged; NA: not applicable; M: male; F: female; C: Caucasian; A: Asian; IPSS: International Prognostic Scoring System; 2016 WHO myelodysplastic syndrome subtypes: MDS with excess blasts (MDS-EB), *UPN09 is multiple transient grade 3- She had 4 AEs (she had tumor lysis syndrome (TLS) grade 3 on C1D2, lasting 2 days); TLS grade 3 on C4D1, lasting 2 days; anemia, grade 3 on C5D1, lasting 8 days; anemia grade 3 on C7D22, lasting 8 days; anemia grade 3 on C6D22, lasting 12 days; anemia grade 3 on C7D8, lasting 4 days; decreased platelet count grade 3 on C4D15, lasting 7 days; decreased platelet count grade 4 on C4D22, lasting 50 days; decreased platelet count grade 3 on C6D15, lasting 2 days; decreased platelet count grade 4 on C6D22, lasting 33 days; hyperglycemia grade 3 on C5D5, lasting 2 days. $$UPN09: Anemia (Hgb 9.7g / dL), thrombocytopenia (Plt 30,000 / μL), intermediate risk karyotype, 47,XX+8, multiple gene mutations (NRAS, ASXL-1, PTPNII, RUNX1, STAG2), progressed to CMML-MPN with absolute monocyte count 17,600 / μL around cycle 10. UPN10: Anemia (Hgb 8.0g / dL), thrombocytopenia (Plt: 9,000 / μL), severe neutropenia (ANC: 0.29×103 / μL), 7.7% blasts in bone marrow, cytogenetics not available. Achieved bone marrow CR with bone marrow blast count 2.4%. UPN-11: Anemia (Hgb 9.3g / dL, thrombocytopenia (Plt: 23,000 / μL) (non-severe neutropenia with -ANC 1.3x103 / μL), intermediate risk cytogenetics with MDS-associated abnormalities 11q- and -7, 11.3% blasts in bone marrow. C2D1 reduced bone marrow blast count to 0% and achieved myeloblast CR.UPN12: No pancytopenia (Hgb 11g / dL, Plt: 100,000μL, ANC: 1.1×103μL), favorable karyotype: 46,XY, 8.2% blasts in bone marrow. C2D1 reduced bone marrow blast count to 2% and achieved bone marrow CR. UPN13: Anemia (Hgb 7.2g / dL), thrombocytopenia (Plt: 8,000 / μL), severe neutropenia (ANC: 0.5×103 / μL), 5% blasts in bone marrow, complex karyotype (poor risk category): 46,XY,der(12;19)(q10;p10), +mar[8] / 46,XY,del(20)q11.2q13.1). Intermediate risk karyotype with 12(p) abnormality on cytogenetics. 5% blasts in bone marrow. UPN14: Anemia (Hgb 9.3 / dL), neutropenia (ANC: 0×103 / μL), intermediate risk karyotype with t(1;2)(p36.3;p21) on cytogenetics, 6% blasts in bone marrow. [Figure 13B]Table 13A shows patient characteristics of AML (FIG. 13A) and MDS (FIG. 13B) patients enrolled in the study described in Example 4. C: cycle number, D: days in a particular cycle; AZA: Azacitidine; MDS: MDS-rf: MDS-related features; abn: abnormal; AZA: azacytidine; MDS: myelodysplastic syndrome; myelodysplastic syndrome; PD: progressive disease; IM-1: intermediate-1, IM-2: intermediate-2; SD: unchanged; NA: not applicable; M: male; F: female; C: Caucasian; A: Asian; IPSS: International Prognostic Scoring System; 2016 WHO myelodysplastic syndrome subtypes: MDS with excess blasts (MDS-EB), *UPN09 is multiple transient grade 3- She had 4 AEs (she had tumor lysis syndrome (TLS) grade 3 on C1D2, lasting 2 days); TLS grade 3 on C4D1, lasting 2 days; anemia, grade 3 on C5D1, lasting 8 days; anemia grade 3 on C7D22, lasting 8 days; anemia grade 3 on C6D22, lasting 12 days; anemia grade 3 on C7D8, lasting 4 days; decreased platelet count grade 3 on C4D15, lasting 7 days; decreased platelet count grade 4 on C4D22, lasting 50 days; decreased platelet count grade 3 on C6D15, lasting 2 days; decreased platelet count grade 4 on C6D22, lasting 33 days; hyperglycemia grade 3 on C5D5, lasting 2 days. $$UPN09: Anemia (Hgb 9.7g / dL), thrombocytopenia (Plt 30,000 / μL), intermediate risk karyotype, 47,XX+8, multiple gene mutations (NRAS, ASXL-1, PTPNII, RUNX1, STAG2), progressed to CMML-MPN with absolute monocyte count 17,600 / μL around cycle 10. UPN10: Anemia (Hgb 8.0g / dL), thrombocytopenia (Plt: 9,000 / μL), severe neutropenia (ANC: 0.29×103 / μL), 7.7% blasts in bone marrow, cytogenetics not available. Achieved bone marrow CR with bone marrow blast count 2.4%. UPN-11: Anemia (Hgb 9.3g / dL, thrombocytopenia (Plt: 23,000 / μL) (non-severe neutropenia with -ANC 1.3x103 / μL), intermediate risk cytogenetics with MDS-associated abnormalities 11q- and -7, 11.3% blasts in bone marrow. C2D1 reduced bone marrow blast count to 0% and achieved myeloblast CR.UPN12: No pancytopenia (Hgb 11g / dL, Plt: 100,000μL, ANC: 1.1×103μL), favorable karyotype: 46,XY, 8.2% blasts in bone marrow. C2D1 reduced bone marrow blast count to 2% and achieved bone marrow CR. UPN13: Anemia (Hgb 7.2g / dL), thrombocytopenia (Plt: 8,000 / μL), severe neutropenia (ANC: 0.5×103 / μL), 5% blasts in bone marrow, complex karyotype (poor risk category): 46,XY,der(12;19)(q10;p10), +mar[8] / 46,XY,del(20)q11.2q13.1). Intermediate risk karyotype with 12(p) abnormality on cytogenetics. 5% blasts in bone marrow. UPN14: Anemia (Hgb 9.3 / dL), neutropenia (ANC: 0×103 / μL), intermediate risk karyotype with t(1;2)(p36.3;p21) on cytogenetics, 6% blasts in bone marrow. [Figure 14] Table showing that TRI130 activates TH1 and TH2 cells from relapsed / refractory AML and MDS patients. E: early samples obtained at the beginning of cycle 2, L: late samples obtained during cycle 2. *IL-10 induction was observed in UPN01 (46-fold), UPN05 (8.3-fold), UPN06 (17.3-fold), and UPN10 (24.5-fold). Only two MDS patients with SD as BOR did not show significant IL-10 induction. **IFN-γ induction was observed in UPN01 and UPN05. ***IL-5 induction was observed only in UPN06. [Figure 15] 1 is a table showing the pharmacodynamic effects of TRI130 on putative circulating LSCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] One of the most important and frequent side effects associated with the use of bispecific T cell binding antibodies is neurotoxicity. Neurotoxicity can be severe, life-threatening, or fatal. This application describes a multispecific protein (including TRI130) that includes a CD123 binding domain and a CD3 binding domain. Clinical data is provided showing that TRI130 is not associated with severe neurotoxicity. Another potential complication associated with treatment using bispecific T cell binding antibodies is a systemic inflammatory syndrome known as cytokine release syndrome (CRS). TRI130 caused CRS in some patients, but this could be managed using standard CRS treatment. TRI130 showed clinical activity, sustained stabilization of leukemia, and signs of response, resulting in partial and complete responses in two difficult-to-treat relapsed / refractory AML patients.

[0018] CD123 is highly expressed on AML blasts, but also on normal bone marrow hematopoietic stem and progenitor cells that give rise to the white blood cells that provide protection against infection. Thus, CD123-targeted therapeutic platforms are often associated with a prolonged and significant reduction in white blood cell count, known as neutropenia, and infections, especially pneumonia. This common side effect, which overlaps with the blood glucose-lowering side effect of standard chemotherapy drugs, is one of the main hurdles preventing the desired incorporation of CD123-targeted drugs into current frontline and second-line standard of care treatment regimens. Thus, there is an urgent need for new drugs effective for treating AML patients that can destroy leukemic cells without damaging normal bone marrow cells. In particular, the inventors unexpectedly discovered that, unlike other CD123-targeted drugs, TRI130 does not cause severe or prolonged neutropenia at doses that result in complete responses, even after several months of weekly infusions. This unique feature allows for the use of TRI130 in combination with one or more additional anticancer drugs, such as chemotherapy drugs.

[0019] These and various aspects of the invention are described in further detail below.

[0020] 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 cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for all purposes. In the event that one or more of the incorporated documents or portions of documents defines a term in conflict with the definition of the term in this application, the definition set forth in this application shall prevail. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, a form of admission or any suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0021] In the present description, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value in the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified. The terms "a" and "an" as used herein are understood to refer to "one or more" of the recited components, unless otherwise specified. The use of alternatives (e.g., "or") is understood to mean either one, both, or any combination of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously. Furthermore, it is understood that polypeptides comprising various combinations of the components (e.g., domains or regions) and substitution elements described herein are disclosed by the present application to the same extent as if each polypeptide were individually described. Thus, the selection of specific components of individual polypeptides is within the scope of this disclosure.

[0022] definition The term "about" immediately preceding a numerical value means ± up to 10% of the numerical value. For example, "about 40" means ± up to 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%, ± up to less than 1% or any other value or range of values ​​within that range.

[0023] As used herein, "substantially" has its normal meaning as used in the art. For example, "substantially" can mean "significantly," "substantially," "largely," 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 99%.

[0024] The term "CD123" may refer to any isoform of CD123, which is also known as cluster of differentiation 123, interleukin 3 receptor alpha chain, and IL3RA. CD123 associates with the beta chain of the interleukin 3 receptor to form the receptor. CD123 is a type I transmembrane glycoprotein with an extracellular domain that includes a predicted Ig-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 signal transduction is mediated 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)).

[0025] CD123 is overexpressed in many hematological malignancies, including acute myeloid leukemia (AML), B-lymphoid leukemia, blastic plasmacytoid dendritic neoplasm (BPDCN), and a subset of hairy cell leukemia. Although most AML patients respond well to initial treatment, most 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 molecules that target CD123 with increased efficiency and efficacy, and reduced adverse effects, and that can be used to treat disorders associated with dysregulation of CD123.

[0026] "CD3" is known in the art as a six-chain multiprotein complex that is a subunit of the T cell receptor complex (see, for example, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999). In mammals, the CD3 subunit of the T cell receptor complex is a homodimer of the CD3γ chain, the CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ chain, the CD3δ chain, and the CD3ε chain are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane domains of the CD3γ chain, the CD3δ chain, and the CD3ε chain are negatively charged, a property that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3 gamma, delta, and epsilon chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3 zeta chain has three. ITAMs are believed to be important for the signaling capacity of the TCR complex. The CD3 used in this disclosure may be derived from a variety of animal species, including human, monkey, mouse, rat, or other mammals.

[0027] "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, sore throat, and difficulty breathing can occur. Some patients may experience severe, life-threatening reactions caused by the massive release of cytokines. "Reduced" cytokine release refers to a reduction in the release of at least one cytokine (e.g., IFN-γ, TNF-α, IL-6, IL-2, IL-8, IL-10, IL-17, GM-CSF, IL-4, IL-12, IL-13, or IL-1β) following administration of the bispecific molecules disclosed herein compared to the OKT3 antibody (which binds CD3) or other CD3-binding bispecific molecules. The reduction in cytokine release can be measured using in vitro or in vivo assays.

[0028] As used herein, the term "step administration" or "step-by-step administration" or similar terms refer to a dosing regimen in which a multispecific polypeptide as described herein is administered to a patient on at least a first and a second day, and the dose administered to the patient remains constant or increases between the first and second days. For example, in some step-by-step dosing regimens, a patient may be administered a first, second, third and fourth dose, each dose being administered on a different day, with the second dose being higher than the first dose. The third dose may be higher than the second dose or the same as the second dose. The fourth dose may be higher than the third dose 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.

[0029] 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 binding domain derived from an antibody, receptor, or ligand that has the ability to specifically recognize and bind to a target molecule, e.g., an antigen, ligand, receptor, substrate, or inhibitor. Exemplary binding domains include antibodies 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., comprising variable heavy and variable light chain sequences, or comprising three heavy chain CDRs from an antibody arranged in three light chain complementarity determining regions (CDRs) and alternative 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 disclosure that specifically bind to a particular target, including Western blot, ELISA, phage display library screening, and BIACORE® interaction analysis.

[0030] The binding domain or a binding protein containing the binding domain is 5 M -1 or higher affinity or K a A binding domain "specifically binds" to a target if it binds to the target with a specific affinity (i.e., the equilibrium association constant for a particular binding interaction in units of 1 / M) while not binding significantly to other components present in the test sample. Binding domains can be classified as "high affinity" binding domains and "low affinity" binding domains. A "high affinity" binding domain is one that has 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 10 12 M -1 , or at least 10 13 M -1 K a A "low affinity" binding domain refers to a binding domain having a maximum affinity of 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 K a Alternatively, affinity is expressed in units of M (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) of the equilibrium dissociation constant of the specific binding interaction (K d The affinity of the 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. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0031] As used herein, "conservative substitution" is recognized in the art as the substitution of one amino acid with another amino acid with similar properties.Exemplary conservative substitutions are well known in the art (see, for example, PCT Application Publication No. WO97 / 09433, filed March 13, 1997, p. 10; 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 substitutions include leucine-serine substitutions.

[0032] 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, with or without enzymatic activity, for example, by glycosylation, alkylation, acylation, ester formation, or amide formation.

[0033] As used herein, a polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, a polypeptide or amino acid sequence is derived from a particular sequence (sometimes referred to as the "starting" or "parent" or "parental" sequence) and has an amino acid sequence that is substantially identical to the parent sequence or a portion thereof, which portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or can be demonstrated by one of skill in the art to be otherwise derived from 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.

[0034] A polypeptide derived from another polypeptide can have one or more mutations or changes relative to the parent polypeptide, for example, one or more amino acid residues substituted with another amino acid residue or having one or more amino acid insertions or deletions. In such an embodiment, a polypeptide derived from a parent polypeptide and containing one or more mutations or changes is referred to as a "variant". As used herein, the term "variant" or "variants" refers to a polynucleotide or polypeptide having a sequence that differs from a reference polynucleotide or polypeptide but retains its essential properties. Generally, a variant polynucleotide or polypeptide sequence is overall very similar and in many regions identical to the reference polynucleotide or polypeptide. For example, a variant polynucleotide or polypeptide can 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 reference polynucleotide or polypeptide. A polypeptide can include an amino acid sequence that does not occur in nature. Such variants will necessarily have less than 100% sequence identity or similarity with the parent polypeptide. In one embodiment, the variant will have an amino acid sequence that has less than about 60%-100% amino acid sequence identity or similarity with the amino acid sequence of the parent polypeptide. In another embodiment, the variant will have an amino acid sequence that has less than about 75%-100%, less than about 80%-100%, less than about 85%-100%, less than about 90%-100%, or less than about 95%-100% amino acid sequence identity or similarity with the amino acid sequence of the parent polypeptide.

[0035] 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 in the corresponding position of the comparison sequence, the sequences are said to be "identical" at that position. Percentage sequence identity is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain 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 sequence identity. Percentage sequence identity is determined by comparing two optimally aligned sequences over the comparison window. A comparison window for polynucleotide 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, about 400, about 500, about 600, about 700, about 800, about 900 or about 1000 or more nucleic acids in length. The comparison window of a polypeptide sequence 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, the portion of the polynucleotide or polypeptide sequence in the comparison window can include additions or deletions, called gaps, while keeping the reference sequence constant. An optimal alignment is one that produces as many "identical" positions as possible between the reference sequence and the comparison sequence, even if it has gaps.The percentage "sequence identity" between two sequences can be determined using the version of the program "BLAST 2 Sequences" available from the National Center for Biotechnology Information on September 1, 2004, which incorporates the programs BLASTN (nucleotide sequence comparison) and BLASTP (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 following parameters can be used: word size (3), open gap penalty (11), extended gap penalty (1), gap dropoff (50), expectation value (10), and any other parameters required, including but not limited to matrix options, as of September 1, 2004. 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% sequence identity to each other.

[0036] As used herein, unless otherwise specified, amino acid residue positions in variable regions of immunoglobulin molecules 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 constant regions of immunoglobulin molecules 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., the Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5 thed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)).

[0037] As used herein, the term "dimer" refers to a biological entity that consists of two subunits that bind 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 non-denaturing and / or non-reducing electrophoretic conditions). 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 a polypeptide, including its characteristics and activities (such as binding and RTCC), should be understood to include the polypeptide in its dimeric form as well as other multimeric forms.

[0038] When the polypeptide of the present disclosure is in the form of a dimer (i.e., a dimeric protein), it contains two binding sites at the amino terminus and two binding sites at the carboxyl terminus. Thus, the binding domain is considered bivalent (i.e., two binding moieties at each end) when the single polypeptide chain dimerizes.

[0039] "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 constituting the constant region are human. In some embodiments, the constant region of the fusion protein of the present disclosure retains the ability to bind to some Fc receptors, such as neonatal Fc receptor (FcRn), and retains a relatively long half-life in vivo, while lacking or having minimal effector function. For example, the constant region of the fusion protein of the present disclosure results in no or substantially reduced induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement activation, and / or complement-dependent cytotoxicity (CDC). In other variations, the 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, the binding domains of the present disclosure are fused to a human IgG1 constant region, and the IgG1 constant region has one or more amino acid mutations of 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), and lysine at position 322 (K322), or any combination thereof (according to 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 is mutated to alanine at each of L234, L235, G237, E318, K320 and K322 (according to EU numbering) (i.e. L234A, L235A, G237A, E318A, K320A and K322A, respectively), and optionally also contains a N297A mutation (i.e. is substantially free of glycosylation of the CH2 domain).

[0040] The "light chain variable region" ("light chain variable domain" or "VL ") and a "heavy chain variable region (also referred to as a "heavy chain variable domain" or "V H The terms "CL" and "CL-L" refer to the variable binding regions from the light and heavy chains of an antibody, respectively. The variable binding regions are composed of separate, well-defined subregions known as "complementarity determining regions" (CDRs) and "framework regions" (FRs). In one embodiment, the FRs are humanized. The term "CL" refers to the "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 the "immunoglobulin heavy chain constant region" or "heavy chain constant region", which can be further divided into CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM), depending on the antibody isotype. "Fab" (Fragment Antigen Binding) is the part 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.

[0041] As used herein, the term "linker" generally refers to a short polypeptide sequence that connects two subdomains of a polypeptide. Non-limiting examples of linkers include flexible linkers that contain 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 that is compatible with the interaction of the two subbinding domains, such that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody that contains the same light chain variable region and heavy chain variable region. In certain embodiments, the linker is composed 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 10-12 amino acid residues) between the C-terminus of the CH3 domain (e.g., wild-type CH3 or mutant CH3) and the N-terminus of the CH1 domain or CL domain (e.g., Cκ).

[0042] In some embodiments, depending on the context, the linker can be: (1) a V H Area and V LThe term may refer to a polypeptide region between a first binding domain and a second binding domain in a multispecific polypeptide comprising two binding domains, or (2) a 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 linkers are referred to herein as "Fc binding domain linkers." In some embodiments, an Fc binding domain linker can directly link or connect two or more binding domains, resulting in a construct comprising the structure binding domain-Fc binding domain linker-binding domain. In some embodiments, a multispecific polypeptide described herein comprises, 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, a multispecific polypeptide comprises, 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, the Fc binding domain linker may link or connect 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 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, and (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) can 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 shown in Table 1.

[0043] In some embodiments, "hinge" or "hinge region" refers to a polypeptide derived from an immunoglobulin hinge region and 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 sequence that is intermediate between and connects the CH1 and CH2 domains (in the case of IgG, IgA, and IgD) or the CH1 and CH3 domains (in the case of 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 include a human IgG hinge region (e.g., and IgG1, IgG2, IgG3, or IgG4 hinge region).

[0044] "Altered immunoglobulin hinge region" or "mutated immunoglobulin hinge region" refers to a hinge region polypeptide having one or more mutations, substitutions, insertions, or deletions compared to a corresponding parent wild-type immunoglobulin hinge region. In certain embodiments, the altered 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%, or 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 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 25 amino acids). Typically, modified immunoglobulin hinge regions that are fragments of the hinge region of a wild-type immunoglobulin comprise an IgG core hinge region (e.g., comprising the sequence CXXC, where X is any amino acid (SEQ ID NO: 390)) as disclosed in U.S. Patent Application Publication Nos. 2013 / 0129723 and 2013 / 0095097. Non-limiting examples of hinges are shown in Table 2.

[0045] As used herein, the term "humanization" refers to the process of using genetic engineering techniques to render antibodies or immunoglobulin binding proteins and polypeptides derived from non-human species (e.g., mouse or rat) less immunogenic to humans while retaining the antigen-binding properties of the original antibody. In some embodiments, the binding domain(s) of the antibodies or immunoglobulin binding proteins and polypeptides (e.g., light and heavy chain variable regions, Fab, scFv) are humanized. Non-human binding domains can be humanized using techniques known as CDR grafting (Jones et al., Nature 321:522 (1986)) and modifications thereof, 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 Immuno l148:1149-1154), and "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312). When of non-human origin, other regions of antibodies or immunoglobulin binding proteins and polypeptides can also be humanized, such as hinge regions and constant region domains.

[0046] As used herein, an "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain" refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or binds with a distinct immunoglobulin domain of a second polypeptide chain, where the interaction of the distinct immunoglobulin heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of a first and second polypeptide chain (i.e., the formation of a dimer between two distinct polypeptide chains, also referred to as a "heterodimer"). An interaction between immunoglobulin heterodimerization regions "substantially contributes to or efficiently promotes" heterodimerization of a first and second polypeptide chain if there is a statistically significant decrease 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 99% of the first and second polypeptide chains form heterodimers with each other. 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 altered (or mutated) immunoglobulin CH1 and CL domains provided therein.

[0047] 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 amenable to treatment or amelioration with a binding protein or multispecific polypeptide or composition thereof provided herein. As used herein, "patient" and "subject" are used interchangeably.

[0048] As used herein, the term "pharmacologically acceptable" refers to molecular entities and compositions that do not generally cause allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that have been approved by a federal or state regulatory agency or for use in animals, and more specifically in humans, in the United States Pharmacopeia or other generally recognized pharmacopoeias are considered "pharmacologically acceptable."

[0049] As used herein, the term "nucleic acid", "nucleic acid molecule" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, 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 generating 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, and mRNA encoded by a gene. As used herein, the term "nucleic acid," "nucleic acid molecule," or "polynucleotide" is 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, derivatives, fragments, and homologs thereof.

[0050] 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 includes the transcription of the nucleic acid segment into mRNA and the translation of mRNA into one or more polypeptides.

[0051] The terms "expression unit" and "expression cassette" are used interchangeably herein to refer to a nucleic acid segment that encodes a polypeptide of interest and is capable of effecting expression of the nucleic acid segment in a host cell. An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in an operable configuration. In addition to a transcription promoter and terminator, an expression unit may further comprise other nucleic acid segments, such as, for example, an enhancer or a polyadenylation signal.

[0052] The term "expression vector" as used herein refers to a linear or circular nucleic acid molecule that contains one or more expression units. In addition to one or more expression units, expression vectors can also contain additional nucleic acid segments, such as one or more origins of replication or one or more selectable markers. Expression vectors can generally be derived from plasmid or viral DNA, or contain elements of both.

[0053] As used herein, "polypeptide", "polypeptide chain" or "protein" refers to a contiguous arrangement of covalently linked amino acids. A polypeptide may form one or more intrachain disulfide bonds. With respect to the polypeptides described herein, reference to a modification or alteration of an amino acid residue corresponding to one identified by a SEQ ID NO includes post-translational modification of such a residue. The terms polypeptide and protein also encompass embodiments in which two polypeptide chains are linked together in a non-linear manner, such as through an interchain disulfide bond. For example, a native immunoglobulin molecule is composed of two heavy and two light polypeptide chains.

[0054] As used herein, a "multispecific polypeptide" refers to a polypeptide that comprises two or more binding domains, each capable of specifically binding to a target antigen. For example, the polypeptides described herein may comprise two, three, four or more binding domains and may 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 is capable of binding 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 may be derived from an antibody (e.g., variable heavy chain and / or variable light chain, scFv), a ligand, or a receptor.

[0055] Multispecific polypeptides are disclosed, for example, in PCT Publication Nos. WO2007 / 146968, WO2010 / 040105, WO2010 / 003108, WO2016 / 094873, WO2017 / 053469; U.S. Patent Application Publication No. 2006 / 0051844, and U.S. Patent Nos. 7,166,707 and 8,409,577, each of which is incorporated herein by reference in its entirety. In certain embodiments, the multispecific polypeptides described herein are bispecific polypeptides and may comprise an scFv-Fc-scFv structure, also referred to herein as ADAPTIR™ polypeptides. The structure of a polypeptide containing such a structure comprises from N-terminus to C-terminus: first scFv binding domain-immunoglobulin (Ig) hinge region-Ig constant region-second scFv binding domain.

[0056] 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 chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma. Other multivalent formats that can be used include, for example, quadromas, Kλ-bodies, dAbs, bispecific antibodies, TandAbs, nanobodies, Small Modular ImmunoPharmaceutials (SMIPs™), DOCK-AND-LOCK® (DNL®), CrossMab Fab, CrossMab VH-VL, strand-exchange engineered domain bodies (SEED bodies), affibodies, finomers, Kunitz Domains, Albu-dabs, two engineered Fv fragments with swapped VHs (e.g., dual affinity retargeting molecules (DARTs)), scFv x Exemplary bispecific formats include scFv (e.g., BiTE), DVD-IG, Covx bodies, peptibodies, scFv-Ig, SVD-Ig, dAb-Ig, knob-in-hole, IgG1 antibodies with matching mutations in the CH3 domain (e.g., DuoBody antibodies), and triomAb. Exemplary bispecific formats are described in Garber et al., Nature Reviews Drug Discovery 13:799-801 (2014), which is incorporated herein by reference in its entirety. Further exemplary bispecific formats are described 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, a bispecific antibody may be a F(ab')2 fragment, which comprises the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0057] As will be understood by those of skill in the art, proteins and polypeptides are defined herein by the amino acid sequences of the individual polypeptide chains, as indicated by the SEQ ID NOs referenced throughout this disclosure. For example, in some embodiments, a scFv-Fc-scFv protein or polypeptide described herein is composed 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 include non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents can be added to proteins or polypeptides by the cell in which the protein is produced and vary depending on the type of cell. Proteins and polypeptides are defined herein by their amino acid backbone structures, and substituents such as carbohydrate groups may be present, even if not generally specified.

[0058] "Light chain variable region" ("light chain variable domain" or "VL" or "V L and a "heavy chain variable region" (also referred to as a "heavy chain variable domain" or "VH" or V HThe terms "CL" and "CL-FR" refer to the "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 the "immunoglobulin heavy chain constant region" or "heavy chain constant region", which can be further divided into CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM), depending on the isotype of the antibody. "Fab" (Fragment Antigen Binding) is the part of an antibody that binds an antigen and contains the variable region and CH1 domain of the heavy chain linked to a light chain via an interchain disulfide bond.

[0059] The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote locations within a polypeptide. Where the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative location. For example, a particular sequence located carboxyl-terminal to a reference sequence within a polypeptide is located adjacent to the carboxyl-terminus of the reference sequence, but is not necessarily located at the carboxyl-terminus of the entire polypeptide.

[0060] As used herein, the terms "transformation," "transfection," 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 transferred nucleic acid can be introduced into the cell via an expression vector.

[0061] As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated process in which non-specific 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 and subsequently cause lysis of the target cells. Essentially, any effector cell with an activated FcγR can be activated to mediate ADCC. The primary cells for mediating ADCC are NK cells, which express only FcγRIII, whereas monocytes can express FcγRI, FcγRII, and FcγRIII depending on their state of activation, localization, or differentiation. For a review of FcγR expression on hematopoietic cells, see, e.g., Ravetch et al., 1991, Annu. Rev. Immunol., 9:457-92.

[0062] The term "having ADCC activity" as used herein with respect to a polypeptide or protein means that a polypeptide or protein, e.g., one that comprises an Fc domain (e.g., an immunoglobulin hinge region, and an immunoglobulin constant region having CH2 and CH3 domains), such as from an IgG (e.g., IgG1), is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) via a cytolytic Fc receptor (e.g., FcγRIII) on cytolytic immune effector cells expressing an Fc receptor (e.g., NK cells). In some embodiments, a multispecific polypeptide or protein that comprises an Fc domain may lack effector function (e.g., null ADCC activity) as a result of mutations in the CH2 and / or CH3 domains.

[0063] As used herein, "complement dependent cytotoxicity" and "CDC" refer to the process by which a component in normal serum ("complement"), together with antibodies or other C1q-complement binding proteins bound to a target antigen, exhibits lysis of a target cell expressing the target antigen. Complement consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect.

[0064] 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 orderly activation of nine major protein components designated C1-C9. For some 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.

[0065] The term "having CDC activity" as used herein with respect to a polypeptide or protein means that a polypeptide or protein, such as one that comprises an Fc domain (e.g., an immunoglobulin constant region having an immunoglobulin hinge region and CH2 and CH3 domains) from an IgG (e.g., IgG1), is capable of mediating 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.

[0066] As used herein, "enhanced effector cell activation" refers to an increase, prolongation, and / or enhancement 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 an increase in cytokine production, cell proliferation, or changes in the expression of cell surface molecules such that the ability of an effector cell to lyse a target cell is enhanced.

[0067] 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. As used herein, effector cell can refer to a lymphocyte, such as a T cell, a natural killer (NK) cell, or a NKT cell, a monocyte, a macrophage, a dendritic cell, or a granulocyte. In certain embodiments, the term effector cell refers to a T cell, a NK cell, or a NKT cell.

[0068] As used herein, the terms "treatment," "treat," or "ameliorate" refer to either curative or prophylactic / preventative treatment. A treatment is curative if at least one symptom of a disease in an individual receiving the treatment improves, or the treatment may slow the progression of a progressive disease or prevent the onset of additional related diseases in an individual.

[0069] As used herein, the term "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a polypeptide or protein or composition thereof described herein refers to an amount of a compound sufficient to ameliorate one or more symptoms of the disease being treated in a statistically significant manner or to cause a statistically significant improvement in organ function. With respect to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When referring to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered sequentially or simultaneously (either in the same formulation or simultaneously in separate formulations).

[0070] Pharmaceutical Compositions Described herein are stable pharmaceutical formulations of protein therapeutics, such as multispecific polypeptides, that prevent denaturation and / or prevent or substantially reduce the formation of aggregates, 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 composition comprises, consists of, or consists essentially of a buffer, an excipient, and a surfactant, the multispecific 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, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a second binding domain, and the buffer consists of succinic acid or a pharma- ceutically acceptable salt or acid thereof.

[0071] In some embodiments, the composition comprises from about 0.1 mg / ml to about 10 mg / ml of the multispecific protein. In some embodiments, the composition comprises from 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 succinic acid, about 6.5% weight / volume (w / v) sucrose and about 0.02% w / v polysorbate 80.

[0072] In some embodiments, the composition substantially prevents degradation of the multispecific protein. In some embodiments, the composition retards or reduces degradation of the multispecific polypeptide compared to the same multispecific polypeptide stored in histidine buffer under the same storage conditions. In some embodiments, the composition is substantially stable at 4° C. for at least one year. In some embodiments, the composition is substantially resistant to the formation of aggregates of the multispecific protein.

[0073] 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 a multispecific polypeptide stored in a histidine buffer under the same freeze-thaw conditions.

[0074] In other embodiments, multispecific polypeptides targeting CD123xCD3, when prepared as disclosed herein, exhibit little or no degradation after lyophilization. For example, multispecific polypeptides targeting CD123xCD3 may be prepared in succinic acid and sucrose formulations that reduce degradation after lyophilization compared to the same polypeptides prepared with a histidine buffer. Also provided herein are lyophilized anti-CD123xanti-CD3 multispecific polypeptides, including but not limited to TRI130 and TRI129, prepared in about 5 mM succinic acid, about 6.5% weight / volume (w / v) sucrose, and about 0.02% w / v polysorbate 80. In some embodiments, the compositions are lyophilized.

[0075] Buffer 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 from changes in pH upon addition of acid or alkali, or upon dilution with a solvent.

[0076] In some embodiments, the buffer comprises, consists of, or essentially consists of any pharma- ceutically acceptable buffer.For example, the buffer can be potassium phosphate, acetic acid / sodium acetate, citric acid / sodium citrate, succinic acid / sodium succinate, tartaric acid / sodium tartrate, histidine / histidine HCl, glycine, Tris, glutamic acid, acetate, mixtures thereof, or pharma-ceutically acceptable salts or acids thereof.In certain embodiments, the pre-buffer comprises, consists of, or essentially consists of succinic acid or its pharma-ceutically acceptable salts or acids.

[0077] 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, or about 5 mM to about 20 mM, about 5 mM to about 10 mM. In some embodiments, the composition comprises about 1 mM to about 10 mM succinic acid or a pharma- ceutically acceptable salt or acid thereof. In some embodiments, the composition comprises about 5 mM succinic acid or a pharma- ceutically acceptable salt or acid thereof.

[0078] 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 composition has a pH of about 4.0 to about 5.5. In some embodiments, the pH of the composition is about 4.8.

[0079] Excipients As referred to herein, excipients are pharmacologically inactive substances formulated with the active pharmaceutical ingredient of the composition. Excipients may aid in lubrication, flowability, disintegration, or taste, and may impart some form of antimicrobial functionality.

[0080] Exemplary 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 AH Kibbe, American Pharmaceutical Association and Pharmaceutical Press (2000), and ETCole 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 oxystearate glycerol, saturated polyglycolized glycerides, polyethylene polypropylene glycol, vitamin E, and vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate).

[0081] In some embodiments, the composition comprises about 1% to about 20% weight / volume (w / v), about 1% to about 10% w / v, about 5% to about 15% w / v, or about 10% w / v. In some embodiments, the composition comprises about 1% to about 12% w / v, e.g., about 6.5% w / v.

[0082] 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.

[0083] Surfactants 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, a carboxyl group and a carboxylate group).

[0084] Suitable surfactants for use in the compositions described herein include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and derivatives; Triton; sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetadine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT™ series (Mona Polysorbate 80 (Polysorbate 80) is a surfactant that can be used in a wide range of applications, including, but not limited to, polysorbate 80 (Polysorbate 80), ...

[0085] 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 of a surfactant, hi some embodiments, the composition comprises about 0.02% w / v of a surfactant.

[0086] 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 polysorbate 80. In some embodiments, the composition comprises about 0.02% w / v polysorbate 80.

[0087] Therapeutic Proteins The compositions described herein can be used with many of the different protein therapeutics described herein.

[0088] Binding domain In some embodiments, the therapeutic protein comprises a binding domain. The binding domain may provide specific binding to at least one cell surface molecule (e.g., a cell surface receptor). The binding domain may be in the form of an antibody or a fragment thereof, or may be a fusion protein in any of a variety of different formats (e.g., the fusion protein may be in the form of a bispecific or multispecific molecule). In other embodiments, the binding domain may comprise, for example, a specific cytokine, or a molecule that targets the binding domain polypeptide to, for example, a specific cell type, a toxin, an additional cell receptor, or an antibody.

[0089] In some embodiments, the binding domains described herein are derived from antibodies and comprise a variable heavy chain (V H ) and variable light chain (V L ) for example, V H and V L The binding domains and variable chains may be arranged in any order that still retains binding to the target(s). 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 L ).

[0090] In some embodiments, the polypeptides and proteins described herein comprise a binding domain that is an scFv. In such embodiments, the binding domain may be referred to as an scFv domain. In some embodiments, the binding domain comprises a VFv domain specific for a target of interest. H and V L In certain embodiments, the V H and V L The region is human or humanized. In some variations, the binding domains are V L Area and V H It is a single-chain Fv (scFv) containing the

[0091] In certain embodiments, the binding domain of the polypeptide described herein comprises (i) an immunoglobulin light chain variable region (VLCDR1), comprising the CDRs LCDR1, LCDR2 and LCDR3. L ), and (ii) an immunoglobulin heavy chain variable region (V 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).

[0092] In certain embodiments, a known V L and / or V H When compared to the sequence, the 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(e.g., 1618 / 1619 as described in PCT Publication No. WO2016 / 185016), and optionally includes 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 insertion(s), deletion(s) or substitution(s) can be at or near the amino or carboxyl terminus or both ends of this region. L and / or V H Anywhere within the region, provided that each CDR contains zero or a maximum of one, two, or three alterations. L and / or V H A binding domain that includes the region can still specifically bind to a target with an affinity similar to or greater than that of the parent binding domain.

[0093] V L and V H The use of peptide linkers to link domains is well known in the art and there are numerous publications in this particular field. In some embodiments, the peptide linker is a 15-mer consisting 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 techniques as well as selectively infecting phage techniques have been used to diversify and select suitable 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 is of the formula (Gly4Ser) n(wherein 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 via random mutagenesis. In some embodiments, the V of the scFv described herein H In some embodiments, the V region may be located N-terminal to the linker sequence. L The region may be located C-terminal to the linker sequence.

[0094] 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 domain 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 4-1BB and OX40.

[0095] 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 in the wild-type immunoglobulin hinge region are replaced with one or more other 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 replaced with one, two, or three different amino acid residues (e.g., serine or alanine). The modified immunoglobulin hinge may further comprise a proline replaced with another amino acid (e.g., serine or alanine). For example, the modified human IgG1 hinge described above may further comprise a proline replaced with another amino acid residue (e.g., serine, alanine) that is located carboxyl-terminal to the three cysteines in 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 an identical sequence 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, 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.

[0096] Immunoglobulin constant domains The therapeutic protein may also comprise an immunoglobulin constant (Fc) domain (also referred to herein as constant region, Fc domain, Fc region, etc.). In some embodiments, the constant region comprises the CH2 and CH3 domains of IgG, e.g., the CH2 and CH3 domains of IgG1. In some embodiments, the constant region does not comprise the 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 that make up 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 binding 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 prevent or reduce 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.

[0097] 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 region 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 region comprises L234A, L235A, G237A, and K322A mutations in the CH2 domain according to the EU numbering system. In some embodiments, the Fc region comprises L234A, L235A, G237A, E318A, K320A, and K322A mutations in the CH2 domain according to the EU numbering system. In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising the following substitutions according to the EU numbering system: E233P, L234A, L235A, G237A, and K322A, and a deletion of G236. In some embodiments, the Fc region 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.

[0098] In some embodiments, the immunoglobulin constant region comprises an amino acid sequence of any one of SEQ ID NOs: 32-35 or a variant thereof. The inclusion of an immunoglobulin constant region slows the clearance of the polypeptides and proteins of the present disclosure from the circulation after administration to a subject. By mutation or other modification, the immunoglobulin constant region further allows for relatively easy modulation of the polypeptide effector functions (e.g., ADCC, ADCP, CDC, complement fixation, and binding to Fc receptors), which may 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 region of a polypeptide or protein described in the present disclosure compared to the corresponding wild-type immunoglobulin constant region.

[0099] The immunoglobulin constant region 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, the 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 a portion of a CH2 domain and a CH3 domain. In certain embodiments, the polypeptides or proteins described herein do not comprise a CH1 domain.

[0100] A polypeptide or protein described herein may comprise a wild-type or modified immunoglobulin CH2 domain from a particular immunoglobulin class or subclass (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, as set forth in SEQ ID NOs: 115, 199-201, and 195-197, respectively, as described in U.S. Patent Application Publication No. 2013 / 0129723, which sequences are incorporated herein by reference. In a particular 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. US2013 / 0129723, which sequence is incorporated herein by reference.

[0101] In certain embodiments, an altered 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 mouse IgG2a (e.g., IGHG2c).

[0102] The modified immunoglobulin CH2 region in the polypeptide or protein of the present disclosure may be derived from the CH2 region of various immunoglobulin isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgD, from various species, including human, mouse, rat, and other mammals. In certain embodiments, the immunoglobulin CH2 region in the fusion protein of the present disclosure may 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 US Patent Application Publication No. 2013 / 0129723, which sequences are incorporated herein by reference. In certain embodiments, the modified CH2 domain of the polypeptide or protein described herein is a modified human IgG1 CH2 domain with mutations known in the art that enhance or reduce immune activity (i.e., effector function), such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.

[0103] 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 includes one or more amino acid deletions or substitutions. In some embodiments, the CH2 domain includes an amino acid substitution at asparagine at position 297 (e.g., asparagine to alanine). Such an amino acid substitution reduces or eliminates glycosylation at this site and inhibits 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 shown in SEQ ID NO: 324 of US Patent Application Publication No. 2013 / 0129723 (said sequence is incorporated herein by reference). In some embodiments, the modified CH2 domain includes at least one substitution or deletion at positions 234-238. For example, the immunoglobulin CH2 region can include substitutions of 2, 3, 4, or 5 amino acids at positions 234, 235, 236, 237, or 238; 234 and 235; 234 and 236; 234 and 237; 234 and 238; 234-236, 234, 235, and 237; 234, 236, and 238; 234, 235, 237, and 238; 236-238; or any other combination of 2, 3, 4, or 5 amino acid deletions at one of positions 234-238, e.g., 2, 3, 4, or 5, at positions 234-238, e.g., 236 or 237, and the other position is substituted. In certain embodiments, the amino acid residue at one or more of positions 234-238 is substituted with one or more alanine residues. In a further embodiment, only one of the amino acid residues at positions 234-238 may be deleted, and one or more of the remaining amino acids at positions 234-238 may be substituted with another amino acid (eg, alanine or serine).

[0104] In some embodiments, the above mutation(s) reduce or eliminate ADCC activity or Fc receptor binding ability of the polypeptide comprising the altered CH2 domain.

[0105] In certain other embodiments, the CH2 domain of a polypeptide or protein described herein is an altered immunoglobulin CH2 region (e.g., an altered 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 comprises substitutions in any combination of two, three, four, five, or six amino acids at positions 253, 310, 318, 320, 322, 318, 320, 322, or 331. In such embodiments, the above mutation(s) reduce or eliminate the CDC activity of the polypeptide comprising the altered CH2 domain.

[0106] In certain other 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., 2, 3, 4, or 5) additional substitutions at positions 234-238. For example, the immunoglobulin CH2 region comprises substitutions at positions 234 and 297, 234, 235 and 297, 234, 236 and 297, 234-236 and 297, 234, 235, 237 and 297, 234, 236, 238 and 297, 234, 235,237,238 and 297, 236-238 and 297, or any combination of 2, 3, 4, or 5 at positions 234-238 in addition to position 297. Additionally or alternatively, the modified CH2 region may include one or more (e.g., 2, 3, 4, or 5) amino acid deletions at positions 234-238, such as positions 236 or 237. The additional mutation(s) reduce or eliminate the ADCC activity or Fc receptor binding ability of a polypeptide comprising the modified CH2 domain. 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 of the amino acid residues at positions 234-238 is deleted, and one or more of the remaining amino acids at positions 234-238 may be substituted with another amino acid (e.g., alanine or serine).

[0107] In certain embodiments, in addition to one or more (e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region of a polypeptide or protein described herein in a fusion protein of the disclosure (e.g., a modified human IgG1 CH2 domain) can include one or more (e.g., 2, 3, 4, 5, or 6) 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 a murine IGHG2c CH2 region having alanine substitutions at L234, L235, G237, E318, K320 and K322.

[0108] In yet another embodiment, in addition to the amino acid substitution at position 297 and the further deletion(s) or substitution(s) at 234-238, 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., 2, 3, 4, 5, or 6) additional substitutions at positions 253, 310, 318, 320, 322, and 331. For example, the immunoglobulin CH2 region comprises (1) a substitution at position 297, (2) one or more substitutions or deletions or combinations thereof at positions 234-238, and one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions at I253, H310, E318, K320, K322, and P331, such as 1, 2, 3 substitutions at E318, K320, and K322. The amino acids at the above positions may be substituted with alanine or serine.

[0109] In certain embodiments, the immunoglobulin CH2 region of a polypeptide or protein described herein comprises: (i) an amino acid substitution at asparagine at position 297 and one amino acid substitution at positions 234, 235, 236 or 237; (ii) an amino acid substitution at asparagine at position 297 and two amino acid substitutions at positions 234 to 237; (iii) an amino acid substitution at asparagine at position 297 and three amino acid substitutions at positions 234 to 237; (iv) an amino acid substitution at asparagine at position 297, amino acid substitutions at positions 234, 235 and 237 and an amino acid deletion at position 236; (v) an amino acid substitution at three positions 234 to 237 and amino acid substitutions at positions 318, 320 and 322; or (vi) an amino acid substitution at three positions 234 to 237, an amino acid deletion at position 236 and amino acid substitutions at positions 318, 320 and 322.

[0110] Exemplary modified immunoglobulin CH2 regions having an amino acid substitution at asparagine at position 297 include a human IgG1 CH2 region having alanine substitutions at L234, L235, G237, and N297, and a deletion at G236 (SEQ ID NO: 325 of US Patent Application Publication No. 2013 / 0129723, which is incorporated by reference); a human IgG2 CH2 region having alanine substitutions at V234, G236, and N297 (SEQ ID NO: 326 of US Patent Application Publication No. 2013 / 0129723, which is incorporated by reference); a human IgG4 CH2 region having alanine substitutions at F234, L235, G237, and N297, and a deletion at G236 (SEQ ID NO: 322 of US Patent Application Publication No. 2013 / 0129723, which is incorporated by reference); CH2 (SEQ ID NO: 343 of US Patent Application Publication No. US2013 / 0129723, which is incorporated by reference), a human IgG4 CH2 region with alanine substitutions at L235 and N297 (SEQ ID NO: 344 of US Patent Application Publication No. US2013 / 0129723, which is incorporated by reference), a human IgG4 CH2 region with alanine substitutions at G236 and N297 (SEQ ID NO: 345 of US Patent Application Publication No. US2013 / 0129723, which is incorporated by reference), and a human IgG4 CH2 region with alanine substitutions at G237 and N297 (SEQ ID NO: 346 of US Patent Application Publication No. US2013 / 0129723, which is incorporated by reference). These CH2 regions can be used in the polypeptides of the present disclosure.

[0111] In certain embodiments, in addition to the amino acid substitutions described above, 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, P233 may be changed to E233 in the modified IgG2 CH2 region (see, for example, SEQ ID NO: 326 in US Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference). Additionally or alternatively, in certain embodiments, the modified CH2 region may include one or more amino acid insertions, deletions, or both. The insertion(s), deletion(s), or substitution(s) may be anywhere within the immunoglobulin CH2 region, such as at the N-terminus or C-terminus of the wild-type immunoglobulin CH2 region resulting from linking the CH2 region to another region (e.g., a binding domain or an immunoglobulin heterodimerization domain) via a hinge.

[0112] In certain embodiments, the modified CH2 domain of a polypeptide or protein described herein is a human IgG1 CH2 domain having alanine substitutions at positions 235, 318, 320 and 322 (i.e., a human IgG1 CH2 domain having L235A, E318A, K320A and K322A substitutions) (SEQ ID NO: 595 of US Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference), optionally having an N297 mutation (e.g., to alanine). In another specific embodiment, the modified CH2 domain is a human IgG1 CH2 domain with alanine substitutions at positions 234, 235, 318, 237, 320 and 322 (i.e. a human IgG1 CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO: 596 of US Patent Application Publication No. 2013 / 0129723, which sequence is incorporated herein by reference), optionally with a N297 mutation (e.g., to alanine).

[0113] 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.

[0114] 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 human, mouse, rat, 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 CH2 domains of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM as set forth in SEQ ID NOs: 116, 208-210, 204-207, and 212, respectively, of US Patent Application Publication No. 2013 / 0129723, which sequences are incorporated herein by reference. In a particular 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. US2013 / 0129723, which sequence is incorporated herein by reference.

[0115] In certain embodiments, the CH3 domain of the polypeptides 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 a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM antibody. For example, the modified CH3 domain can be a human IgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions according to EU numbering). Mutations at such positions can be involved in complement binding. In certain other embodiments, the modified CH3 domain of the polypeptides described herein can be a human IgG1 CH3 domain, but with one or two amino acid substitutions at positions F405 or Y407. Amino acids at such positions are involved in interactions with another CH3 domain. In certain embodiments, the modified CH3 domain of the polypeptides described herein can be a modified human IgG1 CH3 domain with the 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, which sequence is incorporated herein by reference.

[0116] In certain embodiments, the polypeptides or proteins described herein comprise a CH3 domain that comprises a so-called "knob-into-hole" mutation (see Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al., Protein Engineering 9:617-21, 1966). More specifically, a mutation can be introduced into each of the CH3 domains of each polypeptide chain, such that the steric complementarity required for the CH3 / CH3 bond is essential for these two CH3 domains to pair with each other. For example, the CH3 domain in one single-chain polypeptide of a polypeptide heterodimer can comprise a T366W mutation (a "knob" mutation that substitutes a smaller amino acid for a larger amino acid), and the CH3 domain in the other single-chain polypeptide of the polypeptide heterodimer can comprise a Y407A mutation (a "hole" mutation that substitutes a larger amino acid for a smaller amino acid). Additional 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.

[0117] The CH4 domain that can form the immunoglobulin constant region of the polypeptide or protein described herein can be a wild-type immunoglobulin CH4 domain from an IgE or IgM molecule or a modified immunoglobulin CH4 domain thereof. In certain embodiments, the CH4 domain of the polypeptide described herein is a wild-type human immunoglobulin CH4 domain, such as the wild-type CH4 domain of human IgE and IgM molecules shown in SEQ ID NOs: 213 and 214, respectively, of US Patent Application Publication No. 2013 / 0129723 (the foregoing sequences are incorporated herein by reference). In certain embodiments, the CH4 domain of the polypeptide described herein is a modified human immunoglobulin CH4 domain, such as a modified human immunoglobulin CH4 domain based on or derived from the CH4 domain of a human IgE or IgM molecule, which has a mutation that increases or decreases the immune activity known to be associated with the Fc region of IgE or IgM.

[0118] 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, the immunoglobulin constant region can comprise CH2 and CH3 domains, or CH3 and CH4 domains. In certain other embodiments, the immunoglobulin constant region can comprise two CH3 domains and no CH2 or CH4 domains (i.e., only two or more CH3s). 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 the same class or immunoglobulin molecule. 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 can be human (e.g., human IgG1, IgG2 and IgG4) CH2CH3. 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) human IgG1 CH2 with an N297A substitution (i.e., CH2(N297A)) and wild-type human IgG1 CH3; or (3) human IgG1 CH2(N297A) and a human IgG1 CH3 with the last lysine 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 different classes or subclasses of immunoglobulin molecules. 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 directly linked together or can be linked to each other via one or more (e.g., about 2-10) amino acids.

[0119] Exemplary immunoglobulin constant regions that may 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, which sequences are incorporated herein by reference. Further exemplary immunoglobulin constant regions that may be used in the polypeptides or proteins described herein are provided in the table below. [Table 1] JPEG2024519964000003.jpg232159JPEG2024519964000004.jpg21159

[0120] In certain embodiments, the immunoglobulin constant regions of each polypeptide chain of the homodimeric or heterodimeric proteins described herein are identical to each other. In certain other embodiments, the immunoglobulin constant region of one polypeptide chain of the heterodimeric protein is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer. For example, the immunoglobulin constant region of one of the heterodimeric proteins can comprise a CH3 domain with a "knob" mutation, and the immunoglobulin constant region of the other of the heterodimeric proteins can comprise a CH3 domain with a "hole" mutation.

[0121] Fc-binding domain linker In some embodiments, the polypeptide may further comprise an Fc binding domain linker. In some embodiments, an Fc binding domain linker may 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 may 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 consisting 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 SEQ ID NOs: 50-70. Other linkers have been used, and phage display techniques as well as selectively infecting phage techniques have been used to diversify and select suitable linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., Protein Eng. 11, 405-410, 1998). L and V H The region is of the formula (Gly4Ser) n (wherein n=1-5 (SEQ ID NO: 129)). Other suitable linkers can be obtained by optimizing simple linkers by random mutation. In some embodiments, the bispecific molecule does not include a hinge region or a constant region.

[0122] In certain embodiments, the Fc binding domain linker is a flexible linker sequence comprising glycine-serine (e.g., Gly4Ser, SEQ ID NO: 128) repeats. 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.

[0123] Some exemplary hinge and Fc binding domain linker sequences suitable for use according to the present disclosure are shown in Table 2. Additional exemplary hinge and linker regions are shown 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] JPEG2024519964000006.jpg106159

[0124] In addition to the aforementioned domains, 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.

[0125] Bispecific / multispecific proteins In some embodiments, the therapeutic protein may 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 may 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.

[0126] In some embodiments, the multispecific protein may comprise, from N-terminus to C-terminus, a CD3 binding domain, a hinge region, an immunoglobulin constant region, and a tumor antigen binding domain. The tumor antigen binding domain may bind, for example, to CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0127] In some embodiments, the multispecific protein may comprise, from N-terminus 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 may bind, for example, to CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0128] In some embodiments, the multispecific protein may comprise, 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. The tumor antigen binding domain may bind, for example, to CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0129] In some embodiments, the multispecific protein may comprise, 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. The tumor antigen binding domain may bind, for example, to CD123, PSMA, CD19, CD33, 5T4, or HER2.

[0130] Homodimer / heterodimer 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 polypeptide 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, and (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 polypeptide 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 bispecific antibody.

[0131] 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 embodiments, the hinge of one polypeptide chain of the 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 (in its length or sequence) from the hinge of the other chain. Different hinges in different chains allow for different engineering of the binding affinities of the binding domains to which the hinges are connected, such that the heterodimer can preferentially bind the target of one binding domain over the target of the other binding domain.

[0132] In other embodiments, the polypeptides and proteins described herein contain a heterodimerization domain, which allows heterodimerization 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 polypeptide containing a first binding domain and the other optionally containing a second binding domain, dimerize to form a heterodimeric binding protein. Dimerization / heterodimerization domains can be used when it is desired to form a heterodimer from two non-identical polypeptide chains, one or both polypeptide chains containing a binding domain. In certain embodiments, one polypeptide chain member of a particular heterodimer 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 International PCT Publication No. WO2016 / 094873.

[0133] In certain embodiments, the first and second polypeptide chains dimerize by comprising an "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain." An "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain," as used herein, refers to an immunoglobulin domain of a first polypeptide chain that preferentially interacts or binds with a distinct immunoglobulin domain of a second polypeptide chain, where the interaction of the distinct 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 distinct polypeptide chains, also referred to as a "heterodimer"). The immunoglobulin heterodimerization domains in the polypeptide chains of a heterodimer are distinct from one another and can therefore be engineered separately to promote heterodimerization of both chains and minimize homodimerization of either chain. The immunoglobulin heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting heterodimeric proteins.

[0134] 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 wild-type IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain, e.g., as set forth in SEQ ID NOs: 114, 186-192, and 194 of US Patent Application Publication No. 2013 / 0129723, respectively, or SEQ ID NO: 114 of US Patent Application Publication No. 2013 / 0129723 (these sequences are incorporated herein by reference). In a further embodiment, cysteine ​​residues in the wild-type CH1 domain (e.g., human CH1) that are involved in disulfide bond formation with the wild-type immunoglobulin CL domain (e.g., human CL) are deleted or substituted in the modified immunoglobulin CH1 domain such that disulfide bonds are not formed between the modified CH1 domain and the wild-type CL domain.

[0135] The polypeptides and proteins described herein can be produced using scaffolds as generally disclosed in US Patent Publication Nos. 2013 / 0129723 and 2013 / 0095097, each of which is incorporated herein by reference in its entirety. The polypeptides described herein can comprise two non-identical polypeptide chains, each of which comprises an immunoglobulin heterodimerization domain. The heterodimerization domains of the opposing immunoglobulins are different. In one embodiment, the immunoglobulin heterodimerization domain comprises a CH1 domain or a derivative thereof. In one embodiment, the immunoglobulin heterodimerization domain comprises a CL domain or a derivative thereof. In one embodiment, the CL domain is of the Cκ or Cλ isotype or a derivative thereof.

[0136] Exemplary Protein Therapeutics: Anti-CD123 x Anti-CD3 Polypeptides and Dimers Thereof Exemplary protein therapeutics can bind to both CD123-expressing cells and the T cell receptor complex on T cells, inducing target-dependent T cell cytotoxicity, activation and proliferation.

[0137] Thus, in certain embodiments, the 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 binding to the target(s). 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)-(VH CD123); or (VL CD3)-(VH CD3)-(VH The pair of VH and VL regions in the binding domain that binds to CD3 may be in the format of a single chain antibody (scFv). The VH and VL regions may be arranged in the order VH-VL or VL-VH. In some embodiments, an scFv may bind to CD123 more effectively than an antibody that contains the same VH and VL region sequences in the same orientation. In certain embodiments, an scFv may bind to CD123 more effectively in a VL-VH orientation than in a VH-VL orientation, or vice versa. The VH region may be located N-terminal to the linker sequence. The VL region may be located C-terminal to the linker sequence. The domain sequence in the CD3 binding domain of the bispecific single chain molecule may be VH-VL, with the CD3 binding domain being located C-terminal to the CD123 binding domain. The bispecific molecule may comprise an scFv that binds CD123 linked to an scFv that binds CD3.These scFvs may be linked to short peptides. In some embodiments, the bispecific single chain molecules do not include a hinge or constant region (see, e.g., US2013 / 0295121, WO2010 / 037836, WO2004 / 106381, and WO2011 / 121110, each of which is incorporated by reference in its entirety).

[0138] 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] JPEG2024519964000008.jpg227159JPEG2024519964000009.jpg225159JPEG2024519 964000010.jpg225159JPEG2024519964000011.jpg217159JPEG2024519964000012.j pg221159JPEG2024519964000013.jpg221159JPEG2024519964000014.jpg221159JPE G2024519964000015.jpg220159JPEG2024519964000016.jpg217159JPEG2024519964 000017.jpg217159JPEG2024519964000018.jpg220159JPEG2024519964000019.jpg2 30159JPEG2024519964000020.jpg221159JPEG2024519964000021.jpg217159JPEG20 24519964000022.jpg225159JPEG2024519964000023.jpg218159JPEG2024519964000 024.jpg223159JPEG2024519964000025.jpg221159JPEG2024519964000026.jpg33159 [Table 4]

Table 5

[0139] In certain embodiments, the CD123 binding domain comprises (i) an immunoglobulin light chain variable region (VL) comprising the CDRs LCDR1, LCDR2 and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) comprising the CDRs HCDR1, HCDR2 and HCDR3, where 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 the CDRs LCDR1, LCDR2 and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) comprising the CDRs HCDR1, HCDR2 and HCDR3. In some such embodiments, (i) LCDR1 has an 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 an 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 an 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 an 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 an 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 an 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-mentioned amino acid substitutions may be conservative or non-conservative amino acid substitutions. In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the depicted sequence.In certain embodiments, the CDRs of the disclosure 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) compared to the CDR sequences of known monoclonal antibodies, or a combination of the above changes. For example, the present disclosure encompasses a recombinant polypeptide, in which (i) LCDR1 has an 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) LCDR2 has an 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) LCDR3 has an 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) HCDR1 has an 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) HCDR2 has an 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) HCDR3 has an 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 above-mentioned amino acid substitutions may be conservative or non-conservative amino acid substitutions.

[0140] 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) (e.g., SEQ ID NO: 136), or both. In one embodiment, the CD123 binding domain of the recombinant polypeptide is an scFv comprising a variable heavy chain comprising SEQ ID NO: 136 in a VHVL orientation 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 a variable light chain comprising SEQ ID NO: 134 in a VHVL orientation 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.

[0141] In certain embodiments, the CD123 binding domain comprises (i) an immunoglobulin light chain variable region (VLCDR1), comprising CDRs LCDR1, LCDR2, and LCDR3. L ), and (ii) an immunoglobulin heavy chain variable region (V 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 above-mentioned 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 described sequences by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the CDRs of the disclosure 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, compared to the CDR sequences of known monoclonal antibodies.

[0142] 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.

[0143] In certain embodiments, the CD123 binding domain is a humanized immunoglobulin V L and / or V H Contains the region Immunoglobulin V L and V H Techniques for humanizing regions are well known in the art and are described, 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.

[0144] Essentially, humanization by CDR grafting involves replacing only the CDRs of a non-human antibody with 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 allotypic differences). However, it has been reported that some original antibody framework residues may also need to be preserved (Reichmann et al., Nature, 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10,029 (1989)).

[0145] Framework residues that need to be conserved are subject to identification by computer modeling. Alternatively, important framework residues may be identified by comparison to known antigen-binding site structures (Padlan, Molec. Immunol., 31(3):169-217 (1994), incorporated by reference).

[0146] Residues that may affect antigen binding are divided into several groups. The first group includes residues that are contiguous with the surface of the antigenic site and therefore may be in direct contact with the antigen. These residues include the amino-terminal residues and residues adjacent to the CDRs. The second group includes residues that may change the structure or relative alignment of the CDRs by contacting either the CDRs or another peptide chain in the antibody. 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 vary 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).

[0147] Information regarding humanized antibodies in the art is applicable to polypeptides according to the present disclosure, even if the polypeptide is not an antibody.

[0148] In some embodiments, the anti-CD123 scFv comprises an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HDCR3 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 domain 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 a C anti-D123 scFv, wherein the scFv comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:18.

[0149] In some embodiments, the present disclosure relates to a CD123 binding domain, in which (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.

[0150] 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 a fragment or derivative thereof, that specifically binds to a target of interest (e.g., CD123).

[0151] In certain embodiments, the CD123 binding domain does not inhibit IL-3 binding to CD123.

[0152] In certain embodiments, a CD123 binding molecule or protein may comprise a T 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 a 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 virtually any binding domain that binds to T cells, e.g., an antibody-derived binding domain. Exemplary anti-CD3 antibodies from which a 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), V-seq. SEQ ID NO: 341 (QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITR) and SEQ ID NO: 342 (QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWGQGTLVTVSA), respectively. L and V HThe amino acid sequence, HuM291 (Chau et al. (2001) Transplantation 71:941-950, SEQ ID NO: 343 (DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK) and SEQ ID NO: 344 (QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSS) are shown in SEQ ID NO: 344, respectively. L and V HAmino acid sequences, 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), humanized Fc variants 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 ... al. (1988) J. Immunol. 140:3766) and I2C monoclonal antibodies (see, e.g., US2011 / 0293619 and US20120244162). For example, the CD3 binding domain may comprise a CD3 binding domain disclosed in US Patent Application Publication No. 2012 / 0244162, including 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 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 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 US2012 / 0244162. In some embodiments, the CD3 binding domain is one described in WO2004 / 106380, WO2005 / 040220A1, US2014 / 0099318, or a CD3 binding domain derived therefrom.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 WO2013 / 158856, which is incorporated herein by reference in its entirety.

[0153] 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, HCDR1, HCDR2 and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 351, 352 and 353, respectively, 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 described in this paragraph is humanized.

[0154] In some embodiments of a CD123 binding protein that comprises a second binding domain that specifically binds 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 (VLCV) 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 can be humanized variable regions comprising the light and heavy chain CDRs of a monoclonal antibody, respectively). The second binding domain can comprise the light chain variable region, the heavy chain variable region, or both, of a DRA222, TSC455, or TSC456 CD3 binding domain. The amino acid sequences of DRA222, TSC455, and TSC456 are shown in Table 4. The DRA222 binding domain is also described in WO2013 / 158856. TSC455 can also be referred to as TSC394 F87Y. TSC455 can also be referred to as TSC394 E86D F87Y or TSC394 DY.

[0155] 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, wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO:384, or an amino acid sequence that is at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO:385, and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence in SEQ ID NO:383.

[0156] In some embodiments, the second binding domain is a CD3 binding domain comprising HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 20, and HDCR3 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. In some embodiments, the CD3 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.

[0157] In some embodiments, the CD123 binding polypeptide or protein further comprising a CD3 binding domain may have a low level of high molecular weight aggregates produced during recombinant expression of the polypeptide or protein.The CD123 binding polypeptide or protein further comprising a CD3 binding domain may exhibit a relatively long stability in human serum depending on the CD3 binding domain present in the polypeptide or protein.

[0158] In certain variations, the CD3 binding domain comprises one or more (e.g., CDRs or variable regions) of the CD3 binding sequences disclosed in US2013 / 0129730, US2011 / 0293619, US7,635,472, WO2010 / 037836, WO2004 / 106381, or WO2011 / 121110 (each of which is incorporated by reference in its entirety herein). In some embodiments, the CD3 binding domain comprises one or more of the sequences shown in Table 6. [Table 6]

[0159] In various embodiments, the CD3 binding domain comprises one or more of the sequences shown in Table 7. [Table 7]

[0160] 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 immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the first 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, HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, and HDCR3 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, HDCR3 comprises SEQ ID NO: 12, 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 (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, HCDR1 comprises SEQ ID NO: 19, HCDR2 comprises SEQ ID NO: 20, and HDCR3 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, HDCR3 comprises SEQ ID NO: 21, 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 that is 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.

[0161] 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 alternative structural formats. Without being bound by any theory, the polypeptide structural formats disclosed herein (e.g., in order from amino terminus to carboxyl terminus, (a) a first 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 to T cells, CD3, CD3ε, or T cell receptor (TCR) complex) induce moderate T receptor (TCR) stimulation compared to other T cell engagers. It has been widely reported that the strength or magnitude of the TCR signal regulates the outcome of T cell activation. TCR stimulation induces many cellular events, including the initiation of effector functions (e.g., cytolytic granzymes), as well as cytokine secretion and cell division (Corse, Gottschalk and Allison. J Immunol 2011, 186:5039-5045). These distinct cellular events may proceed with different kinetics and reach a maximum level that varies 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 several days, yet moderate enough to limit the amount of cytokine secretion.

[0162] 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 T cells compared to an OKT3 antibody control. In some embodiments, a multispecific polypeptide comprising a CD123 binding domain and a CD3 binding domain induces reduced cytokine release from 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 that is 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 a 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 a CD3 binding domain derived from I2C in a bispecific T cell engager (scFv-scFv) format or a dual affinity retargeting format.

[0163] Also provided herein are pharmaceutical compositions comprising a therapeutic protein described herein. In some embodiments, the composition comprises 1-20 mg / ml, 2.5-12 mg / ml, or 5-10 mg / ml of therapeutic protein. In some embodiments, the composition comprises 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 composition comprises about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, or about 12 mg / ml of therapeutic protein. In some embodiments, the composition comprises about 5 mg / ml of therapeutic protein.

[0164] How to use The present disclosure provides methods of treating a subject having a disease or disorder, the methods comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure.

[0165] In some embodiments, the disease or disorder may be cancer, which 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).

[0166] In some embodiments, the disease or disorder may be an inflammatory disease or disorder. In some embodiments, the disease or disorder may be an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder may be 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 sporadic encephalomyelitis (ADEM), Addison's disease, ankylosing hepatitis, antiphospholipid syndrome (aPL), 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, vitiligo, autoimmune inner ear disease, and myasthenia gravis. In some embodiments, the inflammatory disease or disorder is psoriasis.

[0167] In some embodiments, the inflammatory disease or disorder may be a "neuroimmune disease," such as neuropathic pain, osteoarthritis, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease.

[0168] 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.

[0169] In some embodiments, for the therapeutic methods and uses described herein, the proteins or polypeptides described herein are delivered consistent with conventional methodologies associated with treating the disease or disorder in need of treatment. In accordance with the 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.

[0170] The subjects for administering the protein of the present disclosure include patients who are at high risk of developing a particular disorder, as well as patients who have such existing disorders.Typically, the subject has been diagnosed with the disorder that requires treatment.Furthermore, the subject can be monitored during the course of treatment for any changes in the disorder (e.g., for the increase or decrease of the clinical symptoms of the disorder).In addition, in some variations, the subject does not suffer from other disorders that require treatment.

[0171] In preventive applications, pharmaceutical compositions or medicaments comprising the proteins of the present disclosure are administered to patients susceptible to or otherwise at risk of a particular disorder in an amount sufficient to reduce the risk or delay the onset of the disorder. In therapeutic applications, pharmaceutical compositions or medicaments comprising the proteins of the present disclosure can be administered to patients suspected of or already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to achieve this is defined as a therapeutically effective amount or dose. In both preventive and therapeutic regimens, the agent is usually administered in several doses until a sufficient response (e.g., inhibition of inappropriate angiogenic activity) is achieved. Typically, the response is monitored and repeated administration is performed if the desired response begins to fade.

[0172] To identify a subject patient to be treated according to the method of the present disclosure, accepted screening methods can be employed 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 a relative diagnosed with a particular disorder. Screening methods can also include, for example, routine workup to determine familial status of a particular disorder known to have a genetic component. For example, various cancers are also known to have a particular genetic component. Genetic components of cancer include, for example, mutations in multiple transforming genes (e.g., Ras, Raf, EGFR, cMet, etc.), the presence or absence of certain HLA and killer inhibitory receptor (KIR) molecules, or mechanisms by which cancer cells can modulate, either directly or indirectly, 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 with genetic markers associated with a particular disease of interest. In addition, a wide variety of immunological methods are known in the art that are useful for identifying markers for particular disorders. For example, a variety of ELISA immunoassays that use monoclonal antibody probes to detect antigens associated with specific tumors are available and well known in the art. Screening can be performed as indicated by known patient symptoms, age factors, associated risk factors, and the like. These methods allow clinicians to routinely select patients who require the methods described herein for treatment.

[0173] For administration, the pharmaceutical compositions of the present disclosure may include (i) a therapeutic protein / polypeptide, and (ii) a pharma- ceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition may include (i) a therapeutic protein / peptide, (ii) a buffer, (iii) an excipient, and (iv) a surfactant.

[0174] Pharmaceutical compositions comprising the polypeptides or proteins described herein can be formulated in 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 can be selected from the group consisting of tablets, pills, pellets, capsules, powders, lozenges, granules, liquids, suspensions, emulsions, syrups, elixirs, sustained release formulations, aerosols, and sprays.

[0175] Pharmaceutical compositions comprising the polypeptide or protein described herein can be administered to a subject in a therapeutically effective amount. According to the methods of the present disclosure, the polypeptide or protein described herein can be administered to a subject by various administration forms, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrathoracic, intrathecal, and oral administration routes. For prophylactic and therapeutic purposes, the antagonist can be administered to a subject in a single bolus delivery, via continuous delivery over time (e.g., continuous transdermal delivery), or in a repeated administration protocol (e.g., hourly, daily, weekly, or monthly).

[0176] In this regard, the determination of effective dosage is typically based on animal model studies followed by human clinical trials, and is guided by determining the effective dosage and administration protocol that significantly reduces the occurrence or severity of the disease of interest in the model subject. The effective dosage of the composition of the present disclosure will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, whether the treatment is prophylactic or therapeutic, as well as the specific activity of the composition itself and its ability to induce the desired response in an individual. Usually, the patient is a human, but for some diseases, the patient may be a non-human mammal. Typically, the dosage regimen is adjusted to provide the optimal therapeutic response, i.e., to optimize safety and efficacy.

[0177] Also provided herein is the use of the compositions of the present disclosure in the manufacture of a medicament for treating cancer. For example, the compositions of the present disclosure can be used to treat acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Further provided is a method, the 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 by IV infusion. Typically, a patient is treated once or twice a week for 4-6 weeks. The patient receives the same dose every week or the dose can be increased, for example, every week.

[0178] In some embodiments, the dosage is increased every week, with the first dose being less than the patient is expected to tolerate. This type of step-up treatment regimen reduces the risk of the patient developing infusion-related reactions or cytokine release syndrome. In some embodiments, a multispecific protein (e.g., TRI130 or TRI129) comprising a CD123 binding domain and a CD3 binding domain may be administered intravenously to a patient in increasing dosages every week for at least the first two or first three doses. For example, the composition of the present disclosure may 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 dose and subsequent doses: 12 μg. In some embodiments, a patient may 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 dose and subsequent doses: 18 μg. In some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: dose 6 μg in week 1 and dose 9 μg in week 2 and subsequent weeks, in some embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: dose 9 μg in week 1 and dose 12 μg in week 2 and subsequent weeks, in other embodiments, the composition is administered intravenously to a patient according to a weekly treatment schedule: dose 12 μg in week 1 and dose 18 μg in week 2 and subsequent weeks.

[0179] 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 dose and subsequent doses: 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 dose and subsequent doses: 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 dose and subsequent doses: 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: 12 μg; week 3 dose: 18 μg; and week 4 dose and subsequent weeks dose: 24 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 dose and subsequent weeks dose: 36 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 dose 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 dose and subsequent weeks dose: 60 μg.

[0180] In some embodiments, patients may receive a composition 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 administration schedule of 36 μg.

[0181] In some embodiments, patients may receive 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 administration schedule of 48 μg.

[0182] 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 a weekly administration schedule of 60 μg.

[0183] 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 a weekly administration schedule of 100 μg.

[0184] 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 is administered on day 8, 12 μg is administered on day 15, and 12 μg is administered on day 22. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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.

[0185] 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 by 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 prior to treatment.

[0186] In some embodiments, patients treated according to the methods of the present disclosure exhibit a complete response (CR). As used herein, a complete response is defined as a reduction in bone marrow blasts to less than about 5%, absence of circulating blasts and blasts with Auer rods, absence of extramedullary disease, and an absolute neutrophil count (ANC) of ≧1.0×10 9 / L (1,000 / μL) and PLT ≥ 100 × 109 / L (100,000 / μL). In some embodiments, patients treated according to the methods of the present disclosure achieve CR without minimal residual disease (CR MRD 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 have a CR with incomplete hematological recovery (CR i ) CR i Residual neutropenia (ANC < 1.0 × 10 9 / L [1,000 / μL]) or thrombocytopenia (PLT < 100 × 10 9 Includes all criteria for CR listed above except for CR (100,000 / μL [100,000 / μL]).

[0187] 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 less than 5% bone marrow blasts (i.e., the bone marrow should not simply be "aplastic" but should have at least 200 cells counted or at least 10% cellularity), no blasts with Auer rods, and no extramedullary disease. Hematological recovery is not required.

[0188] In some embodiments, patients treated according to the methods of the present disclosure exhibit a partial response (PR). As used herein, PR includes all of the hematological criteria of CR described above, with the additional reduction in bone marrow blast percentage to 5-25% and at least a 50% reduction in bone marrow blast percentage from pre-treatment.

[0189] In some embodiments, patients treated according to the methods of the present disclosure exhibit stable disease (SD), which is consistent with CR. MRD , C.R., C.R. i, PR, and MLFS, but is characterized by the absence of progressive disease (i.e., increasing bone marrow blast percentage and / or increasing absolute blast counts in the blood).

[0190] Patients treated with a multispecific polypeptide targeting CD123xCD3 (e.g., TRI130 or TRI129) at the same weekly dose or in a step-up treatment regimen may also vary the infusion time (i.e., length of infusion) 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., 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 is administered over a period of about 8 hours, the third dose is administered over a period of about 6 hours, and the fourth and subsequent doses are administered over a period of about 4 hours. In some embodiments, a first dose of the composition is administered over a period of about 20-24 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, wherein the first, second, third, and fourth doses are the same. The composition can also be administered to a subject by continuous IV infusion, e.g., continuous IV infusion for up to about 72 hours.

[0191] Patients treated with a multispecific polypeptide targeting CD123xCD3 (e.g., TRI130 or TRI129) can also be treated with one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered at the same time or close to the time of the multispecific polypeptide targeting CD123xCD3. In some embodiments, the one or more additional therapeutic agents are administered prior to administration of the multispecific polypeptide (i.e., as a "premedication"), for example, about 1-3 hours before administration thereof. In some embodiments, the one or more additional therapeutic agents are administered after administration of one or more doses of the multispecific polypeptide.

[0192] 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 dose of about 10-20 mg. In some embodiments, methylprednisolone may be administered at a dose of about 1 mg / kg. In some embodiments, acetaminophen may be administered at a dose of about 650 mg or about 1,000 mg. In some embodiments, acetaminophen may be administered three times a day, with the first dose being administered 1-3 hours prior to administering the multispecific polypeptide targeting CD123×CD3. In some embodiments, the one or more additional therapeutic agents may include an antihistamine, such as diphenhydramine. Diphenhydramine may be administered at a dose of about 50 mg. In some embodiments, the one or more therapeutic agents may include allopurinol. In some embodiments, allopurinol is administered at least 2 days prior to administration of the multispecific polypeptide targeting CD123xCD3. In some embodiments, the one or more additional therapeutic agents may include tocilizumab.

[0193] In some embodiments, a method of 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) in any of the doses or regimens described herein. In some embodiments, a method of 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), wherein administering the pharmaceutical composition reduces the level of cytokines induced in the subject compared to (a) administration of a dual affinity retargeting antibody comprising a CD123 binding domain and a CD3 binding domain of the recombinant polypeptide, or (b) administration of a bispecific T cell engager molecule comprising a CD123 binding domain and a CD3 binding domain of the recombinant polypeptide. In some embodiments, the disorder is a cancer, such as AML or MDS. In some embodiments, the subject has been previously treated with a different CD123 binding molecule and the subject experienced an adverse event following the previous treatment. In some embodiments, the adverse event is excessive cytokine release. In some embodiments, the cytokine levels are levels of IFN-γ, TNF-α, 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 are levels of IFN-γ, IL-2, TNF-α, and IL-10. In some embodiments, the cytokine levels are measured in an in vitro activated T cell assay.

[0194] Pharmaceutical compositions comprising the proteins and polypeptides described herein may be supplied as a kit comprising a container containing the pharmaceutical composition described herein. The pharmaceutical composition may be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted prior to injection. Such kits may further comprise information describing the indications and uses of the pharmaceutical composition. In some embodiments, the kit comprises 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 possibility of the multispecific polypeptide sticking to plastic tubes and bags).

[0195] Combination therapy Combination therapy is also provided herein. As used herein, the term "combination therapy" refers to administering a first therapeutic agent and a second therapeutic agent simultaneously or sequentially to achieve a therapeutic effect. For example, in some embodiments, the combination therapy includes administration of a multispecific protein comprising a CD123 binding domain and a CD3 binding domain and a second anti-cancer agent. The multispecific protein may be referred to as the "first anti-cancer agent." The multispecific protein and the second anti-cancer agent may be administered simultaneously or sequentially, and may be administered in a single composition or separate compositions.

[0196] In some embodiments, the method for treating cancer comprises administering to a subject in need thereof i) a multispecific protein comprising a CD123 binding domain and a CD3 binding domain, and ii) a second anti-cancer agent, hi some embodiments, the method for treating cancer comprises administering a third, fourth, fifth, sixth, seventh, or eighth anti-cancer agent.

[0197] The anti-cancer drug can be any drug that is effective in treating malignant or cancerous disease. There are several major classes of anti-cancer drugs, including monoclonal antibodies, alkylating agents, antimetabolites, and hormones. In some embodiments, the anti-cancer drug is a chemotherapeutic drug. For example, the chemotherapeutic drug can be venetoclax, azacitidine, decitabine, daunorubicin, cytarabine, idarubicin, mitoxantrone, or etoposide. In some embodiments, a combination of one or more chemotherapeutic drugs is administered to the subject.

[0198] Venetoclax is a chemotherapy drug typically used to treat chronic lymphocytic leukemia, small lymphocytic lymphoma, or acute myeloid leukemia. Venetoclax is a highly selective BCL-2 inhibitor. It blocks the anti-apoptotic activity of BCL-2, thereby promoting cell death. In some embodiments, venetoclax may be administered at a dose of about 100 mg to about 400 mg per day, e.g., about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, or any range therebetween.

[0199] Azacitidine is a chemical analog of cytidine, a nucleoside of DNA and RNA. It is believed to have antitumor activity by two mechanisms: at low doses by inhibiting DNA methyltransferase (causing hypermethylation of DNA), and at high doses by its direct cytotoxicity to abnormal hematopoietic cells in the bone marrow by incorporation into DNA and RNA, resulting in cell death. Azacitidine and its deoxy derivative decitabine (also called 5-aza-2'-deoxycytidine) are often used to treat myelodysplastic syndromes. In some embodiments, azacitidine is administered at a dose of about 50 to about 100 mg / m 2 / day, e.g., about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg / m 2 In some embodiments, the azacitidine is administered at a dose of about 75 mg / m 2 It is administered at a dose of / day.

[0200] Decitabine is a cytidine analogue that acts as a nucleotide synthesis inhibitor. It is incorporated into DNA strands during replication, and then when a DNA methyltransferase (DNMT) such as DNMT1 contacts and binds to DNA to replicate the methylation to the daughter strand, the DNMT irreversibly binds to decitabine and cannot be detached. It is typically used to treat myelodysplastic syndromes and acute myeloid leukemia. In some embodiments, decitabine is administered at a dose of about 10 to about 30 mg / m 2 / day dose, e.g., about 10, about 15, about 20, about 25, or about 30 mg / m 2 In some embodiments, decitabine is administered at a dose of about 20 mg / m 2 It is administered at a dose of / day.

[0201] Daunorubicin, also known as daunomycin, is a chemotherapy drug used to treat acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and Kaposi's sarcoma. It is typically administered intravenously. Daunorubicin intercalates into DNA, inhibiting macromolecular biosynthesis and blocking the function of topoisomerase II. In addition, daunorubicin stabilizes the topoisomerase II complex after cleaving the DNA strand for replication, preventing resealing of the DNA double helix, thereby halting the replication process. In some embodiments, daunorubicin is administered at a dose of about 30-90 mg / m 2 , for example about 30, about 35, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 mg / m 2 In some embodiments, daunorubicin is administered at a dose of about 60 mg / m 2 is administered at a dose of

[0202] Cytarabine, also known as cytosine arabinoside (Ara-C), is a chemotherapy drug typically used to treat various forms of leukemia and lymphoma, including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and non-Hodgkin's lymphoma. It can be administered intravenously, subcutaneously, or into the spinal fluid. Cytarabine is an antimetabolite drug and acts by inhibiting the function of DNA polymerase. In some embodiments, cytarabine is administered at a dose of about 2 to about 3 g / m 2 In some embodiments, cytarabine is administered in doses of about 1 g / m 2 In some embodiments, cytarabine is administered in doses of 1.5 g / m 2 In some embodiments, cytarabine may be administered in doses of about 100 mg to about 400 mg / m 2 It may be administered in body surface area doses.

[0203] Idarubicin (also called 4-demethoxydanorubicin) is a chemotherapy drug that blocks DNA unwinding by interfering with the enzyme topoisomerase II. It is an analog of danorubicin, but the absence of a methoxy group increases its lipid solubility and cellular uptake. It also causes histone ejection from chromatin. It is used to treat acute lymphoblastic leukemia and chronic myeloid leukemia. In some embodiments, idarubicin is administered at a dose of about 1 to about 20 mg / m per day. 2 , for example, about 1, about 2, about 3, about 4, 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, or about 20 mg / m per day. 2 In some embodiments, idarubicin is administered at a dose of about 12 mg / m per day. 2 is administered at a dose of

[0204] Mitoxantrone is an anthracenedione antibiotic with antitumor activity. Mitoxantrone intercalates and crosslinks DNA, thereby disrupting DNA and RNA replication. The drug also binds to topoisomerase II, resulting in DNA strand breaks and inhibition of DNA repair. It is used in the treatment of acute leukemia, lymphoma, prostate and breast cancer, but also in late-stage severe multiple sclerosis. In some embodiments, mitoxantrone is administered at a dose of about 1 to about 20 mg / m 2 / day, e.g., about 1, about 2, about 3, about 4, 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, or about 20 mg / m 2 In some embodiments, mitoxantrone is administered at a dose of about 6 mg / m 2 It is administered at a dose of / day.

[0205] Etoposide is used to treat testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer, as well as hemophagocytic lymphohistiocytosis. It can be administered orally or intravenously. Etoposide forms a ternary complex with DNA and topoisomerase II enzyme, preventing religation of DNA strands and causing DNA strand breaks. This results in errors in DNA synthesis and apoptosis of cancer cells. In some embodiments, etoposide is administered at a dose of about 10 to about 100 mg / m 2 / day, e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mg / m 2 It is administered at a dose of / day.

[0206] In some embodiments, the method for treating cancer comprises administering to a subject in need thereof a multispecific protein as described herein and one or more additional anti-cancer agents. The one or more additional anti-cancer agents may be individually selected from venetoclax, azacitidine, decitabine, daunorubicin, cytarabine, idarubicin, mitoxantrone, and etoposide. The cancer may be, for example, a carcinoma or sarcoma. In some embodiments, the cancer is melanoma, kidney cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, or blood cancer. In some embodiments, the cancer is 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). In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is myelodysplastic syndrome (MDS).

[0207] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof a multispecific protein described herein, mitoxantrone, etoposide, and cytarabine. This combination of chemotherapy drugs is also referred to herein as "MEC." In some embodiments, mitoxantrone, etoposide, and cytarabine are administered to a subject, wherein mitoxantrone is administered at a dose of about 6 mg / m 2 / day, and etoposide is administered at a dose of approximately 80 mg / m 2 / day, and cytarabine is administered at a dose of approximately 1 g / m 2 / day. Mitoxantrone, etoposide and cytarabine may be administered simultaneously or sequentially.

[0208] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof a multispecific protein described herein, azacitidine, and venetoclax. In some embodiments, the azacitidine is at about 75 mg / m 2Azacitidine and venetoclax are administered at a dose of about 100 to about 400 mg / day. Azacitidine and venetoclax may be administered simultaneously or sequentially.

[0209] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof a multispecific protein described herein, decitabine, and venetoclax. In some embodiments, decitabine is at a dose of about 20 mg / m 2 Decitabine and venetoclax are administered at a dose of about 100 to about 400 mg / day. Decitabine and venetoclax may be administered simultaneously or sequentially.

[0210] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof a multispecific protein described herein, daunorubicin, and cytarabine. In some embodiments, daunorubicin is administered at a dose of about 30-90 mg / m 2 and cytarabine is administered at a dose of about 100 to about 200 mg / m 2 Daunorubicin and cytarabine may be administered simultaneously or sequentially.

[0211] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof a multispecific protein described herein, idarubicin, and cytarabine. In some embodiments, idarubicin is administered at a dose of about 12 mg / m 2 and cytarabine is administered at a dose of about 100 to about 200 mg / m 2 Idarubicin and cytarabine may be administered simultaneously or sequentially.

[0212] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof i) a multispecific protein comprising a CD123 binding domain and a CD3 binding domain, and ii) a second anti-cancer agent, wherein the multispecific protein is administered by intravenous infusion and the second anti-cancer agent is administered either intravenously or orally. In some embodiments, the multispecific protein is administered to the 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. In some embodiments, the multispecific protein is administered once or twice weekly.

[0213] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle. In some embodiments, cytarabine is administered intravenously on days 1-5 of the first 28 day cycle and days 1-5 of at least one additional 28 day cycle. In some embodiments, the dose of cytarabine is about 1 g / m 2 In some embodiments, mitoxantrone, etoposide, and cytarabine are administered intravenously on days 1-6 of a first 28-day cycle and at least one additional 28-day cycle. In some embodiments, the dose of mitoxantrone is about 6 mg / m 2 / day and the dose of etoposide is approximately 80 mg / m 2 / day, and the dose of cytarabine is approximately 1 g / m 2 / day.

[0214] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 15 and 12 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle. In some embodiments, venetoclax is administered orally on days 1-21 of the first 28 day cycle and days 1-21 of at least one additional 28 day cycle. In some embodiments, the dose of venetoclax is about 100 to about 400 mg / day. In some embodiments, azacitidine is administered intravenously on days 1-7 of the first 28 day cycle and at least one additional 28 day cycle. In some embodiments, the dose of azacitidine is about 75 mg / m 2 It is.

[0215] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22. In some embodiments, cytarabine is administered intravenously on days 1-7 of the first 28 day cycle and days 1-7 of at least one additional 28 day cycle. In some embodiments, the dose of cytarabine is about 100 to about 200 mg / m 2In some embodiments, idarubicin is administered by intravenous infusion on days 1-3 of a first 28-day cycle and days 1-3 of at least one additional 28-day cycle. In some embodiments, the dose of idarubicin is about 12 mg / m 2 It is.

[0216] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle. In some embodiments, azacitidine is administered orally on days 1-14 of the first 28 day cycle and at least one additional 28 day cycle. In some embodiments, the dose of azacitidine is about 300 mg / day.

[0217] Monotherapy Also provided herein are compositions and methods for the use of multispecific proteins comprising a CD123 binding domain and a CD3 binding domain as a monotherapy to treat cancer.

[0218] In some embodiments, the multispecific protein is administered to the subject as a monotherapy, i.e., without a second anticancer drug. In some embodiments, the multispecific protein is administered once a week, for example, at a dose of 1, 3, 9, 12, 18, 24, 36, 48 or 60 μg. In some embodiments, the weekly target dose level for the multispecific protein is 1, 3, 9, 12, 18, 24, 36, 48 or 60 μg. In some embodiments, the multispecific protein is administered once a week at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 μg / kg.

[0219] In some embodiments, the multispecific protein is administered twice weekly during the first 28 day cycle and at least one additional 28 day cycle. For example, in some embodiments, the multispecific protein is administered to the subject by IV infusion during the first 28 day cycle, and during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 6 μg of the multispecific protein is administered on day 4, 12 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 11, 18 μg of the multispecific protein is administered on day 15, 18 μg of the multispecific protein is administered on day 18, 18 μg of the multispecific protein is administered on day 22, and 18 μg of the multispecific protein is administered on day 11. In some embodiments, the multispecific protein is administered to the subject as monotherapy for at least one additional 28 day cycle, such as 1, 2, 3, 4, 5, 6, 7, 8, or more additional 28 day cycles.

[0220] In some embodiments, a method for treating cancer comprises administering to a subject in need thereof a multispecific protein comprising i) a CD123 binding domain and a CD3 binding domain (i.e., monotherapy). In some embodiments, the multispecific protein comprises a dimer of two identical polypeptides, each polypeptide comprising, from amino-terminus to carboxyl-terminus or carboxyl-terminus to amino-terminus, (i) a CD123 binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a CD3 binding domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD123 binding domain, a hinge region, an immunoglobulin constant region, and a CD3 binding domain. In some embodiments, the CD123 and CD3 binding domains comprise (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, the CD123 binding domain is an scFv comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HDCR3 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 CD123 binding domain is an 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 CD123 binding domain is an scFv, and the scFv comprises a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27. In some embodiments, the CD3 binding domain is an scFv comprising an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HDCR3 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 binding domain is an 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 scFv, and the scFv comprises a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27. In some embodiments, each polypeptide comprises a sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 31. In some embodiments, the multispecific protein is administered to the subject by IV infusion.

[0221] In some embodiments, the multispecific protein is administered to the subject by IV infusion at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 μg / kg. In some embodiments, the multispecific protein is administered to the 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. In some embodiments, the multispecific protein is administered once a week.

[0222] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle.

[0223] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where 6 μg of the multispecific protein is administered on day 15 and 12 μg of the multispecific protein is administered on day 22 during the first 28 day cycle. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle.

[0224] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22.

[0225] In some embodiments, the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, where during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22. In some embodiments, the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, where 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle.

[0226] In some embodiments, the cancer is a carcinoma or sarcoma. In some embodiments, the cancer is a melanoma, kidney cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, or blood cancer. In some embodiments, the cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, B-cell acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is myelodysplastic syndrome (MDS).

[0227] Numbered embodiments Without limiting the scope of the appended claims, the present disclosure describes the following number of embodiments.

[0228] 1. A method for treating cancer, the method comprising administering to a subject in need thereof i) a multispecific protein comprising a CD123 binding domain and a CD3 binding domain, and ii) a second anti-cancer agent.

[0229] 2. The method of embodiment 1, wherein the method further comprises administering to the subject a third anti-cancer agent.

[0230] 3. The method of embodiment 1 or 2, wherein the second anti-cancer agent is a chemotherapeutic agent.

[0231] 4. The method of embodiment 3, wherein said chemotherapy agent is venetoclax, azacitidine, decitabine, daunorubicin, cytarabine, idarubicin, mitoxantrone, or etoposide.

[0232] 5. The method of embodiment 1, wherein said second anticancer agent is cytarabine.

[0233] 6. The cytarabine is administered at a concentration of about 1 g / m 2 6. The method of embodiment 5, wherein the patient is administered at a dose of

[0234] 7. The method of embodiment 2, wherein the second anti-cancer agent is mitoxantrone, the third anti-cancer agent is etoposide, and the method further comprises administering a fourth anti-cancer agent that is cytarabine.

[0235] 8. The mitoxantrone is about 6 mg / m 2 / day, the etoposide being administered at a dose of about 80 mg / m 2 / day, and the cytarabine is administered at a dose of about 1 g / m 2 8. The method of embodiment 7, wherein the dose is administered in a dose of 100 mg / day.

[0236] 9. The method of embodiment 1, wherein the second anticancer agent is venetoclax.

[0237] 10. The method of embodiment 9, wherein the venetoclax is administered at a dose of about 100 to about 400 mg per day.

[0238] 11. The method of embodiment 1, wherein the second anticancer agent is azacitidine.

[0239] 12. The azacitidine is about 75 mg / m 2 The method of embodiment 11, wherein the dose is administered in a dose of 100 mg / day.

[0240] 13. The method of embodiment 2, wherein the second anti-cancer agent is azacitidine and the third anti-cancer agent is venetoclax.

[0241] 14. The azacitidine is about 75 mg / m 2 14. The method of embodiment 13, wherein the venetoclax is administered at a dose of about 100 to about 400 mg / day.

[0242] 15. The method of embodiment 1, wherein the second anticancer agent is decitabine.

[0243] 16. The decitabine is about 20 mg / m 、2The method of embodiment 15, wherein the dose is administered in a dose of 100 mg / day.

[0244] 17. The method of embodiment 2, wherein the second anticancer agent is decitabine and the third anticancer agent is venetoclax.

[0245] 18. The decitabine is about 20 mg / m 2 18. The method of embodiment 17, wherein the venetoclax is administered at a dose of about 100 to about 400 mg / day.

[0246] 19. The method of embodiment 2, wherein the second anti-cancer agent is daunorubicin and the third anti-cancer agent is cytarabine.

[0247] 20. The daunorubicin is about 30 to 90 mg / m 2 and the cytarabine is administered at a dose of about 100 to about 200 mg / m 2 20. The method of embodiment 19, wherein the dose is

[0248] 21. The method of embodiment 1, wherein the anticancer agent is idarubicin.

[0249] 22. The idarubicin is about 12 mg / m 2 22. The method of embodiment 21, wherein the dose is

[0250] 23. The method of embodiment 2, wherein the second anti-cancer agent is idarubicin and the third anti-cancer agent is cytarabine.

[0251] 24. The idarubicin is about 12 mg / m 2 and the cytarabine is administered at a dose of about 100 to about 200 mg / m 2 24. The method of embodiment 23, wherein the dose is

[0252] 25. The method of embodiment 1, wherein the second anticancer agent is azacitidine.

[0253] 26. The azacitidine is administered at a dose of about 75 mg / m 2 The method of embodiment 11, wherein the dose is administered in a dose of 100 mg / day.

[0254] 27. The method of any one of embodiments 1 to 26, wherein the multispecific protein comprises a dimer of two identical polypeptides, each polypeptide comprising, from amino terminus to carboxyl terminus, or from carboxyl terminus to amino terminus, (i) a CD123 binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a CD3 binding domain.

[0255] 28. The method of embodiment 27, wherein the polypeptide comprises, from N-terminus to C-terminus, the CD123 binding domain, the hinge region, the immunoglobulin constant region, and the CD3 binding domain.

[0256] 29. The method of embodiment 28, wherein at least one of the CD123 and CD3 binding domains 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.

[0257] 30. The method of embodiment 29, wherein the CD123 binding domain is an scFv comprising HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, and HDCR3 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.

[0258] 31. The method of embodiment 29, wherein the CD123 binding domain is an 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.

[0259] 32. The method of embodiment 29, wherein the CD123 binding domain is an scFv, and the scFv comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27.

[0260] 33. The method of any one of embodiments 29 to 32, wherein the CD3 binding domain is an scFv comprising an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HDCR3 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.

[0261] 34. The method of any one of embodiments 29 to 32, wherein the CD3 binding domain is an scFv comprising a VH comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 384.

[0262] 35. The method of any one of embodiments 29 to 32, wherein the CD3 binding domain is an scFv comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27.

[0263] 36. The method of embodiment 29, wherein each polypeptide comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:31.

[0264] 37. The method of any one of embodiments 1-36, wherein the multispecific protein is administered to the subject by IV infusion.

[0265] 38. The method of any one of embodiments 1-37, wherein the second anticancer agent is administered to the subject orally or by IV infusion.

[0266] 39. The method of any one of embodiments 1-38, wherein the multispecific protein is administered to the 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.

[0267] 40. The method of any one of embodiments 1-39, wherein the multispecific protein is administered once a week.

[0268] 41. The method of embodiment 40, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22, during the first 28 day cycle.

[0269] 42. The method of embodiment 41, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22 of the at least one additional 28 day cycle.

[0270] 43. The method of embodiment 42, wherein cytarabine is administered intravenously on days 1-5 of said first 28-day cycle and days 1-5 of at least one additional 28-day cycle.

[0271] 44. The dose of cytarabine is about 1 g / m 2 44. The method of embodiment 43, wherein

[0272] 45. The method of embodiment 42, wherein mitoxantrone, etoposide, and cytarabine are administered intravenously on days 1-6 of the first 28-day cycle and at least one additional 28-day cycle.

[0273] 46. ​​The dose of mitoxantrone is about 6 mg / m 2 / day, and the dose of etoposide is about 80 mg / m 2 / day, and the dose of cytarabine is about 1 g / m 2 The method of embodiment 45, wherein the daily dose is 100 mg / day.

[0274] 47. The method of embodiment 40, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 15 and 12 μg of the multispecific protein is administered on day 22 during the first 28 day cycle.

[0275] 48. The method of embodiment 47, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22 of the at least one additional 28 day cycle.

[0276] 49. The method of embodiment 48, wherein venetoclax is administered orally on days 1 to 21 of the first 28-day cycle and days 1 to 21 of at least one additional 28-day cycle.

[0277] 50. The method of embodiment 49, wherein the dose of venetoclax is about 100 to about 400 mg / day.

[0278] 51. The method of any one of embodiments 48-50, wherein azacitidine is administered intravenously on days 1-7 of the first 28-day cycle and at least one additional 28-day cycle.

[0279] 52. The dose of azacitidine is about 75 mg / m 2 52. The method of embodiment 51, wherein

[0280] 53. The method of embodiment 40, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

[0281] 54. The method of embodiment 53, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, wherein 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22.

[0282] 55. The method of embodiment 54, wherein cytarabine is administered intravenously on days 1 to 7 of said first 28-day cycle and days 1 to 7 of at least one additional 28-day cycle.

[0283] 56. The dose of cytarabine is about 100 to about 200 mg / m 2 56. The method of embodiment 55, wherein

[0284] 57. The method of any one of embodiments 53-56, wherein idarubicin is administered by intravenous infusion on days 1-3 of the first 28-day cycle and on days 1-3 of at least one additional 28-day cycle.

[0285] 58. The dose of idarubicin is about 12 mg / m 2 58. The method of embodiment 57, wherein

[0286] 59. The method of embodiment 40, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

[0287] 60. The method of embodiment 59, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28 day cycle.

[0288] 61. The method of embodiment 60, wherein azacitidine is administered orally on days 1 to 14 of said first 28-day cycle and at least one additional 28-day cycle.

[0289] 62. The method of embodiment 61, wherein the dose of azacitidine is about 300 mg / day.

[0290] 63. The method of any one of embodiments 1-62, wherein the cancer is a carcinoma or a sarcoma.

[0291] 64. The method of any one of embodiments 1-62, wherein the cancer is melanoma, kidney cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, or blood cancer.

[0292] 65. The method of any one of embodiments 1-62, wherein the cancer is 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).

[0293] 66. The method of any one of embodiments 1-62, wherein the cancer is acute myeloid leukemia (AML).

[0294] 67. The method of any one of embodiments 1-62, wherein the cancer is myelodysplastic syndrome (MDS).

[0295] 68. A method for treating cancer, the method comprising administering to a subject in need thereof a multispecific protein comprising a CD123 binding domain and a CD3 binding domain.

[0296] 69. The method of embodiment 68, wherein the multispecific protein comprises a dimer of two identical polypeptides, each polypeptide comprising, from amino terminus to carboxyl terminus, or from carboxyl terminus to amino terminus, (i) a CD123 binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a CD3 binding domain.

[0297] 70. The method of embodiment 69, wherein the polypeptide comprises, from N-terminus to C-terminus, the CD123 binding domain, the hinge region, the immunoglobulin constant region, and the CD3 binding domain.

[0298] 71. The method of embodiment 69, wherein at least one of the CD123 and CD3 binding domains 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.

[0299] 72. The method of embodiment 69, wherein the CD123 binding domain is an scFv comprising HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, and HDCR3 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.

[0300] 73. The method of embodiment 69, wherein the CD123 binding domain is an 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.

[0301] 74. The method of embodiment 69, wherein the CD123 binding domain is an scFv, and the scFv comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27.

[0302] 75. The method of any one of embodiments 69 to 74, wherein the CD3 binding domain is an scFv comprising an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HDCR3 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.

[0303] 76. The method of any one of embodiments 69 to 74, wherein the CD3 binding domain is an 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.

[0304] 77. The method of any one of embodiments 69 to 74, wherein the CD3 binding domain is an scFv comprising a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27.

[0305] 78. The method of embodiment 69, wherein each polypeptide comprises a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:31.

[0306] 79. The method of any one of embodiments 68-78, wherein the multispecific protein is administered to the subject by IV infusion.

[0307] 80. The method of any one of embodiments 68-79, wherein the multispecific protein is administered to the subject by IV infusion at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 μg / kg.

[0308] 81. The method of any one of embodiments 68-79, wherein the multispecific protein is administered to the 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.

[0309] 82. The method of any one of embodiments 68-81, wherein the multispecific protein is administered once a week.

[0310] 83. The method of embodiment 82, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein, during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

[0311] 84. The method of embodiment 83, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22 of the at least one additional 28 day cycle.

[0312] 85. The method of embodiment 82, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein 6 μg of the multispecific protein is administered on day 15 and 12 μg of the multispecific protein is administered on day 22 during the first 28 day cycle.

[0313] 86. The method of embodiment 85, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22 of the at least one additional 28 day cycle.

[0314] 87. The method of embodiment 82, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

[0315] 88. The method of embodiment 87, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, wherein 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22.

[0316] 89. The method of embodiment 82, wherein the multispecific protein is administered to the subject by IV infusion during a first 28 day cycle, wherein during the first 28 day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

[0317] 90. The method of embodiment 89, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28 day cycle following the first 28 day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15 and 22 of the at least one additional 28 day cycle.

[0318] 91. The method of any one of embodiments 68 to 90, wherein the cancer is a carcinoma or a sarcoma.

[0319] 92. The method of any one of embodiments 68-90, wherein the cancer is melanoma, kidney cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, or blood cancer.

[0320] 93. The method of any one of embodiments 68-90, wherein the cancer is 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).

[0321] 94. The method of any one of embodiments 68 to 90, wherein the cancer is acute myeloid leukemia (AML).

[0322] 95. The method of any one of embodiments 68-90, wherein the cancer is myelodysplastic syndrome (MDS).

[0323] The present disclosure will be further clarified by the following examples, which are intended to be merely illustrative of the disclosure and are not intended to be limiting in any way. EXAMPLES

[0324] The present invention will be described in further detail by referring to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. 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.

[0325] Without further description, it is believed that one of ordinary skill in the art can, using the above description and the following illustrative examples, make and utilize the compounds of the present invention and practice the methods described in the claims. Accordingly, the following examples specifically illustrate preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0326] Example 1: Determination of the No Observed Adverse Effect Level (NOAEL) and the Minimum Assumed Effective Level (MABEL) for TRI130 This example describes experiments used to determine the NOAEL and MABEL for TRI130, a multispecific protein containing CD123 and CD3 binding domains. This data was used to establish human dosing cohorts for the clinical trials described in the Examples below.

[0327] No observed adverse effect level (NOAEL) A 28-day repeat-dose toxicology study with a 5-week recovery period was conducted in non-human primates (NHPs). Four study groups of animals received weekly doses of vehicle or 0.5, 2.5, or 10 mg / kg TRI130. Parameters measured included safety pharmacology, laboratory evaluation, and necropsy with complete histopathology. There were no clinically adverse findings, changes in animal or organ weights, and no macroscopic or microscopic abnormal findings in histopathology associated with TRI130. Minimal cytokines were detected after dosing, and these decreased after the second dose compared to the first dose. The expected pharmacodynamic effects of the anti-CD3 binding domain of the molecule were observed, along with a transient redistribution of T cells. The elimination half-life was approximately 73 hours at the higher dose. The no observed adverse effect level (NOAEL) was 10 mg / kg in NHPs, which translates to a human equivalent dose (HED) of approximately 3.2 mg / kg.

[0328] Minimum Estimated 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). In order to calculate the estimated minimum pharmacologic effect dose (MABEL), an in vitro activity assay was used instead of in vitro receptor occupancy.

[0329] The effective concentration required to induce 10% activity (EC10) (Muller et al., 2009) in human T cell activation in vitro assays was used to determine the MABEL. Although 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 (E:T) cell ratio, the activation assays are more sensitive assays for the calculation of MABEL compared to the redirected T cytotoxicity (RTCC) assay. Both RTCC and T cell activation were evaluated as possible assays to predict the MABEL for the starting clinical dose. The RTCC assay had a mean EC10 of 5.8 pM when evaluated from five donors (ranging from 4.4 to 6.7 pM across donors evaluated) using purified T cells at an E:T ratio of 10:1 on the CD123+KG1a tumor cell line. 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). After gating on viable CD4+ and CD8+ T cells, upregulation of CD69 and CD25 on T cells was monitored at 20 hours using multicolor flow cytometry. These assays evaluated three donors for activation of both CD4 and CD8 T cells. The average EC10 calculated for these assays was 1.2 pM for CD4 T cells (range 0.7-1.6 pM) and 1.3 pM for CD8 T cells (range 0.9-2.0 pM). This assay represents 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.

[0330] Group 4 clearance and volume estimates 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 estimates of human clearance and volume parameters. The estimates can be used to simulate dosing strategies that result in a Cmax below an EC10 (MABEL) value of 0.7 pM (the lowest EC10 determined from individual donors used in the activation assay). With 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).

[0331] The study uses a fixed or fixed dose instead of weight-based dosing. Several studies have shown that fixed dosing compared with weight-based dosing performs similarly across multiple monoclonal antibodies, and the PK variability introduced by either dosing regimen is modest compared to the commonly observed variability 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.

[0332] Example 2: Administration of TRI130, an anti-CD123 x anti-CD3 therapeutic candidate, to patients Formulated TRI130 (5 mM succinic acid, 6.5% sucrose, 0.02% w / v polysorbate 80, pH 4.8) is being administered to patients in an ongoing Phase 1 / 1b open-label, dose-escalation study in patients with relapsed or refractory acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). The study was conducted in two parts: Part 1 is a Phase 1 open-label, dose-escalation study to determine the recommended dose for Phase 1b. Phase 1b is an open-label extension study to evaluate the clinical activity and safety of the agent at the recommended dose. The study design is shown in Figure 2. Endpoints include safety, immunogenicity, pharmacokinetics, pharmacodynamics, and clinical activity.

[0333] Table 8 is the dosing schedule showing the weekly (IV) dosing for cohorts 1-10 (dose escalation cohorts). In both parts of the study, patients will receive weekly intravenous dosing for six 28-day cycles unless earlier caused by disease progression, intolerable toxicity, or withdrawal of consent. If patients are responding, there is the option of longer treatment. [Table 8]

[0334] 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. To prevent the formulation from adhering to IV bags and IV tubing sets, the formulation is mixed with an IV stabilization solution. The stabilization solution is supplied sterile and refrigerated (2-8°C) in 10 mL vials containing 0.1 M succinate buffer, and 0.08% w / v polysorbate 80, pH 6.0.

[0335] For cohorts 1-4, the first dose (cycle 1, day 1) was administered by IV infusion over 20-24 hours, the second dose (cycle 1, day 8) over 8 hours (± 1 hour), the third dose (cycle 1, day 15) over 6 hours (± 1 hour), and all subsequent doses (cycle 1, day 22 and beyond) over 4 hours (± 1 hour). For cohorts 5 and beyond, the first dose and each dose escalation were administered by IV infusion over 20-24 hours. The second dose was the same dose and was infused over 8 hours (± 30 minutes), the third dose over 6 hours (± 30 minutes), and the fourth and subsequent doses over 4 hours (± 30 minutes).

[0336] If necessary, any dose infusion can be slowed and / or interrupted to manage or prevent adverse events, especially infusion-related reactions (IRR) or cytokine release syndrome (CRS), and the administration time extended for up to 72 hours. If the infusion is extended beyond 60 hours, patients should be observed for 12 hours after completion of the infusion. Table 10 discloses (starting with cohort 5) a step-up dosing regimen that may reduce the likelihood of IRR and / or CRS.

[0337] The frequency of patient dosing was weekly for up to 6 months. The weekly dosing schedule was selected based on a cynomolgus monkey toxicology study with escalating doses of TRI130 (data not shown). The half-life of TRI130 after a single dose ranged from approximately 25 to 113 hours for individual animals dosed at 0.25 to 1 mg / kg. Longer half-life estimates were associated with animals in the high dose group (1 mg / kg).

[0338] Patients received the following premedications to mitigate infusion-related reactions (IRR) and cytokine release syndrome (CRS): diphenhydramine, acetaminophen, and dexamethasone. All premedications were administered 1 to 3 hours before starting the infusion. The dose of any of the premedications could be reduced if, in the opinion of the investigator, comorbidities necessitate it. Dexamethasone is optional after cycle 2, day 15, unless the patient has experienced an IRR or CRS with the previous dose. The doses of the three premedications are as follows: 1. Dexamethasone 10-20 mg IV or methylprednisolone 1 mg / kg IV, or equivalent. Dose determined at the investigator's discretion based on patient comorbidities. 2. Acetaminophen 650 mg or 1,000 mg, or equivalent, orally (PO) three times daily for one day (the dose of 650 mg or 1,000 mg is at the discretion of the investigator). The first dose will be administered 1-3 hours prior to study drug infusion. 3. Antihistamines: 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 initiation of study drug.

[0339] Phase 1 - dose escalation study Dosing was initiated at the estimated minimum effective dose (MABEL) for the patient cohort. Patients enrolled were: 1) with relapsed or refractory AML who refused or were ineligible for intensive chemotherapy or allogeneic stem cell transplantation, or 2) with relapsed or refractory MDS, with >5% blasts in bone marrow or circulating blasts in peripheral blood, and who had failed a previous hypomethylating agent (HMA). Failure was defined as intolerance to HMA, lack of response (no CR by 6 cycles), or progressive disease as defined by the IWG during or after treatment with HMA. Demographic results for the 32 patients enrolled in the ongoing Phase 1 dose escalation study (through cohort 7) are shown in Table 9 below. For patients enrolled through cohort 7, the median age was 67 years, and 79% of patients had AML. The mean number of doses administered per patient was 8.5, and the mean duration of treatment was 54 days. [Table 9]

[0340] Treatment-related adverse events for 32 patients enrolled in the ongoing Phase 1 dose-escalation study are shown in Table 10 below. Thirty-four percent of patients experienced one or more IRR / CRS events (grade 3 or higher 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 two or more patients. Three of the 11 patients who experienced IRR / CRS events were treated with tocilizumab. [Table 10]

[0341] The percentage of blasts in the bone marrow aspirate was monitored over time for patients. As shown in Figures 3A-3D, a reduction in bone marrow blasts was observed in several patients who received the maximum dose of 12 μg or more. Two patients had a reduction in bone marrow blasts from 29% to 0% (cohort 6b, Figure 3C) and from 33% to 4% (cohort 6a, Figure 3B), respectively. Absolute neutrophil and platelet counts met the complete response criteria. Both patients remained on study, as did the one patient who had a reduction in blasts in cohort 7.

[0342] Serum cytokines were assessed in each patient at scheduled time points around the maximum dose, and also at intervals during infusion-related reactions or cytokine release syndrome events. As shown in Figures 4A-4D, cytokines were not elevated during scheduled collections. Elevations in cytokines, especially IL-6, were observed during IRR / CRS adverse events. Notably, no correlation was observed between maximum cytokine concentrations and dose level or event grade in this small data set.

[0343] Pharmacokinetic (PK) data were analyzed for patients in cohorts 6A (Figure 6A) and 6B (Figure 7A). In cohort 6A, quantifiable concentrations of TRI130 were seen in one patient after C3D1 (day 56). All other samples were below the limit of quantification (BLQ) for the current assay format (2.5 ng / mL), although multiple samples were within the detectable range of the assay.

[0344] Anti-drug antibody levels were also determined for patients in cohorts 6A (Figure 6B) and 6B (Figure 7B). In cohort 6A, one patient (patient 4) was screened and confirmed positive, but with low titers. The patient was also positive for pre-treatment with ADA, and titers of such antibodies decreased over time, indicating that the response was not related to treatment. In cohort 6B, patient 6 was positive post-treatment for anti-TRI130 antibodies. In this patient, the titers were also low. Notably, the pre-treatment background was below the cutoff value, but relatively high compared to other negative individuals, indicating that the response may not be related to TRI130 treatment.

[0345] Thus, in this preliminary study, administration of TRI130 via the 24 μg dose was tolerated with a manageable safety profile. As noted above, two patients demonstrated complete responses (CR). Cytokines were not significantly elevated unless an IRR / CRS concurrent adverse event occurred. Preliminary data suggested there was no evidence of treatment-induced anti-drug antibodies (ADA).

[0346] This open-label, multiple dose-escalation dose-escalation phase was designed to determine the recommended dose level of TRI130 for a future Phase 2 study, specifically, a dose of TRI130 that does not result in any acute, clinically significant cytokine release that poses a safety risk to patients. At this stage of the study, TRI130 had a manageable safety profile and established a recommended Phase 2 dose ("RP2D"). Additionally, pharmacokinetic (PK) data from the dose-escalation study demonstrated that treatment as a single agent at RP2D levels below the maximum tolerated dose ("MTD") level achieved long-term stabilization of leukemia, resulting in deepening responses to partial and complete responses ("CR") in two patients with relapsed / refractory AML. Cases of infusion-related reactions and cytokine release syndrome experienced in some Phase 1B studies were manageable with dose interruptions, administration of dexamethasone and / or tocilizumab.

[0347] Phase 1 - Additional Dose Escalation Cohorts After completion of the dose-limiting toxicity (DLT) observation period for cohort 7, patients will be enrolled into four additional sequential cohorts (cohorts A, B, C, and D) while cohorts 8 and above are treated. These cohorts will be sequential and independent of cohorts 8-10 to achieve more rapid dose escalation.

[0348] Patients in cohorts A, B, C, and D received continuous IV dosing (20-24 h / day) for the first 4 days of cycle 1, followed by twice weekly dosing during week 2, and then once weekly dosing during cycle 1 and subsequent cycles. Table 11 shows the dosing schedules for cohorts A, B, C, and D. Dosing during the first week in cohort A uses the doses 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 the second week, a dose of 18 μg is administered on days 8 and 11. In week 3, the dose is increased to 36 μg and maintained at that level. The advantage of increasing the daily dose during the first week is a gradual increase in Cmax, which may reduce the propensity for IRR / CRS. Active treatment with tocilizumab is reserved for grade ≥2 IRR or CRS unresponsive to symptomatic treatment and within 2 hours of dose interruption. [Table 11]

[0349] Cohorts A-D will receive the first dose and each subsequent dose escalation by IV infusion over 20-24 hours. The second dose will be the same dose and will be infused over 8 hours (± 30 minutes), the third dose over 6 hours (± 30 minutes), and the fourth and subsequent doses over 4 hours (± 30 minutes).

[0350] 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 doses thereafter, dexamethasone is optional, but acetaminophen and diphenhydramine are required.

[0351] In Figures 8A-8E, PK simulations are provided for each of cohorts A, B, C, and C, showing how Cmax gradually increases over time. Simulations of additional PK parameters are also shown in Figures 9A, 9B. These simulations are based on modeling low dose days in cynomolgus monkeys to account for a limited amount of TMDD (target-mediated pharmacokinetics).

[0352] Example 3: Phase 1b expansion study of TRI130 in AML and MDS patients The maximum tolerated dose (MTD) of TRI130 was not reached at the dose level of 240 μg / cycle (Cohort 10 in Table 10) in the dose escalation study described in Example 2. The sub-MTD dose level of Cohort 6A was identified as the recommended phase 2 dose (RP2D) level of TRI130 for further evaluation during the expansion phase. This dose level represents a >50% lower amount of TRI13 than that administered in cycle 1 of Cohort 10 without dose-limiting toxicity (DLT) (i.e., 45 μg vs. 96 μg in cycle 1), and a 70% lower amount than the dose level of Cohort 10 in the second and subsequent cycles (72 μg vs. 240 μg in cycles 2-4). Both long-term stable and complete responses of >6 months were observed as the best overall responses for TRI130 as a single agent at this sub-MTD dose level, which was selected for further testing during the expansion phase.

[0353] Thus, this Phase 1b extension study will evaluate the safety and tolerability of TRI130 at the RP2D level when used as an adjunct to standard of care, using dose levels used in Cohort 6A of the dose escalation phase (Example 2). The anti-leukemic activity of TRI130 will be evaluated in this expansion phase as a monotherapy as well as in combination with standard chemotherapy agents.

[0354] The study is an open-label, multicenter, dose-expansion trial that will enroll a total of 90 patients with primary AML into five cohorts of 18 patients each. The treatment arms are designed to evaluate safety and efficacy endpoints. In cohorts 1-4, TRI130 will be administered at a fixed dose of 18 μg, followed by a weekly ramp-up during cycle 1 (cohorts 1, 3, 4) or cycles 1-2 (cohort 2). In cohort 5, TRI130 will be administered at a fixed dose of 18 μg twice weekly, followed by a weekly ramp-up during cycle 1. Each of these cohorts is described in further detail below.

[0355] Cohort 1 - Chemotherapy and induction with TRI130 Patients will be treated with a combination of chemotherapy (ChT) and TRI 130. For ChT, patients may receive intermediate dose cytarabine (IDAC) or a combination of mitoxantrone, etoposide, and cytarabine (MEC).

[0356] Primary or secondary AML patients (age: >18 years) with a previous complete response (CR) <12 months or in first or second relapse of primary refractory disease will receive 4x28-day cycles of two-drug combination immunochemotherapy including (i) TRI130 in combination with intermediate-dose cytarabine (IDAC) or (ii) TRI130 in combination with MEC (mitoxantrone, etoposide, cytarabine). Primary refractory disease is defined by the European LeukemiaNet (ELN) as failure to achieve CR or complete response with incomplete hematologic recovery (CRi) after two intensive induction treatment courses.

[0357] If IDAC is selected as the ChT, patients will receive 4 cycles of IDAC in combination with TRI130. Cytarabine (1g / m 2 ) is administered intravenously over 2 hours daily on days 1-5 during each 28-day cycle (D1-D5). On day 1 of cycle 2 (D29), cycle 3 (D57), and cycle 4 (D85), cytarabine is administered 4 hours after the end of the TRI130 infusion. This dosing regimen is illustrated in Figure 5A.

[0358] If MEC was selected as CHt, patients received mitoxantrone 6 mg / m on days 1–6. 2 / day IV, etoposide 80 mg / m on days 1–6 2 / day IV, cytarabine 1 g / m on days 1-6 2 Patients will receive 2 cycles of 100 mg / day IV. TRI130 will be administered in combination with MEC for 2 cycles followed by 2 cycles as monotherapy. This dosing regimen is diagrammed in Figure 5A.

[0359] TRI130 cycle 1, day 1 (C1D1) will be day 8 after initiating IDAC or MEC chemotherapy. The first TRI130 cycle will include only three doses of TRI130 (C1D1, C1D8, C1D15) at weekly intervals. All subsequent cycles will include four weekly infusions of TRI130. TRI130 will be administered intravenously over 4 hours at a fixed dose of 18 μg on days 1, 8, 15, and 22 of each TRI130 cycle after the weekly ramp-up during TRI130 cycle 1. The escalation will proceed as shown in Table 12. [Table 12]

[0360] The primary endpoints for Cohort 1 were: (1) Safety: cumulative incidence of grade 3-4 adverse events (AEs) and serious adverse events (SAEs) for safety, and incidence of AEs of note (≥ grade 2 cytokine release syndrome (CRS), ≥ grade 2 infusion-related reactions, ≥ 2 cardiac toxicity, ≥ 2 neurotoxicity as a complication of CRS), and (2) Efficacy: leukemia-free survival (LFS), composite CR rate after each cycle (CR / CRi / CR), and minimal residual disease (MRD) status by multicolor / multiparameter flow cytometry (MFC) as measured by the Central Lab for patients who achieved CR.

[0361] Cohort 2 - TRI130 + venetoclax + azacitidine induction - frontline or first relapse Patients (age >18 years) with primary or secondary AML who are poor prognosis, but treatment naïve or in first relapse, receive four 28-day cycles of triple immunochemotherapy including TRI130 plus venetoclax and azacitidine. A minimum of 15 days must have elapsed since the first dose of venetoclax to minimize the risk of tumor lysis syndrome (TLS) while patients are initiating TRI130. The first TRI130 dose in the first TRI130 cycle is administered 15 days after the first dose of venetoclax. In subsequent cycles, TRI130 and the first dose of venetoclax can be administered on the same day without a 15-day interval.

[0362] TRI130 will be administered intravenously over 4 hours weekly at a fixed dose of 18 μg on days 1, 8, 15, and 22 of each TRI130 cycle, following a weekly ramp-up during TRI130 cycle 1. Step-up dosing will proceed as shown in Table 13. [Table 13]

[0363] Venetoclax is administered orally daily on days 1-21 of each cycle at a fixed dose of 400 mg / day following a daily ramp-up (day 1: 100 mg; day 2: 200 mg; days 3-21: 400 mg). The dose of venetoclax will be adjusted according to the standard of care for patients taking azole antifungals. Venetoclax may be administered on days 1-14 only after cycle 2 for patients with less than 5% bone marrow blasts.

[0364] Azacitidine was administered at 75 mg / m2 over 30 minutes daily on days 1-7 of each cycle. 2 It is administered intravenously at a dose of

[0365] The dosing regimen for Cohort 2 is shown in Figure 5B.

[0366] The primary endpoints for cohort 2 are: (1) Safety: cumulative incidence of grade 3-4 AEs and SAEs for safety, and incidence of AES of note (≥ grade 2 CRS, ≥ grade 2 infusion-related reactions, ≥ 2 cardiac toxicity, ≥ 2 neurotoxicity as a complication of CRS), and (2) Efficacy: leukemia-free survival (LFS), combined CR rate (CR / CRi / CRh) after each cycle, MRD status by MFC (Central Laboratory) for patients who achieved CR. HSCT (hematopoietic stem cell transplant) eligible patients can undergo HSCT after 2 cycles of triplet APVA if they are in CR and MRD negative by flow cytometry. If not, 4 TRI130 cycles are strongly recommended according to the protocol. After 4 cycles of TRI130 + venetoclax + azacitidine triple combination therapy, patients will continue venetoclax + azacitidine in follow-up at the discretion of the investigator. Patients who achieve a complete response plus minimal residual disease (CRMRD) status after four cycles of TRI130 but are not eligible to undergo hematopoietic stem cell transplant (HSCT) may receive an additional four cycles of TRI130 monotherapy at the investigator's discretion.

[0367] Cohort 3-7+3 Post-Consolidation-Frontline+1st Relapse Primary AML patients (age: >18 years) with FLT3-negative intermediate or adverse risk AML (including but not limited to TP53, RUNX1 and ASXL1 mutations and / or mixed cytogenetics) who are treatment-naïve or in CR1 for less than 1 year will receive immunotherapy with TRI130 for 4 x 28 day cycles after hematologic recovery (ANC>1,000 μL, Hgb≧9 g / L, Plt≧100,000 / μL) following induction therapy with either (a) or (b) as described below.

[0368] (a) Newly diagnosed patients: Cytarabine 100-200 mg / m 2 12 mg / m2 was administered intravenously daily as a continuous infusion for 7 days on days 1 to 7, and idarubicin 12 mg / m2 was administered intravenously daily as a continuous infusion for 7 days. 2 is given intravenously over 15 minutes on days 1 to 3 (or daunorubicin 60 [30 to 90] mg / m 2 The standard 7+3 regimen consists of 1 cycle of high-dose cytarabine (HiDAC) consolidation or 1-2 cycles of IDAC or CPX-351 after the 7+3 induction regimen (HiDAC may be increased to 1.5 g / m for patients over 60 years of age, depending on institutional practice and / or investigator discretion). CPX-351 induction at standard doses and schedules is also acceptable if preferred by the investigator. Patients are eligible if they received 1 cycle of high-dose cytarabine (HiDAC) consolidation or 1-2 cycles of IDAC or CPX-351 after the 7+3 induction regimen (HiDAC may be increased to 1.5 g / m for patients over 60 years of age, depending on institutional practice and / or investigator discretion). 2 Patients with ≥ 5% blasts in the bone marrow after induction 1 (persistent blasts) should receive a second induction cycle, which does not have to be the same ChT as induction 1. As soon as patients achieve CR / CRi after one or two induction cycles, they should proceed to consolidation therapy.

[0369] (b) For patients with first relapse, a standard cytarabine-based salvage regimen, e.g., mitoxantrone + etoposide + cytarabine (MEC), can be used. When treated with MEC, patients receive mitoxantrone 6 mg / m on days 1–6. 2 / day IV, etoposide 80 mg / m on days 1–6 2 / day IV, cytarabine 1 g / m on days 1-6 2 / day IV. FLAG or FLAG-IDA should not be used due to the potential risk of enhancement of fludarabine-associated neurotoxicity and autoimmune complications with TRI130.

[0370] At least 21 days must have elapsed since the last cytarabine to allow for resolution of side effects related to 7+3 induction or HiDAC consolidation. TRI130 should be initiated after the final intended ChT cycle. Patients must meet all eligibility criteria to receive immunotherapy with TRI130. To be eligible for TRI130 treatment, patients must be in either not in CR or in CR with MRD positivity (≥0.1% level) by Central Laboratory (MFC) after induction / consolidation.

[0371] The first TRI130 consolidation cycle will be initiated ≥21 days (preferably 21-28 days) after 7+3 or MEC induction or HiDAC / IDAC-containing consolidation, at the investigator's option.

[0372] TRI130 will be administered intravenously over 4 hours weekly at a fixed dose of 18 μg on days 1, 8, 15, and 22 of each TRI130 consolidation cycle, following a weekly ramp-up during cycle 1. Step-up dosing will proceed as shown in Table 14. [Table 14]

[0373] The dosing regimen for Cohort 3 is shown in Figure 5C.

[0374] The primary endpoints for Cohort 3 were: (1) Safety: cumulative incidence of grade 3-4 AEs and SAEs for safety, and incidence of AES of note (≥ grade 2 CRS, ≥ grade 2 infusion-related reactions, ≥ 2 cardiac toxicity, and ≥ 2 neurotoxicity as a complication of CRS), and (2) Efficacy: leukemia-free survival (LFS), MRD status by MFC for patients in response at the start of TRI130 consolidation, and composite CR rate for patients not in CR / CRi / CRh at the start of TRI130 consolidation.

[0375] Eligible patients may undergo HSCT after two cycles of TRI130 if MRD-negative with CR and MFC. Otherwise, four TRI130 cycles are strongly recommended depending on the protocol.

[0376] Patients who achieve CRMRD status after four cycles of TRI130 but are not eligible to undergo HSCT may receive four additional cycles of TRI130 monotherapy at the physician's discretion.

[0377] Cohort 4 - MRd positive (MRD+) first response, TRI130 + oral azacitidine Patients older than 18 years with minimal residual disease (levels of 0.1% or greater by multicolor multiparameter flow cytometry [MFC] in the Central Lab) and high-risk first responder AML will be treated with 4 × 28-day cycles of TRI130 plus oral azacitidine (Onureg, CC-486).

[0378] Patients with AML in first response who have received frontline standard 7+3 or other standard induction therapy (including but not limited to hypomethylating agents + venetoclax or low-dose cytarabine + venetoclax) are eligible. If MRD+CR, patients who receive 7+3 induction followed by 1 cycle of high-dose cytarabine (HiDAC) consolidation or 1-2 cycles of IDAC or CPX-351 are eligible.

[0379] Patients who are in MRD+ first response after at least four cycles of venetoclax-based therapy are eligible.

[0380] Cycle 1, Dose 1 of TRI130 must occur ≥21 days after HiDAC- or IDAC-containing consolidation. TRI130 is administered intravenously over 4 hours weekly at a fixed dose of 18 μg on days 1, 8, 15, and 22 of each cycle, following a weekly ramp-up during Cycle 1. Step-up dosing will proceed as shown in Table 15. [Table 15]

[0381] Oral azacitidine is administered at a daily dose level of 300 mg for 14 days. The dosing regimen for Cohort 4 is shown in Figure 5D.

[0382] Eligible patients may undergo HSCT after two cycles of TRI130 + azacitidine if they achieve MRD status by MFC. Otherwise, four cycles of TRI130 + azacitidine are strongly recommended depending on the protocol. Patients who achieve CRMRD status after four cycles of TRI130 but are not eligible to receive HSCT may receive four additional cycles of TRI130 monotherapy at the discretion of the investigator.

[0383] Cohort 5 - MRD+ second response, single agent TRI130 Patients with AML aged >18 years who are MRD+ (MFC level ≥0.1%) in second response after induction therapy with a standard of care regimen will be treated with 4 x 28-day cycles of TRI130 monotherapy.

[0384] Patients with and without consolidation after induction therapy with a standard of care regimen are eligible. In the first 5 patients (= safety lead-in), patients will be enrolled with a staggered period of at least 7 days between consecutive patients 1 to 5.

[0385] Cycle 1, Dose 1 of TRI130 must occur ≥21 days after HiDAC- or IDAC-containing consolidation. TRI130 is administered intravenously at a fixed dose of 18 μg over 4 hours weekly on days 1, 4, 8, 11, 15, 18, and 22 of each cycle, following a weekly ramp-up during cycle 1. (Step-up dosing: TRI130 is administered intravenously at a fixed dose of 18 μg over 4 hours weekly on days 1, 8, 15, and 22 of each cycle, following a weekly ramp-up during cycle 1.

[0386] Stepwise dosing will proceed as shown in Table 16. [Table 16]

[0387] The dosing regimen for Cohort 5 is shown in Figure 5E.

[0388] If twice weekly dosing results in grade 3 IRR or CRS during the safety lead-in, the C1D4 and C1D11 doses of TRI130 may be omitted.

[0389] Eligible patients may undergo post-TRI130 HSCT after 2 cycles of TRI130 if MRD-negative by CR and MFC. Otherwise, 4 cycles are strongly recommended depending on the protocol. Patients who achieve CRMRD status after 4 cycles of TRI130 may receive an additional 4 cycles of TRI130 monotherapy at the investigator's discretion.

[0390] Example 4: Single-agent activity of TRI130 in patients with relapsed / refractory AML or MDS The primary objective of this proof-of-concept study was to evaluate eight patients with relapsed or refractory (R / R) AML who had failed treatment with hypomethylating agents (HMAs, N=2) or venetoclax + HMAs (N=6), and six patients with R / R MDS who had failed treatment with HMAs (N=5) or venetoclax + HMAs (N=1). Clinical activity of TRI130 was tested at submicrogram dose levels above 0.08 μg / kg, which were active in preclinical NOD / SCID mouse xenograft models of AML. This analysis evaluated clinical proof-of-concept conducted using primary data from a recently completed Phase 1B study of TRI130 in R / R AML and MDS patients.

[0391] Clinical Trials This proof-of-concept study was conducted under IND135552 as part of a multicenter Phase 1B clinical dose escalation study of TRI130 in patients with relapsed / refractory AML and high-risk myelodysplastic syndromes (MDS). It is registered in the clinical trials database ClinicalTrials.gov under the identification number NCT03647800. The weekly target dose levels for cohorts 2-10 were 1 mcg in cohort 2, 3 mcg in cohort 3, 9 mcg in cohort 4, 18 mcg in cohort 6A, 12 mcg in cohort 6B, 24 mcg in cohort 7, 36 mcg in cohort 8, 48 mcg in cohort 9, and 60 mcg in cohort 10

[28] (see Example 2, Table 10 above). A 3+3 design was used to guide dose escalation. In each cohort, eligible AML / MDS patients were assigned to receive a predefined fixed dose of TRI130 by weekly intravenous infusion as a single agent. TRI130 was administered at the indicated dose levels by weekly intravenous infusion as a single agent

[28] . TRI130 was administered according to a protocol amendment to the intrapatient escalation strategy implemented in cohort 5 to reduce the risk of cytokine release syndrome (CRS). DNA sequencing for molecular profiling of leukemic blasts was performed using the Genoptix (Carlsbad, CA) platform. All responses were evaluated by the ELN 2017 criteria for AML and the IWG 2006 criteria for MDS. TRI130 demonstrated a favorable safety profile with acceptable tolerability and manageable treatment-emergent AEs

[28] . The MTD was not reached at the weekly fixed dose of 60 μg (60 kg subjects, 1 μg / kg)

[28] . The most common grade 3 AEs suspected to be related to TRI130 were grade 3–4 CRS occurring in 4 of 46 patients (8.7%), grade 3–4 anemia occurring in 2 of 46 patients (4.3%), and infusion-related reactions (IRRs) occurring in 2 of 26 patients

[28] .

[0392] Measurement of serum cytokine levels and flow cytometry A central laboratory instrument used the Meso Scale Discovery (MSD) U-PLEX assay platform and an MSD Meso Quickplex SQ 120 Reader Instrument (Meso Scale Diagnostics, Rockville, MD) to measure serum levels of the proinflammatory cytokines interleukin-5 (IL-5), interleukin-10 (IL-10), and interferon gamma (IFN-γ) by electrochemiluminescence in duplicate serum samples. Immunophenotyping was performed on cryopreserved peripheral blood mononuclear cells from patients by standard flow cytometry. Flow cytometry was performed using a BD LSR II flow cytometer and FACSDiva Software Version 8.0.2 with fluorochrome-conjugated monoclonal antibodies reactive to the CD5 (anti-human CD5, clone REA782 [PE-Vio770]), CD45 (anti-human CD45, clone H130, V500, BD Biosciences #560777), CD34 (anti-human CD34, clone REA1164, VioBright 515, Miltenyl Biotech #130-120-517), and CD123 (anti-human CD123, clone 9F5, AF647, BD Biosciences #563599) antigens.

[0393] statistical analysis Standard statistical methods were applied to analyze clinical data. Survival data were analyzed by the Kaplan-Meier method using the GraphPad Prism 9 statistical program (GraphPad Software, LLC, San Diego, CA). Differences between patient subgroups were compared using the log-rank statistic [29, 30].

[0394] result Patient characteristics: Eight patients (three men and five women), median age 66 years (mean ± SE = 65 ± 6 years), of whom seven were Caucasian and one was African American, had R / RAML and had been previously treated with one to eight AML therapies (Figure 13A). Four men and two women had R / RMDS. Median age was 75 years (mean ± SE = 75 ± 2 years), of whom five were Caucasian and one was Asian, and had been previously treated with one to three MDS therapies (Figure 13B). Of the eight AML patients, four (UPN01, UPN02, UPN04, UPN06) suffered from AML with MDS-related features - one of these patients (UPN04) also had a FLT3-ITD gene mutation, one (UPN03) suffered from AML with recurrent genetic abnormalities, two (UPN07, UPN08) suffered from AML with genetic mutations, and one suffered from AML-NOS (M0-AML) (Figure 13A). All six MDS patients suffered from MDS with excess blasts according to the WHO classification (MDS-EB-1 or MDS-EB-2). Five of these patients had IPSS prediction scores consistent with intermediate-1 (IM-1) or intermediate-2 (IM-2) risk groups, and one suffered from high-risk MDS (Figure 13B). Patient characteristics and treatment outcome data are shown in Figures 13A-13B.

[0395] No dose-limiting toxicity (DLT) or grade 5 adverse events (AE) were observed in any of the 14 cases analyzed. Of the eight AML patients, six (UPN01, UPN02, UPN03, UPN04, UPN-7, and UPN08) were CRS-free, one patient (UPN05) was with grade 1 CRS lasting for 2 days, and one patient (UPN06) was with grade 2 CRS lasting for 2 days (Figures 13A-13B). There were two severe adverse events (SAEs). One SAE was reported for UPN05, who developed sepsis, diarrhea, and vomiting at C6D5 and fully recovered within 5 days. Grade 2 CRS in UPN06 was also reported as an SAE due to hospitalization, but lasted only 2 days and fully resolved (Figures 13A-13B). No SAEs were reported in any of the six MDS patients. One MDS patient (UPN13) had grade 1 CRS that lasted 1 day on C2D1, and another MDS patient (UPN09) had several transient grade 3-4 AEs, including tumor lysis syndrome (TLS), anemia, decreased platelet count, and hyperglycemia (Figures 13A-13B).

[0396] Pharmacodynamic Effects of TRI130: Analysis was performed to determine whether TRI130 could activate T cells by measuring serum levels of three T cell-derived cytokines (IFN-γ, IL-5, and IL-10) as surrogate pharmacokinetic biomarkers: T helper 1 cytokines: IFN-γ, IL-10; T helper 2 cytokines: IL-5, IL-10; effector T cell cytokine: IL-10. In post-treatment blood samples from R / R AML patients (UPN01, UPN05, and UPN06), serum levels of IFN-γ and IL-10 were significantly above baseline, consistent with treatment-induced T cell activation. Similarly, IL-10 levels were above baseline in post-treatment samples from one MDS patient UPN10, but not MDS patients UPN09 or UPN13 (Figure 14).

[0397] We next investigated the effect of TRI130 on tumor burden as reflected by CD123+ target blast cells using immunophenotyping by multiparameter flow cytometry. Unlike normal CD34+CD38 hematopoietic stem cells, putative leukemic stem cell (LSC) populations in AML have been reported to express CD123 (19). The majority of these cells co-express the CD33 antigen (i.e., they are CD33+CD34+CD38-). Flow cytometric analysis of post-treatment blood samples from three of the four AML patients (UPN01, UPN03, UPN06) showed a reduction in the number of circulating CD123+CD34+CD38- and CD33+CD34+CD38- putative LSC cells. (FIG. 15) The maximum reduction in CD123+ LSC cell numbers was 76.1% in UPN01 (C6D1 sample), 57.8% in UPN03 (C1D30 sample), and 99.2% in UPN06 (C4D1 sample). FIG. 10 shows multicolor dot profiles from multiparameter flow cytometry studies, which demonstrate a rapid and significant reduction in CD123+CD34+CD38- and CD33+CD34+CD38- LSC cell numbers with TRI130 monolayer therapy in UPN06. Virtually all CD34+CD38- cells were CD123+ and CD33+, consistent with AML. The size of this CD123+CD33+CD34+CD38- AML LSC population, indicated by the arrow in the third row of panel A, was significantly reduced by TRI130 monotherapy. In contrast to these three AML cases, Figure 15 and Figure 11 show that CD34+CD38-CD123+ cells from a fourth AML patient, UPN05, were not reduced upon TRI130 treatment, but their proliferation was prevented, which was associated with a stable progression-free interval of 238 days. As shown in Figure 15, reductions in CD123+CD34+CD38- and CD33+CD34+CD38- cells were also observed in post-treatment samples of two MDS patients.

[0398] These pharmacodynamic results provide an early proof of concept that the CD3xCD123 bispecific TRI130 can activate T cells and cause a reduction in target CD123+ blasts in patients with MDS and AML. Notably, at progression, there was a significant increase in CD123+ cell numbers in UPN10, and the only cells that did not decrease in UPN03 were CD123+. These results indicate that failure of TRI130 monotherapy can occur independent of CD123 expression, as the antileukemic activity of TRI130 depends on the cytotoxic T cell (CTL) activity of CD3-expressing T cell populations redirected to CD123+ AML / MDS cells.

[0399] Efficacy: Of the eight R / R AML patients, two patients (UPN2 and UPN4) had progressive disease (PD) and one had stable disease (SD) / resistant disease (RES) (UPN3) as best overall response (BOR) and died of leukemia between 75 and 122 days (Figures 13A-13B). Three patients (including UPN01, UPN05, and UPN06) had long-term SD. Their progression-free times ranged from >106 days to 238 days. Among the three AML patients with SD as best overall response, one patient (UPN06; AML with MDS-related features) had complete clearance of peripheral blasts at day 113 (from 21% pretreatment to 14% in cycle 2, 2% in cycle 3, and 0% in cycle 4) with a decrease in pretreatment bone marrow blast percentage of >50% (from 78% pretreatment to 37% posttreatment) followed by sustained SD. This patient also saw pharmacodynamic activation of T cells with TRI130 (Figure 14) and a decrease in target CD123+CD34+CD38- AML cells (Figure 15). Another AML patient (UPN01) with MDS-related features had 29% bone marrow blasts, unfavorable cytogenetics (del5q and monosomy7) and TP53 mutations, whose disease had previously progressed on venetoclax + decitabine therapy, but achieved PR in 31 days and CR in 92 days with complete hematological recovery as BOR (best overall response) after TRI130 monotherapy. This patient also saw pharmacodynamic activation of T cells with TRI130 (Figure 14) and reduction in target CD123+CD34+CD38- AML blasts (Figure 15). The onset and duration of SD, peripheral blood blast count clearance (PBBC-C), PR or CR in these patients are shown by the swimmer plots shown in Figure 12.

[0400] Similarly, at dose levels ranging from 0.1mcg / kg to 0.8mcg / kg, three MDS patients had SD (50%) and three additional MDS patients (50%) had bone marrow CR (Figures 13A-13B). One patient (UPN10 from cohort 9) who showed T cell activation in post-treatment samples (Figure 14) had 8.2% pre-treatment BM blasts with 15% bone marrow cellularity and 2% BM blasts with 20% cellularity after C2D1 treatment. Another patient (2000003 from cohort 7) had 11.3% baseline BM blasts with 20-30% cellularity and 0% BM blast percentage with 50% cellularity after C2D1 treatment. The third patient (2190001 in cohort 6) had a pretreatment BM blast percentage of 7.5% with 10% cellularity and a BM blast percentage of 2.4% with 20% cellularity after C2D1 treatment (Figures 13A-13B). The onset of bone marrow CR and progression-free time in these MDS patients are shown by the swimmer plots shown in Figure 12. One MDS patient with an ASXL-1 mutation, UPN09 in cohort 4, showed a decrease in the percentage of myeloblasts in the bone marrow, which was part of the case's conversion to high-risk chronic myelomonocytic leukemia-myeloproliferative neoplasm (CMMN-MPN) with an absolute monocyte count of 17,600 / μL around cycle 10 (Figures 13A-13B).

[0401] Summary: The data presented herein demonstrate that TRI130 activated T cells in R / R AML and MDS patients, as evidenced by a significant increase in serum Th1 / Th2 cytokine IL-10 and a decrease in the number of circulating CD123+CD34+ and CD33+CD34+ peripheral blasts. Single-drug activity was observed in four R / R AML patients (50%), including three patients with stable disease (SD) and one patient with complete response (CR), at dose levels ranging from 0.1 μg / kg to 0.7 μg / kg. Similarly, three MDS patients had SD (50%) and three additional MDS patients (50%) had bone marrow CR at dose levels ranging from 0.1 μg / kg to 0.8 μg / kg. These data provide proof of concept in support of the in vivo immunomodulatory and antileukemic activity of TRI130.

[0402] This analysis also provides clinical proof-of-concept supporting the mechanism of action of TRI130 using primary data from a recently completed Phase 1B study in patients with R / R AML and MDS

[28] . These results informed the design of the currently accruing cohort 2 of the expansion phase of the Phase 1B study (NCT03647800). The data demonstrate that TRI130 (administered intravenously weekly) has promising early single-agent activity and immunomodulatory effects in patients who have failed prior treatment with venetoclax + HMA for AML or HMA alone for MDS. Although single-agent TRI130 was not associated with dose-limiting myelosuppression, the tolerability and efficacy of TRI130 in combination with a venetoclax + azacitidine backbone will be formally evaluated in a Phase IB study in patients with newly diagnosed AML ≥75 years or in patients >60 years who are unsuitable for intensive chemotherapy or HSCT (Clinicaltial.gov Identifier: NCT04973618). There is a 2-week induction phase with venetoclax + azacitidine to reduce the risk of TLS and cytokine release syndrome (CRS). We believe that the addition of TRI130 will eliminate residual CD123+ blasts and leukemia stem cells that are resistant to venetoclax, leading to more durable responses.

[0403] Treatment-naïve patients with AML who are elderly (≥75 years) or unfit to undergo intensive chemotherapy have improved survival when treated with venetoclax and HMAs compared to HMAs alone [7,31]. The Food and Drug Administration (FDA) has approved venetoclax in combination with HMAs for this population

[15] . However, the use of venetoclax requires more clinical experience, and several challenges and limitations have emerged for venetoclax-based therapy, as highlighted in recent publications [13,32].

[0404] Responses induced by venetoclax in treatment-naïve elderly / unfit AML patients last less than 12 months (median) even after combination of venetoclax with HMAs. Furthermore, secondary AML patients and those previously treated with HMAs have a significantly reduced CR rate and an overall survival of less than 6 months, and are less responsive to venetoclax-based treatment regimens

[13] . Similarly, some AML patient populations in adverse risk categories, such as those with TP53 and RTK mutations, may exhibit inherent venetoclax resistance

[13] . Unlike its remarkable activity in treatment-naïve AML patients, venetoclax is less effective in relapsed AML patients. The reported overall response rate was only 21% in relapsed or refractory AML patients treated with venetoclax in combination with HMAs, LDAC, or other agents such as cladribine or midostaurin

[33] . Therefore, there is an urgent need for new agents that can be combined with venetoclax-based treatment regimens for elderly AML patients and potentially improve clinical outcomes.

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Claims

**Claim 1** A method for treating cancer, the method comprising administering to a subject in need of cancer treatment i) a multispecific protein comprising a CD123 binding domain and a CD3 binding domain, and ii) a second anti-cancer agent The method as described above. **Claim 2** The method according to claim 1, further comprising administering to the subject a third anti-cancer agent. **Claim 3** The method according to claim 1, wherein the second anti-cancer agent is a chemotherapeutic agent. **Claim 4** The method according to claim 3, wherein the chemotherapeutic agent is venetoclax, azacitidine, decitabine, daunorubicin, cytarabine, idarubicin, mitoxantrone, or etoposide.

5. The second anticancer agent is cytarabine, and the cytarabine is administered at a dose of about 1 g / m ２ 2, according to the method of claim 4. **Claim 6** The method according to claim 2, wherein the second anti-cancer agent is mitoxantrone, the third anti-cancer agent is etoposide, and the method further comprises administering a fourth anti-cancer agent which is cytarabine. **Claim 7** The mitoxantrone is administered at a dose of about 6 mg / m ２ / day, the etoposide is administered at a dose of about 80 mg / m ２ / day, and the cytarabine is administered at a dose of about 1 g / m ２ / day, the method according to claim 6. **Claim 8** The method according to claim 4, wherein the second anti-cancer agent is venetoclax and the venetoclax is administered at a dose of about 100 to about 400 mg per day. **Claim 9**: The second anti-cancer agent is azacitidine, and the azacitidine is administered at a dose of about 75 mg / m ２ / day. The method according to claim 4. **Claim 10** The method according to claim 2, wherein the second anti-cancer agent is azacitidine and the third anti-cancer agent is venetoclax. **Claim 11** The azacitidine is administered at a dose of about 75 mg / m ２ / day, and the venetoclax is administered at a dose of about 100 to about 400 mg / day. The method according to claim 10.

12. The second anti-cancer agent is decitabine, and the decitabine is administered at a dose of about 20 mg / m ２ / day. The method according to claim 4. **Claim 13** The method according to claim 2, wherein the second anti-cancer agent is decitabine and the third anti-cancer agent is venetoclax. **Claim 14** The decitabine is administered at a dose of about 20 mg / m ２ / day, and the venetoclax is administered at a dose of about 100 to about 400 mg / day, the method according to claim 13. **Claim 15** The method according to claim 2, wherein the second anti-cancer agent is daunorubicin and the third anti-cancer agent is cytarabine. **Claim 16** The daunorubicin is administered at a dose of about 30 to 90 mg / m ２ and the cytarabine is administered at a dose of about 100 to about 200 mg / m ２ The method according to claim 15, wherein the method is administered at a dose of

17. The anticancer agent is idarubicin, and the idarubicin is administered at a dose of about 12 mg / m ２ 2, according to the method of claim 21. **Claim 18** The method according to claim 2, wherein the second anti-cancer agent is idarubicin and the third anti-cancer agent is cytarabine. **Claim 19** The idarubicin is administered at a dose of about 12 mg / m ２ and the cytarabine is administered at a dose of about 100 to about 200 mg / m ２ The method according to claim 18, wherein the method is administered at a dose of

20. The second anticancer agent is azacitidine, and the azacitidine is administered at a dose of about 75 mg / m ２ / day. The method according to claim 5. **Claim 21** The multispecific protein comprises a dimer of two identical polypeptides, and each polypeptide, from the amino terminus to the carboxyl terminus or from the carboxyl terminus to the amino terminus, (i) a CD123 binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, and (iv) a CD3 binding domain The method according to claim 1. **Claim 22** The method according to claim 21, wherein the polypeptide comprises the CD123 binding domain, the hinge region, the immunoglobulin constant region, and the CD3 binding domain from the N-terminus to the C-terminus.

23. wherein at least one of said CD123 binding domain and said CD3 binding domain is (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 method according to claim 22, comprising.

24. wherein said CD123 binding domain is a) HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, and HDCR3 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, b) 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, or c) a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27 The method according to claim 23, which is an scFv comprising.

25. wherein said CD3 binding domain is a) HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 20, and HDCR3 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, b) a VH comprising a sequence that is at least 90%, at least 95% or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence that is at least 90%, at least 95% or 100% identical to SEQ ID NO: 384, or c) a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27 The method according to claim 23, which is an scFv comprising.

26. The method according to claim 23, wherein each polypeptide comprises a sequence that is at least 90%, at least 95% or 100% identical to SEQ ID NO:

31.

27. The method according to claim 1, wherein said multispecific protein is administered to said subject by IV injection.

28. The method according to claim 1, wherein said second anti-cancer agent is administered to said subject orally or by IV injection.

29. The method according to claim 1, wherein said multispecific protein is administered to said subject by IV injection at a dose of 0.3, 1, 3, 6, 9, 12, 18, 20, 24, 30, 36, 48, 50, 60, 75 or 100 μg.

30. The method according to claim 1, wherein the multispecific protein is administered once a week.

31. The method according to claim 30, wherein the multispecific protein is administered to the subject by IV infusion during a first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

32. The method according to claim 31, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28-day cycle.

33. The method according to claim 32, wherein cytarabine is administered intravenously on days 1 to 5 of the first 28-day cycle and on days 1 to 5 of at least one additional 28-day cycle.

34. The dosage of the cytarabine is about 1 g / m ２ The method according to claim 33, wherein it is such.

35. The method according to claim 32, wherein mitoxantrone, etoposide, and cytarabine are administered intravenously on days 1 to 6 of the first 28-day cycle and on days 1 to 6 of at least one additional 28-day cycle.

36. The dosage of the mitoxantrone is about 6 mg / m ２ / day, the dosage of the etoposide is about 80 mg / m ２ / day, and the dosage of the cytarabine is about 1 g / m ２ / day, the method according to claim 35.

37. The method according to claim 30, wherein the multispecific protein is administered to the subject by IV infusion during a first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 15, and 12 μg of the multispecific protein is administered on day 22.

38. The method according to claim 37, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28-day cycle.

39. The method according to claim 38, wherein venetoclax is administered orally on days 1 to 21 of the first 28-day cycle and on days 1 to 21 of at least one additional 28-day cycle.

40. The method according to claim 39, wherein the dose of venetoclax is about 100 to about 400 mg / day.

41. The method according to claim 38, wherein azacitidine is administered intravenously on days 1 to 7 of the first 28-day cycle and at least one additional 28-day cycle.

42. The dosage of said azacitidine is about 75 mg / m ２ The method according to claim 41, wherein the dosage is as such.

43. The method according to claim 30, wherein the multispecific protein is administered to the subject by IV infusion during the first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

44. The method according to claim 43, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, and during the at least one additional 28-day cycle, 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22.

45. The method according to claim 44, wherein cytarabine is administered intravenously on days 1 to 7 of the first 28-day cycle and days 1 to 7 of at least one additional 28-day cycle.

46. The dosage of the cytarabine is about 100 to about 200 mg / m ２ The method according to claim 45, wherein the dosage is as described above.

47. The method according to claim 43, wherein idarubicin is administered by intravenous infusion on days 1 to 3 of the first 28-day cycle and days 1 to 3 of at least one additional 28-day cycle.

48. The dosage of said idarubicin is about 12 mg / m ２ The method according to claim 47, wherein it is such.

49. The method according to claim 30, wherein the multispecific protein is administered to the subject by IV infusion during the first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22.

50. The method according to claim 49, wherein the multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28-day cycle.

51. The method according to claim 50, wherein azacitidine is administered orally on days 1 to 14 of the first 28-day cycle and at least one additional 28-day cycle.

52. The method according to claim 51, wherein the dosage of said azacitidine is about 300 mg / day.

53. The method according to claim 1, wherein said cancer is carcinoma or sarcoma.

54. The method according to claim 1, wherein said cancer is melanoma, renal cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, blood cancer, 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).

55. The method according to claim 1, wherein said cancer is acute myeloid leukemia (AML).

56. The method according to claim 1, wherein said cancer is myelodysplastic syndrome (MDS).

57. A method for treating cancer, said method comprising administering to a subject in need thereof a multispecific protein comprising a CD123 binding domain and a CD3 binding domain.

58. Said multispecific protein comprises a dimer of two identical polypeptides, and each polypeptide, from amino terminus to carboxyl terminus or from carboxyl terminus to amino terminus, (i)a CD123 binding domain, (ii)a hinge region, (iii)an immunoglobulin constant region, and (iv)a CD3 binding domain The method according to claim 57.

59. The method according to claim 58, wherein said polypeptide comprises said CD123 binding domain, said hinge region, said immunoglobulin constant region, and said CD3 binding domain from N-terminus to C-terminus.

60. At least one of said CD123 binding domain and said CD3 binding domain is (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 method according to claim 58.

61. Said CD123 binding domain is a)HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, and HDCR3 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, b) 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, or c) a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27 The method according to claim 58, which is an scFv comprising the same.

62. The CD3 binding domain is a) an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO: 20, and an HDCR3 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, b) a VH comprising a sequence that is at least 90%, at least 95% or 100% identical to SEQ ID NO: 383 or 387, and a VL comprising a sequence that is at least 90%, at least 95% or 100% identical to SEQ ID NO: 384, or c) a sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 27 The method according to claim 58, which is an scFv comprising the same.

63. The method according to claim 58, wherein each polypeptide comprises a sequence that is at least 90%, at least 95% or 100% identical to SEQ ID NO:

31.

64. The method according to claim 57, wherein the multispecific protein is administered to the subject by IV injection.

65. The method according to claim 57, wherein the multispecific protein is administered to the subject by IV injection at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 μg / kg.

66. The method according to claim 57, wherein the multispecific protein is administered to the subject by IV injection at a dose of 0.3, 1, 3, 6, 9, 12, 18, 20, 24, 30, 36, 48, 50, 60, 75 or 100 μg.

67. The method according to claim 57, wherein the multispecific protein is administered once a week.

68. The multispecific protein is administered to the subject by IV injection during a first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 8, 12 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22. The multispecific protein is administered to the subject by IV infusion during the first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 15, and 12 μg of the multispecific protein is administered on day 22. The multispecific protein is administered to the subject by IV infusion during the first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22, or The multispecific protein is administered to the subject by IV infusion during the first 28-day cycle, and during the first 28-day cycle, 6 μg of the multispecific protein is administered on day 1, 8 μg of the multispecific protein is administered on day 12, 18 μg of the multispecific protein is administered on day 15, and 18 μg of the multispecific protein is administered on day 22. The method according to claim 67.

69. The multispecific protein is administered to the subject by IV infusion during at least one additional 28-day cycle following the first 28-day cycle, and 18 μg of the multispecific protein is administered on days 1, 8, 15, and 22 of the at least one additional 28-day cycle. The method according to claim 68.

70. The method according to claim 57, wherein the cancer is a carcinoma or a sarcoma.

71. The method according to claim 57, wherein the cancer is melanoma, renal cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, colon cancer, ovarian cancer, blood cancer, 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).

72. The method according to claim 57, wherein the cancer is acute myeloid leukemia (AML).

73. The method according to claim 57, wherein the cancer is myelodysplastic syndrome (MDS).

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