Method and composition for preventing the adsorption of therapeutic proteins to drug delivery system components.

Abstract

The present invention provides compositions and methods for reducing protein loss during drug delivery caused by protein adsorption onto one or more drug delivery system components. [Solution] In some embodiments, the present disclosure provides a composition for preventing the adsorption of proteins to one or more drug delivery system components, the composition comprising succinate and polysorbate 80. In some embodiments, the composition further comprises a therapeutic protein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 960,602, filed on January 13, 2020, which is hereby incorporated by reference in its entirety into this specification.

[0002] Field of the Disclosure This disclosure relates to the intravenous delivery of therapeutic proteins. More specifically, this disclosure relates to methods and compositions for preventing the adsorption of therapeutic proteins to one or more components of an intravenous drug delivery system. This disclosure also relates to methods of intravenous treatment of patients using therapeutic proteins.

[0003] Sequence Listing This application includes an electronically - filed sequence listing, which is hereby incorporated by reference in its entirety into this specification. The sequence listing was recorded on January 13, 2021, named APVO_060_01WO_SeqList_ST25.txt, and is approximately 301 kilobytes in size.

Background Art

[0004] Protein - based therapeutic agents have achieved great success in medical treatment. There are hundreds of therapeutic proteins approved for clinical use in the United States and Europe. Examples of approved therapeutic proteins include, for example, antibody - based drugs, Fc - fusion proteins, anticoagulants, blood factors, bone - forming proteins, modified scaffold proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

[0005] Many therapeutic proteins are administered via intravenous routes. When proteins are administered intravenously (IV), contact surfaces are of particular concern because proteins tend to adsorb to such surfaces due to their amphiphilic nature. With the widespread use of various plastic polymers for injectors, IV containers (e.g., IV bags), and tubing, the risk of protein loss due to adsorption is a practical problem, especially at low concentrations. Protein adsorption impairs the desired therapeutic effect, necessitates higher dosage levels, and increases treatment costs.

[0006] There remains in the art a need for improved compositions and methods for intravenous delivery of therapeutic proteins that reduce protein loss due to adsorption of one or more drug delivery system components. [Overview of the project]

[0007] This disclosure provides compositions that can be used to reduce or remove protein adsorption to one or more drug delivery system components. The compositions can be applied to the surface of one or more drug delivery system components before they come into contact with therapeutic proteins. The compositions described herein may also be referred to as IVSS (intravenous infusion solution stabilizer) compositions.

[0008] Provided herein are compositions for reducing the adsorption of therapeutic proteins to one or more intravenous drug delivery system components, the compositions comprising succinate and polysorbate 80. In some embodiments, the composition comprises about 1 to about 10 mM succinate and about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80. In some embodiments, the composition comprises about 4 mM to about 6 mM succinate, for example, about 5 mM succinate. In some embodiments, the composition comprises about 0.002 (w / v)% to about 0.008 (w / v)% polysorbate 80, for example, about 0.004 (w / v)% polysorbate 80. In some embodiments, the pH of the composition is about 5.0 to about 7.0, for example, about 6.0. In some embodiments, the composition comprises about 5 mM succinate in water and about 0.0004 (w / v)% polysorbate 80, the pH of the composition is about 6.0, and the composition is formulated for injection.

[0009] The compositions disclosed herein can be used with any therapeutic protein that, due to its size, charge, and / or other properties, has a tendency to adhere to plastic tubing and bags used for intravenous drug delivery. Accordingly, in some embodiments, the compositions include a therapeutic protein. The therapeutic protein may be a monospecific or multispecific binding protein. In some embodiments, the therapeutic protein forms a homodimer. In some embodiments, the therapeutic protein forms a heterodimer. In some embodiments, the therapeutic protein is scFv-Fc-scFv (e.g., ADAPTIR®), quadroma, Kλ body, dAb, diabody, TandAb, nanobody, DOCK-AND-LOCK® (DNL®), CrossMab Fab, CrossMab VH-VL, strand-exchange engineered domain body (SEEDbody), affibody, finomer, Knitz domain, Albu-dab, two engineered Fv fragments with exchanged VHs (e.g., dual-affinity re-targeting molecule (DART)), scFv x The format is selected from the group consisting of scFv (e.g., BiTE), SVD-IG, Covx-body, peptide-body, scFv-Ig, SVD-Ig, dAb-Ig, Knob-in-Holes, IgG1 antibody comprising matched mutation in the CH3 domain (e.g., duobody antibody), and triomabs.

[0010] In some embodiments, the therapeutic protein comprises at least a first binding domain, the first binding domain being a single-chain variable fragment (scFv), and the second binding domain being an scFv. In some embodiments, the therapeutic protein comprises at least a first binding domain and a second binding domain, the first binding domain being a single-chain variable fragment (scFv), and the second binding domain being an scFv. In some embodiments, the first binding domain specifically binds to tumor antigens, and the second binding domain specifically binds to CD3 (e.g., CD3ε). In some embodiments, the first binding domain specifically binds to CD3, and the second binding domain specifically binds to tumor antigens.

[0011] In some embodiments, the first binding domain specifically binds to the tumor antigen, and the second binding domain specifically binds to 4-1BB or OX40. In some embodiments, the first binding domain specifically binds to 4-1BB or OX40, and the second binding domain specifically binds to the tumor antigen. For example, in some embodiments, the binding domain specifically binds to 4-1BB, and the second binding domain specifically binds to the tumor antigen.

[0012] Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a 4-1BB binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a tumor antigen domain, or in order from the amino terminus to the carboxyl terminus, a tumor antigen binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a 4-1BB binding domain. In some embodiments, the binding domain specifically binds to OX40, and the second binding domain specifically binds to the tumor antigen.

[0013] Also provided is a composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, an OX40 binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a tumor antigen domain, or in order from the amino terminus to the carboxyl terminus, a tumor antigen binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and an OX40 binding domain.

[0014] In some embodiments, the first binding domain specifically binds to 4-1BB and the second binding domain specifically binds to OX40, or the first binding domain specifically binds to OX40 and the second binding domain specifically binds to 4-1BB. Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 / 0 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a 4-1BB binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and an OX40 binding domain, or in order from the amino terminus to the carboxyl terminus, an OX40 binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a 4-1BB binding domain.

[0015] In some embodiments, the first binding domain specifically binds to CD123, and / or the second binding domain specifically binds to CD3ε. In some embodiments, the therapeutic protein comprises, from the amino terminus to the carboxyl terminus, the first binding domain, a hinge region, an immunoglobulin constant region, and a second binding domain. In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the first binding domain comprises an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 includes SEQ ID NO: 10, HCDR2 includes SEQ ID NO: 11, and HCDR3 includes SEQ ID NO: 12. In some embodiments, LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15. In some embodiments, HCDR1 includes sequence number 10, HCDR2 includes sequence number 11, and HCDR3 includes sequence number 12; and LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15. In some embodiments, the first binding domain includes a sequence at least 95% identical to sequence number 18. In some embodiments, the second binding domain includes an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 includes sequence number 19, HCDR2 includes sequence number 20, and HCDR3 includes sequence number 21. In some embodiments, LCDR1 includes sequence number 22, LCDR2 includes sequence number 23, and LCDR3 includes sequence number 24. In some embodiments, HCDR1 includes sequence number 19, HCDR2 includes sequence number 20, HCDR3 includes sequence number 21, LCDR1 includes sequence number 22, LCDR2 includes sequence number 23, and LCDR3 includes sequence number 24.In some embodiments, the second binding domain contains a sequence that is at least 95% or 100% identical to sequence number 27. In some embodiments, the therapeutic protein contains the sequence of sequence number 31.

[0016] In some embodiments, the concentration of the therapeutic protein is about 0.01 μg / mL to about 2.0 μg / mL. In some embodiments, the concentration of the therapeutic protein is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, or about 0.09 μg / mL. In some embodiments, the concentration of the therapeutic protein is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 μg / mL. In some embodiments, the concentration of the therapeutic protein is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, or about 1.9, or about 2.0 μg / mL.

[0017] In some embodiments, the composition contains about 25 to about 150 mM succinate and about 0.01 (w / v)% to about 0.1 (w / v)% polysorbate 80. The composition may have a concentration of, for example, 10X to 50X. In some embodiments, the composition has a concentration of 20X. In some embodiments, the composition contains about 75 mM to about 125 mM succinate, for example, about 100 mM succinate. In some embodiments, the composition contains about 0.05 (w / v)% to about 0.1 (w / v)% polysorbate 80, for example, about 0.08 (w / v)% polysorbate 80. In some embodiments, the pH of the composition is about 5.0 to about 7.0, for example, about 6.0. In some embodiments, the composition comprises about 100 mM succinate in water and about 0.08 (w / v)% polysorbate 80, the pH of the composition is about 6.0, and the composition is formulated for injection.

[0018] Also provided herein are compositions for reducing the adsorption of therapeutic proteins to one or more intravenous drug delivery system components, the compositions comprising about 100 mM succinate, about 0.08 (w / v)% polysorbate 80, and a therapeutically effective amount of therapeutic protein.

[0019] Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a first binding domain that specifically binds to a first target, a hinge region, an immunoglobulin constant region, and a second binding domain that specifically binds to a second target. In some embodiments, the first target is CD86. In some embodiments, the first target is CD123. In some embodiments, the second target is the IL-10 receptor. In some embodiments, the second target is CD3ε. In some embodiments, the first target is CD86 and the second target is the IL-10 receptor. In some embodiments, the first target is CD123 and the second target is CD3ε.

[0020] Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a first binding domain, a hinge region, an immunoglobulin constant region, and a second binding domain, the first binding domain comprising an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region containing LCDR1, LCDR2, and LCDR3. The second binding domain includes an immunoglobulin heavy chain variable region (VL) comprising HCDR1, HCDR2, and HCDR3; HCDR1 includes SEQ ID NO: 10, HCDR2 includes SEQ ID NO: 11, and HCDR3 includes SEQ ID NO: 12; and LCDR1 includes SEQ ID NO: 13, LCDR2 includes SEQ ID NO: 14, and LCDR3 includes SEQ ID NO: 15; the second binding domain includes an immunoglobulin heavy chain variable region (VL) comprising HCDR1, HCDR2, and HCDR3; HCDR1 includes SEQ ID NO: 19, HCDR2 includes SEQ ID NO: 20, and HCDR3 includes SEQ ID NO: 21; and LCDR1 includes SEQ ID NO: 22, LCDR2 includes SEQ ID NO: 23, and LCDR3 includes SEQ ID NO: 24.

[0021] Also provided is a composition that reduces the adsorption of proteins to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2.0 μg / mL of a therapeutic protein, the therapeutic protein comprising the sequence of SEQ ID NO: 31.

[0022] Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, the CD86-binding domain comprising a variable heavy chain and a variable light chain that specifically bind to CD86, and the immunoglobulin Fc domain comprising two or more mutations that prevent or significantly reduce binding to the Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIb. The Fc domain, the monomeric IL-10 domain, contains two human IL-10 subunits separated by a short linker, and the therapeutic protein is homodimer.

[0023] Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, the CD86-binding domain being HCD The immunoglobulin heavy chain variable region (VH) comprises R1, HCDR2, and HCDR3, and the immunoglobulin light chain variable region (VL) comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO: 6, and the monomer IL-10 domain has the amino acid sequence of SEQ ID NO: 28.

[0024] Also provided is a composition for reducing protein adsorption to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, the CD86-binding domain comprising the amino acid sequence of SEQ ID NO: 9, and the monomer IL-10 domain comprising the amino acid sequence of SEQ ID NO: 28.

[0025] Also provided is a composition that reduces the adsorption of proteins to one or more intravenous drug delivery system components, the composition comprising about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2.0 μg / mL of a therapeutic protein, the therapeutic protein comprising the amino acid sequence of SEQ ID NO: 30.

[0026] The Disclosure also provides a container adapted for holding a therapeutic protein, wherein the inner surface of the container is first in contact with the composition of the Disclosure, and then in contact with the composition containing the therapeutic protein. In some embodiments, the container is substantially latex-free. In some embodiments, the container is substantially bis(2-ethylhexyl)phthalate (DEHP)-free. In some embodiments, the container is selected from the group consisting of IV bags, injectors, and tubing.

[0027] This disclosure also provides a method for constructing an intravenous drug delivery system for delivering a therapeutic protein, the method comprising: preparing at least one container adapted to hold the therapeutic protein; and contacting the inner surface of at least one container with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80 before the therapeutic protein is added to at least one container. In some embodiments, the composition coats the inner surface of at least one container to prevent the therapeutic protein from binding to the inner surface of the container. In some embodiments, at least one container is substantially latex-free. In some embodiments, at least one container is substantially bis(2-ethylhexyl) phthalate (DEHP)-free. In some embodiments, at least one container is selected from the group consisting of IV bags, injectors, and tubing.

[0028] This disclosure also provides a method for treating a subject by intravenous administration of a therapeutic protein, the method comprising: preparing at least one container adapted to hold the therapeutic protein; contacting the inner surface of the container with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80; contacting the inner surface of the container with the composition comprising the therapeutic protein; and intravenously administering the therapeutic protein to the subject. In some embodiments, the therapeutic protein comprises at least a first binding domain. In some embodiments, the first binding domain is a single-chain variable fragment (scFv). In some embodiments, the therapeutic protein comprises at least a first binding domain and a second binding domain. In some embodiments, the first binding domain is a single-chain variable fragment (scFv) and the second binding domain is an scFv. In some embodiments, the first binding domain specifically binds to CD123. In some embodiments, the second binding domain specifically binds to CD3ε. In some embodiments, the therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a first binding domain, a hinge region, an immunoglobulin constant region, and a second binding domain. In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the first binding domain comprises an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 includes SEQ ID NO: 10, HCDR2 includes SEQ ID NO: 11, and HCDR3 includes SEQ ID NO: 12. In some embodiments, LCDR1 includes SEQ ID NO: 13, LCDR2 includes SEQ ID NO: 14, and LCDR3 includes SEQ ID NO: 15. In some embodiments, HCDR1 includes sequence number 10, HCDR2 includes sequence number 11, HCDR3 includes sequence number 12, and LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15.

[0029] In some embodiments, the first binding domain includes a sequence that is at least 95% or 100% identical to SEQ ID NO: 18. In some embodiments, the second binding domain includes an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 includes SEQ ID NO: 19, HCDR2 includes SEQ ID NO: 20, and HCDR3 includes SEQ ID NO: 21. In some embodiments, LCDR1 includes SEQ ID NO: 22, LCDR2 includes SEQ ID NO: 23, and LCDR3 includes SEQ ID NO: 24. In some embodiments, HCDR1 includes SEQ ID NO: 19, HCDR2 includes SEQ ID NO: 20, and HCDR3 includes SEQ ID NO: 21; and LCDR1 includes SEQ ID NO: 22, LCDR2 includes SEQ ID NO: 23, and LCDR3 includes SEQ ID NO: 24. In some embodiments, the second binding domain includes a sequence that is at least 95% identical to SEQ ID NO: 27. In some embodiments, the therapeutic protein includes the sequence of SEQ ID NO: 31. In some embodiments, the therapeutic protein comprises, from amino-terminus to carboxyl-terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, wherein the CD86-binding domain comprises a variable heavy chain and a variable light chain that specifically bind to CD86, the immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIb, and the monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker, and the therapeutic protein is a homodimer. In some embodiments, the CD86-binding domain comprises an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3.In some embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO: 6. In some embodiments, the CD86-binding domain includes a variable heavy chain having an amino acid sequence that is at least 95% or 100% identical to SEQ ID NO: 7, and a variable light chain having an amino acid sequence that is at least 95% or 100% identical to SEQ ID NO: 8. In some embodiments, the CD86-binding domain includes an amino acid sequence that is at least about 95% or 100% identical to SEQ ID NO: 9. In some embodiments, the monomer IL-10 domain includes an amino acid sequence that is at least 95% or 100% identical to SEQ ID NO: 28. In some embodiments, the therapeutic protein includes the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to SEQ ID NO: 30. In some embodiments, the therapeutic protein is administered by intravenous infusion. In some embodiments, the composition coats the inner surface of at least one container to prevent the therapeutic protein from binding to the inner surface of the container. In some embodiments, the subject is a mammal such as a human.

[0030] Also provided is a drug delivery system for delivering a therapeutic protein to a patient, the system comprising at least one container adapted to hold the therapeutic protein, the inner surface of at least one container being in contact with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80, and then in contact with the composition containing the therapeutic protein.

[0031] These and other embodiments will be discussed in more detail in the following detailed description. [Brief explanation of the drawing]

[0032] [Figure 1A-D]The spectral scan profiles of IVSS solutions with wavelengths from 200 nm to 600 nm are shown. The spectral scan profiles were generated 3 days (Figure 1A), 41 days (Figure 1B), 77 days (Figure 1C), and 144 days (Figure 1D) after solution preparation. [Figure 2] This is a schematic diagram illustrating the structure of an exemplary therapeutic protein for use in the compositions and methods of the present invention. The therapeutic protein, referred to herein as Q0128, is a homodimer protein comprising two identical polypeptides linked by a disulfide bond. Each polypeptide comprises a CD86-binding domain, an Fc domain, and the monomer IL-10. [Figure 3A-B] This is a schematic diagram showing the structure of an exemplary therapeutic protein for use in the compositions and methods of the present invention. Figure 3A shows a homodimer protein containing two identical polypeptides, each containing a CD3-binding domain and an Fc domain, respectively. Figure 3B shows a homodimer protein containing two identical polypeptides, each containing a tumor-binding domain (e.g., a CD123-binding domain), an Fc domain, and a CD3-binding domain, respectively. An exemplary CD123 x CD3 bispecific therapeutic protein is referred to herein as TRI130. [Figure 4] This schematic diagram illustrates an exemplary protocol for using IVSS solution to coat the inner surface of an IV bag before placing a therapeutic protein into the IV bag for administration to a subject in need of treatment. [Modes for carrying out the invention]

[0033] This disclosure provides compositions and methods for reducing protein loss during drug delivery due to protein adsorption onto one or more drug delivery system components. This disclosure is based on the finding that protein adsorption onto a surface (e.g., the surface of a drug delivery system component) can be reduced or removed by contacting the surface with a composition comprising succinate and polysorbate 80 before drug administration. Accordingly, in some embodiments, this disclosure provides compositions for preventing protein adsorption onto one or more drug delivery system components, the compositions comprising succinate and polysorbate 80.

[0034] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter. However, all documents or parts of documents cited herein, including patent applications, articles, books, and commentaries, are thus explicitly, as appropriate, incorporated into this specification by reference. If one or more incorporated documents or parts of documents define a term that conflicts with the definitions provided in this application, the definitions provided in this application shall prevail. However, any references to references, articles, publications, patents, patent gazettes, and patent applications cited herein do not constitute, and should not be interpreted as, an endorsement or suggestion in any way that they constitute valid prior art or form part of common knowledge in any country of the world.

[0035] In this specification, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the indicated range and, where applicable, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified. Where used herein, the terms “a” and “an” should be understood to mean “one or more” of the enumerated components, unless otherwise indicated. The use of a disjunctive conjunction (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. Where used herein, the terms “include” and “comprise” are used synonymously. In addition, it should be understood that polypeptides containing various combinations of components (e.g., domains or regions) and substituents described herein are disclosed to the same extent as each polypeptide is described individually. Thus, the selection of specific components of individual polypeptides is included within the scope of this disclosure.

[0036] definition The term "approximately" preceding a numerical average means that the average is within ±10% of that range. For example, "approximately 40" means ±10% of 40 (i.e., 36 to 44), up to ±10%, up to ±9%, up to ±8%, up to ±7%, up to ±6%, up to ±5%, up to ±4%, up to ±3%, up to ±2%, up to ±1%, less than ±1%, or any other value or range.

[0037] In this specification, the terms "mcg" and "μg" are used interchangeably and refer to micrograms.

[0038] As used herein, “substantially” has its ordinary meaning as used in the art. For example, “substantially” can mean “significantly,” “considerably,” “largely,” “mostly,” or “essentially.” In some embodiments, “substantially” can mean at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0039] As used herein, the terms “binding domain” or “binding region” refer to a domain, region, portion, or site of a binding domain in a protein, polypeptide, oligopeptide, or peptide or antibody or antibody-derived binding domain that has the ability to specifically recognize and bind to a target molecule such as an antigen, ligand, receptor, substrate, or inhibitor. Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab), receptor extradomains, and ligands (e.g., cytokines, chemokines). In certain embodiments, the binding domain includes or comprises an antigen-binding site (e.g., three antibody-derived light chain complementarity-determining regions (CDRs) and three heavy chain CDRs located within a variable heavy chain sequence and a variable light chain sequence or alternating framework region (FR) (e.g., optionally, human FRs containing one or more amino acid substitutions)). Various assays are known for identifying binding domains of this disclosure that specifically bind to a particular target, including Western blotting, ELISA, phage display library screening, and Biacore® interaction analysis.

[0040] The binding domain or protein is 10 5 M -1 The above affinity or K a(That is, it "specifically binds" to the target if it binds to the target with an equilibrium binding constant in units of 1 / M of a specific binding interaction and does not significantly bind 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 has a K 7 M -1 of at least 10 8 M -1 of at least 10 9 M -1 of at least 10 10 M -1 of at least 10 11 M -1 of at least 10 12 M -1 of at least 10 13 M -1 or a binding domain having a K a of at least 10 7 M -1 A "low affinity" binding domain refers to a binding domain having a K 6 M -1 of at most 10 5 [[ID=四十]]M -1 of at most 10 a Alternatively, affinity can be defined as the equilibrium dissociation constant (K d ) in units of M of a specific binding interaction (e.g., 10 -5 M to 10 -13 M). The affinity of the binding domain polypeptides and single-chain polypeptides according to the present disclosure can be readily measured using conventional techniques (see, for example, Scatchard et al., (1949) Ann. N.Y. Acad. Sci. 51:660, and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalent documents).

[0041] As used herein, “conservative substitution” is clearly understood in the art as the substitution of one amino acid for another amino acid having similar properties. Exemplary conservative substitutions [like substitutions] are well known in the art (see, for example, International Publication No. 97 / 09433, p. 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8). In certain embodiments, a conservative substitution includes the substitution of leucine for serine.

[0042] 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 enzymes, such as glycosylation, alkylation, acylation, esterification, or amide formation.

[0043] 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 derived from a particular sequence (sometimes referred to as a "starting" or "parental" sequence) has an amino acid sequence that is essentially identical to the starting sequence or a portion thereof, the portion consisting of at least 10–20 amino acids, at least 20–30 amino acids, at least 30–50 amino acids, or at least 50–150 amino acids, or it can be otherwise identified by those skilled in the art as having its origin in the starting 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.

[0044] A polypeptide derived from another polypeptide may have one or more mutations compared to the starting polypeptide, such as substitution with another amino acid residue or insertion or deletion of one or more amino acid residues. The polypeptide may contain an amino acid sequence that is not of natural origin. Such changes inevitably result in less than 100% sequence identity or similarity with the starting polypeptide. In one embodiment, the variant has about 60% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide. In another embodiment, the variant has about 75% to less than 100%, about 80% to less than 100%, about 85% to less than 100%, about 90% to less than 100%, and about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.

[0045] Where used herein, unless otherwise specified, the positions of amino acid residues in the variable region of immunoglobulin molecules are numbered according to the IMGT numbering rules (Brochet, X, et al, Nucl. Acids Res. (2008) 36, W503-508), and the positions of amino acid residues in the constant region of immunoglobulin molecules are numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2: 77-94). Other numbering rules are known in the art (e.g., Kabat numbering rules (Kabat, Sequences of Proteins of Immunological Interest, 5th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991))).

[0046] As used herein, the term “dimer” refers to a biological entity consisting of two subunits linked to each other via 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), which is stable under appropriate conditions (e.g., physiological conditions, in aqueous solutions suitable for recombinant protein expression, purification, and / or storage, or under conditions for non-denaturation and / or non-reducing electrophoresis). As used herein, “heterodimer” or “heterodimer protein” refers to a dimer formed from two different polypeptides. A heterodimer does not contain an antibody formed from four polypeptides (i.e., two light chains and two heavy chains). As used herein, “homodimer” or “homodimer protein” refers to a dimer formed from two identical polypeptides. The full disclosure of a polypeptide, including its properties and activities (such as binding and RTCC), should be understood to include its dimer form as well as other multimer forms.

[0047] If the polypeptide of the present invention is dimerized (i.e., dimerized protein), it contains two binding sites at the amino terminus and two binding sites at the carboxyl terminus. The binding domain is therefore considered bivalent (i.e., two binding sites at each end) when a single-chain polypeptide is dimerized.

[0048] The “wild-type immunoglobulin hinge region” refers to naturally occurring upper and intermediate hinge amino acid sequences that are inserted between and link the CH1 and CH2 domains (for IgG, IgA, and IgD) or between and link the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, the wild-type immunoglobulin hinge region sequence is human and may include the human IgG hinge region.

[0049] A "modified wild-type immunoglobulin hinge region" or "modified immunoglobulin hinge region" is (a) a wild-type immunoglobulin hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) a range of approximately 5 amino acids (e.g., approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to approximately 120 amino acids (e.g., approximately 10- This refers to a portion of a wild-type immunoglobulin hinge region having an IgG core hinge region disclosed in U.S. Patent Applications Publication Nos. 2013 / 0129723 and 2013 / 0095097, having a length of approximately 40 amino acids or approximately 15 to approximately 30 amino acids or approximately 15 to approximately 20 amino acids or approximately 20 to approximately 25 amino acids, 20% , 15% , 10% , 5% , 4% , 3% , 2% or 1% amino acid substitutions or deletions or combinations thereof, and having a length of approximately 40 amino acids or approximately 15 to approximately 30 amino acids or approximately 15 to approximately 20 amino acids or 20 to approximately 25% or 20% , 15% , 10% , 5% , 4% , 3% , 2% or 1%.

[0050] As used herein, the term “humanization” refers to the process of making antibodies or immunoglobulin-binding proteins and polypeptides derived from non-human species (e.g., mouse or rat) less immunogenic to humans, while still retaining the original antibody-binding properties, using genetic engineering techniques. In some embodiments, the binding domains of the antibody or immunoglobulin-binding proteins and polypeptides (e.g., light chain and heavy chain variable regions, Fab, scFv) are humanized. The non-human binding domain is associated with "reshaping" (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 1991 9:266-271), "hyperchimerization" (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18") Antibodies can be humanized using techniques known as CDR grafting (Jones et al., Nature 321:522 (1986)) and its variations, including Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312). When derived from non-human sources, other regions of antibodies or immunoglobulin-binding proteins and polypeptides, such as hinge regions and constant region domains, can also be humanized.

[0051] As used herein, “immunoglobulin dimerizing domain” or “immunoglobulin heterodimerizing domain” refers to an immunoglobulin domain of a polypeptide chain that selectively interacts with or binds to a different immunoglobulin domain of a second polypeptide chain, and the interaction of different immunoglobulin heterodimerizing domains substantially contributes to or efficiently promotes the heterodimerization of the first and second polypeptide chains (i.e., the formation of a dimer between two different polypeptide chains, also called a “heterodimer”). The interaction between immunoglobulin heterodimerizing domains “substantially contributes to or efficiently promotes” the heterodimerization of the first and second polypeptide chains if there is a statistically significant reduction in dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimerizing domain of the first polypeptide chain and / or the immunoglobulin heterodimerizing domain of the second polypeptide chain. In certain embodiments, when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the first and second polypeptide chains form heterodimers with each other. A typical immunoglobulin heterodimer domain includes an immunoglobulin CH1 domain, an immunoglobulin CL domain (e.g., Cκ or Cλ isotype), or a derivative thereof, including wild-type immunoglobulin CH1 and CL domains and modified (or mutant) immunoglobulin CH1 and CL domains provided therein.

[0052] "Immunoglobulin constant region" or "constant region" is a term defined herein to refer to a peptide or polypeptide sequence that corresponds to or is derived from some or all of one or more constant region domains. In certain embodiments, the immunoglobulin constant region corresponds to or is derived from some or all of one or more constant region domains of the original antibody, but not all of them. In certain embodiments, the constant region includes IgG CH2 and CH3 domains, e.g., IgG1 CH2 and CH3 domains. In certain embodiments, the constant region does not include a CH1 domain. In certain embodiments, the constant region domains constituting the constant region are human. In some embodiments (for example, in certain modifications of a CD123-binding polypeptide or protein including a second binding domain that specifically binds to CD3 or another T cell surface antigen), the constant domain of the fusion protein of the Disclosure lacks or has minimal effector function for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation and complement-dependent cell-mediated cytotoxicity (CDC), but retains the ability to bind to several Fc receptors (such as FcRn, neonatal Fc receptor) and retains a relatively long in vivo half-life. In other modifications, the fusion protein of the Disclosure includes a constant domain that retains one or both of such effector functions for ADCC and CDC. In certain embodiments, the binding domain of the present disclosure is fused to the human IgG1 constant region, the IgG1 constant region having one or more of the following mutated amino acids: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamic acid at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (as numbered by the EU). For example, one or more of these amino acids can be replaced with alanine.In further embodiments, the IgG1 Fc domain has one of the L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to alanine (i.e., L234A, L235A, G237A, E318A, K320A, and K322A, respectively), and optionally also has the N297A mutation (i.e., essentially removing glycosylation of the CH2 domain). In another embodiment, the IgG1 Fc domain has the L234A, L235A, G237A, and K322A mutations, respectively.

[0053] The "Fc region" or "Fc domain" refers to a polypeptide sequence that corresponds to or is derived from the original antibody and is involved in binding to antibody receptors and the C1q component of complement on cells. Fc stands for "crystalline fragment," meaning a fragment of an antibody that readily forms protein crystals. Originally described by proteolytic digestion, separate protein fragments can define the overall structure of an immunoglobulin protein. As first defined in the literature, an Fc fragment consists of a disulfide bond heavy chain hinge region, CH2, and CH3 domains. More recently, however, the term has been applied to single chains consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide bond dimer with a second such chain. For an overview of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31: 169-217, 1994. As used herein, the term Fc includes variants of the naturally occurring sequence.

[0054] The terms patient and subject are used synonymously. As used herein, the terms “patient in need” or “subject in need” refer to a subject at risk of, or suffering from, a disease, disorder, or condition that is suitable for treatment or improvement with the therapeutic protein or composition provided herein. A subject in need may be, for example, a patient diagnosed with a CD123 expression-related disease such as acute myeloid leukemia (AML), B lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), pilocytic cell leukemia (HCL), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), refractory anemia with supervast blasts (RAEB), chronic myeloid leukemia, and Hodgkin lymphoma.

[0055] As used herein, the term “pharmaceutically acceptable” means molecular entities and compositions that, when administered by routes known in the art, do not typically cause allergic or other serious adverse reactions. Molecular entities and compositions that are approved by federal or state regulatory authorities or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals and more specifically in humans are considered “pharmaceutically acceptable.”

[0056] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides, and their polymers in single-stranded or double-stranded forms. Unless otherwise specified, the terms encompass nucleic acids, including known analogues of native nucleotides, which have similar binding properties to the reference nucleic acid and are metabolized similarly to native nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitution can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (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 genes, the cDNA encoded by genes, and mRNA. As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs, and their derivatives, fragments, and homologs.

[0057] The term "expression" refers to the biosynthesis of products encoded by nucleic acids. 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 the mRNA into one or more polypeptides.

[0058] The terms “expression unit” and “expression cassette” are used synonymously herein and refer to a nucleic acid segment encoding a polypeptide of interest that can provide expression of the nucleic acid segment in a host cell. An expression unit typically includes a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all of which are functional components. In addition to the transcription promoter and terminator, an expression unit may further include other nucleic acid segments, such as enhancers or polyadenylation signals.

[0059] As used herein, the term “expression vector” refers to a linear or cyclic nucleic acid molecule containing one or more expression units. In addition to one or more expression units, an expression vector may also contain additional nucleic acid segments, such as one or more origins of replication or one or more selectable markers. An expression vector is typically derived from plasmid or viral DNA, or may contain elements of both.

[0060] As used herein, the term “sequence identity” refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. A sequence is said to be “identical” at a position if that position in the sequence is occupied by the same nucleic acid base or amino acid residue as the corresponding position in the comparison sequence. The percentage of “sequence identity” is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues occur, thereby obtaining the number of “identical” positions. The number of “identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of “sequence identity.” The percentage of “sequence identity” is determined by comparing two best-aligned sequences across the comparison window. The comparison window for nucleic acid sequences may be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleic acid lengths, or greater. The comparison window for polypeptide sequences may be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 300 amino acid lengths, or greater. To optimally align sequences for comparison, portions of the polynucleotide or polypeptide sequences within the comparison window may contain deletions called additions or gaps, while simultaneously maintaining the reference sequence constant. Optimal alignment is the alignment that, even with gaps, produces the maximum possible number of "identical" positions between the reference and comparison sequences.The percentage of sequence identity between two sequences can be determined using the program version "BLAST 2 Sequences," which, as of September 1, 2004, is available from the National Center for Biotechnology Information. This program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), based on the algorithm of Karlin and Altschul (Proc.Natl.Acad.Sci.USA 90(12):5873-5877,1993). When using "BLAST 2 Sequences," the default parameters as of September 1, 2004, were word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff value (50), expected value (10), and any other necessary parameters, including matrix options, are available, although they are not limited to these. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantially similar sequence identity" if the two sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one another.

[0061] "CD3" is known in the art as a six-chain polyprotein complex (see, e.g., Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999), and is a subunit of the T cell receptor complex. In mammals, the CD3 subunit of the T cell receptor complex is a homodimer of the CD3γ, CD3δ, two CD3ε, and CD3ζ chains. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which gives them the property of enabling them to bind to the positively charged T cell receptor chain. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif or ITAM known as the immune receptor tyrosine activation motif, while each CD3ζ chain has three ITAMs. ITAMs are thought to be important for the signaling ability of the TCR complex. The CD3 used in this disclosure may be derived from various animal species, including humans, monkeys, mice, rats, or other mammals.

[0062] The term "CD123" may refer to any isoform of CD123, also known as surface antigen classification 123, interleukin-3 receptor alpha chain, and IL3RA. CD123 binds to the β chain of the interleukin-3 receptor to form the receptor. CD123 is a type I transmembrane glycoprotein and has an extracellular domain containing a predictive immunoglobulin-like domain and two FnIII domains. The CD123-binding domain of this disclosure binds to the extracellular domain of CD123.

[0063] 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 its beta chain (CD131). IL-3 binds to CD123, and signaling is provided by CD131. IL-3 regulates the function and production of hematopoietic and immune cells and stimulates endothelial cell proliferation (Testa et al., Biomark Res. 2:4 (2014)).

[0064] CD123 is overexpressed in many hematological malignancies, including acute myeloid leukemia (AML), B lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms (BPDCN), and subsets of pilocytic cell leukemia. While most AML patients respond well to initial treatment, the majority of AML patients are eventually diagnosed with relapsed or refractory disease (Ramos et al., J. Clin. Med. 4:665-695 (2015)). There is a need for CD123-targeted molecules that have enhanced efficiency and potency and low adverse effects, and that can be used to treat disorders associated with CD123 dysregulation.

[0065] CD86 is known in the art as a surface molecule belonging to the B7 receptor subfamily and functioning as a T cell costimulator (Lu et al. 1997; Vicenti et al. 2008). It is typically expressed on antigen-presenting cells (APCs) such as dendritic cells, monocytes, and activated, non-quiescent B cells (Lu et al. 1997; Vicenti et al. 2008). It is highly expressed in naive human monocytes and DCs, and its expression is further upregulated under certain activation conditions (Hathcock et al. 1994; Sansom et al. 2003). CD86 expression on naive monocytes is estimated to range from 2,000 to 5,000 copies per cell (Wolk et al. 2007). High levels of CD86 expression are associated with inflammatory tissues in certain pathological conditions (Vuckovic et al. 2001; Nakazawa et al. 1999), and CD86 and CD80 (the latter being the second member of the B7 family) promote T cell activation through interaction with the T cell coreceptor CD28.

[0066] The "CD86" binding domain specifically binds to CD86. In some embodiments, the CD86 binding domain binds to an epitope located in the extracellular domain of CD86 (e.g., human CD86). In certain embodiments, this epitope is discontinuous and / or structurally distinct. In some embodiments, the CD86 binding domain binds to CD86 but not to CD80. In some embodiments, the CD86 binding domain binds to human CD86. In some embodiments, the CD86 binding domain binds to non-human primate CD86. In some embodiments, the CD86 binding domain binds to human CD86 and also cross-reacts with cynomolgus monkey CD86. In some embodiments, the CD86 binding domain binds to cynomolgus monkey monocytes and lineage-negative populations (DCs). In some embodiments, the CD86 binding domain is humanized.

[0067] A “protein” is a macromolecule comprising one or more polypeptide chains. Proteins may also contain non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents can be added to proteins by the cell in which they are produced, and vary depending on the cell type. Proteins are defined herein by their amino acid backbone structure; substituents such as carbohydrate groups are usually not specified but may nevertheless be present. The terms “protein,” “polypeptide,” “therapeutic protein,” and “therapeutic polypeptide” are used synonymously herein.

[0068] The therapeutic protein may be an antibody or an antigen-binding fragment of an antibody. In some embodiments, the therapeutic protein may also be an scFv-Fc-scFv molecule, a bispecific T cell engager (scFv-scFv) molecule, or a biaffinity retargeting molecule. In some embodiments, the therapeutic 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 polyvalent formats that can be used for therapeutic proteins include, for example, scFv-Fc-scFv (e.g., ADAPTIR®), quadroma, Kλ body, dAb, diabody, TandAb, nanobody, Small Modular ImmunoPharmaceutials (SMIP®), DOCK-AND-LOCK® (DNL®), CrossMab Fab, CrossMab VH-VL, strand-exchange engineered domain body (SEEDbody), aphibody, finomer, Knitz domain, Albu-dab, two engineered Fv fragments with exchanged VHs (e.g., dual-affinity re-targeting molecule (DART)), scFv x This includes scFv (e.g., BiTE), SVD-IG, Covx-body, peptide-body, scFv-Ig, SVD-Ig, dAb-Ig, Knob-in-Hole, IgG1 antibody comprising matched mutation in the CH3 domain (e.g., duobody antibody), and triomabs. An exemplary bispecific format is discussed in Garber et al., Nature Reviews Drug Discovery 13:799-801 (2014).This document is incorporated herein by reference in its entirety. Additional exemplary bispecific formats are discussed in Liu et al. Front.Immunol.8:38 doi:10.2289 / fimmu.2017.00038, and in Brinkmann and Kontermann, MABS 9:2,182-212(2017). Each of these is incorporated herein by reference in its entirety. In certain embodiments, the bispecific antibody may be an F(ab')2 fragment. The F(ab')2 fragment contains two antigen-binding arms of a tetramer antibody molecule linked by a disulfide bond at a hinge region.

[0069] The terms “amino terminus” and “carboxyl terminus” are used herein to indicate locations within a polypeptide. Where context permits, these terms are used relative to a specific sequence or portion of a polypeptide to indicate approximation or relative position. For example, a particular sequence located at the carboxyl terminus relative to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but not necessarily at the carboxyl terminus of the entire polypeptide.

[0070] As used herein, the terms “treatment,” “treating,” and “ameliorating” refer to therapeutic or prophylactic / preventive treatments. A treatment is therapeutic if it improves at least one symptom of a disease in the individual receiving the treatment, or if it can slow the progression of a progressive disease in the individual, or prevent the onset of additional related diseases.

[0071] As used herein, the terms “therapeutic effective dose” or “effective dose” of a specific binding molecule or compound refer to an amount of the compound sufficient to produce a statistically significant recovery of one or more symptoms of a disease being treated, or in terms of a statistically significant improvement in organ function. When referring to an individual active ingredient administered alone, the therapeutic effective dose refers to that ingredient alone. When referring to a combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, whether administered sequentially or simultaneously (in the same formulation or simultaneously in different formulations).

[0072] The term "light chain variable domain" ("light chain variable domain", "VL", or "V"). L Also called "heavy chain variable region" ("heavy chain variable domain", "VH", or "V") and "heavy chain variable region" ("heavy chain variable domain", "VH", or "V") H The terms "CL" and "CH" refer to the variable binding regions derived from the antibody light and heavy chains, respectively. The variable binding regions typically consist of separate, distinct subregions known as the "complementarity-determining regions" (CDRs) and the "framework regions" (FRs), which are typically located from the amino terminus to the carboxyl terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. 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 derived from the 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" (antigen-binding fragment) is a part of an antibody that binds to an antigen and includes a variable region of the heavy chain and a CH1 domain attached to the light chain via interchain disulfide bonds.

[0073] As used herein, “container adapted to hold therapeutic proteins” refers to any clinically acceptable container suitable for holding and / or transporting therapeutic proteins. Non-limiting examples of such containers include, for example, IV bags, injectors, and tubing / piping. In some embodiments, the container is substantially free of latex and / or bis(2-ethylhexyl) phthalate (DEHP).

[0074] "Intravenous drug delivery system" may refer to any clinically acceptable system used to prepare (e.g., dilute, mix, etc.) and / or deliver a drug intravenously to a subject or patient. Such systems may include, for example, IV bags, injectors, tubing / piping, pumps, needles, etc.

[0075] Composition for preventing protein adsorption Therapeutic proteins are often administered intravenously using drug delivery systems. For example, a sterile solution containing a protein therapy drug is provided in an IV bag or other container and injected / infused into the patient's body via a tube attached to a needle inserted into the patient's vein. Thus, during the administration of a therapeutic protein, the protein comes into contact with one or more surfaces of the drug delivery system, such as the inner surface of the IV bag or tube. Therapeutic proteins are known to adsorb to such surfaces, for example, when charged amino acids on the protein surface interact with the surface. The tendency of a protein to remain attached to a surface depends largely on material properties such as surface energy, structure, and relative charge distribution. Larger proteins are more likely to adsorb and remain attached to a surface due to a greater number of contact sites between the amino acids and the surface.

[0076] Protein adsorption can be a major concern during the administration of therapeutic proteins to patients. For example, adsorption of therapeutic proteins to the surface of drug delivery systems can reduce the amount of protein delivered to the patient. Protein adsorption can be particularly problematic during the administration of protein-based therapeutics at low doses and / or low concentrations (i.e., ≤10 mcg / mL).

[0077] This disclosure provides compositions that can be used to reduce or remove protein adsorption to one or more drug delivery system components. The compositions may be brought into contact with the surface of one or more drug delivery system components before administration of a therapeutic protein. In some embodiments, the compositions coat the inner surface of at least one component of the drug delivery system to prevent the therapeutic protein from binding to the inner surface of that component.

[0078] The composition for preventing protein adsorption may comprise a buffer and a surfactant. In some embodiments, the composition may further comprise a therapeutic protein. The pH of the composition may range from about 5.0 to about 7.0, for example, about 5.0, about 5.25, about 5.5, about 5.75, about 6.0, about 6.25, about 6.5, about 6.75, or about 7.0.

[0079] In some embodiments, the composition comprises about 1 to about 10 mM buffer and about 0.001 (w / v)% to about 0.01 (w / v)% of surfactant. In some embodiments, the composition comprises about 4 mM to about 6 mM buffer, for example, about 5 mM buffer.

[0080] In further embodiments, the composition comprises about 25 mM to about 150 mM of buffer. In some embodiments, the composition comprises about 75 mM to about 125 mM of buffer, for example, about 100 mM of buffer.

[0081] In some embodiments, the composition contains about 0.002 (w / v)% to about 0.008 (w / v)% of surfactant. In some embodiments, the composition contains about 0.004 (w / v)% of surfactant.

[0082] In some embodiments, the composition contains about 0.05% to about 0.1% of surfactant. For example, the composition may contain about 0.05% to about 0.1% of surfactant. In some embodiments, the composition contains about 0.08% of surfactant.

[0083] In some embodiments, the buffer is a succinate buffer. In some embodiments, the surfactant may be polysorbate 80. In further embodiments, the buffer is succinate and the surfactant may be polysorbate 80. Succinate is a salt or ester of succinic acid. Polysorbate 80 is a nonionic surfactant and emulsifier.

[0084] In some embodiments, a composition for reducing the adsorption of therapeutic proteins to one or more intravenous drug delivery system components comprises succinate and polysorbate 80. In some embodiments, the composition comprises about 25 mM to about 150 mM succinate. In some embodiments, the composition comprises about 75 mM to about 125 mM succinate, for example, about 100 mM succinate. In some embodiments, the composition comprises about 0.002 (w / v)% to about 0.008 (w / v)% polysorbate 80. In some embodiments, the composition comprises about 0.004 (w / v)% polysorbate 80. In some embodiments, the composition comprises about 0.05 (w / v)% to about 0.1 (w / v)% polysorbate 80. For example, the composition may comprise about 0.05 (w / v)% to about 0.1 (w / v)% polysorbate 80. In some embodiments, the composition contains about 0.08 (w / v)% of polysorbate 80.

[0085] In some embodiments, the composition contains about 4 mM to about 6 mM succinate, for example, about 5 mM succinate. In some embodiments, the composition contains about 0.002 (w / v)% to about 0.008 (w / v)% polysorbate 80, for example, about 0.004 (w / v)% polysorbate 80. In some embodiments, the composition contains about 1 to about 10 mM succinate and about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80. In some embodiments, the composition contains about 5 mM succinate in water and about 0.0004 (w / v)% polysorbate 80, the pH of the composition is about 6.0, and the composition is formulated for injection.

[0086] In some embodiments, the composition may further contain a therapeutic protein. The concentration of the therapeutic protein may range from about 0.01 μg / mL to about 2.0 μg / mL. In some embodiments, the concentration of the therapeutic protein is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, or about 0.09 μg / mL. In some embodiments, the concentration of the therapeutic protein is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 μg / mL. In some embodiments, the concentration of the therapeutic protein is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, or about 1.9, or about 2.0 μg / mL.

[0087] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 100 mM succinate, about 0.08 (w / v)% polysorbate 80, and a therapeutically effective amount of therapeutic protein.

[0088] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a first binding domain that specifically binds to a first target, a hinge region, an immunoglobulin constant region, and a second binding domain that specifically binds to a second target. In some embodiments, the first target is CD86. In some embodiments, the first target is CD123. In some embodiments, the second target is the IL-10 receptor. In some embodiments, the second target is CD3ε. In some embodiments, the first target is CD86 and the second target is the IL-10 receptor. In some embodiments, the first target is CD123 and the second target is CD3ε.

[0089] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, wherein the therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a first binding domain, a hinge region, an immunoglobulin constant region, and a second binding domain, wherein the first binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3. The second binding domain includes (i) an immunoglobulin heavy chain variable region including HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3; HCDR1 includes an immunoglobulin heavy chain variable region including HCDR1, HCDR2, and HCDR3; HCDR1 includes an immunoglobulin heavy chain variable region including HCDR1, HCDR2, and HCDR3; HCDR1 includes an immunoglobulin heavy chain variable region including HCDR1, HCDR2, and HCDR3; and LCDR1 includes an immunoglobulin heavy chain variable region including HCDR1, LCDR2, and LCDR3; HCDR1 includes an immunoglobulin heavy chain variable region including HCDR1, HCDR2, and HCDR3 includes an immunoglobulin heavy chain variable region including HCDR1, LCDR2, and LCDR

[0090] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2.0 μg / mL of a therapeutic protein, wherein the therapeutic protein comprises the sequence of SEQ ID NO: 31.

[0091] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL therapeutic protein, wherein the therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, wherein the CD86-binding domain comprises a variable heavy chain and a variable light chain that specifically bind to CD86, the immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIb, and the monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker, and the therapeutic protein is a homodimer.

[0092] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, wherein the CD86-binding domain is HCDR1, It comprises an immunoglobulin heavy chain variable region (VH) containing HCDR2 and HCDR3, and an immunoglobulin light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO: 6, and the monomer IL-10 domain has the amino acid sequence of SEQ ID NO: 28.

[0093] In some embodiments, a composition for reducing protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2 μg / mL of a therapeutic protein, the therapeutic protein comprising, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, the CD86-binding domain comprising the amino acid sequence of SEQ ID NO: 9, and the monomer IL-10 domain comprising the amino acid sequence of SEQ ID NO: 28.

[0094] In some embodiments, a composition that reduces protein adsorption to one or more intravenous drug delivery system components comprises about 1 to about 10 mM succinate, about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80, and about 0.01 μg / mL to about 2.0 μg / mL of a therapeutic protein, the therapeutic protein comprising the amino acid sequence of SEQ ID NO: 30.

[0095] In some embodiments, the composition may be provided at concentrations greater than 1X. For example, the composition may be at concentrations of 10X to 50X. In some embodiments, the composition may be at concentrations of 2X, 5X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, or 50X. In some embodiments, the composition is at a concentration of 20X. Where used in this context, "X" indicates that the solution is in a concentrated form that typically needs to be diluted to a concentration of 1X for use. For example, a 5X concentrate needs to be diluted 5-fold, while a 100X concentrate needs to be diluted 100-fold. Dilution may be carried out, for example, with water or saline solution.

[0096] In some embodiments, the composition comprises about 25 to about 150 mM succinate and about 0.01 (w / v)% to about 0.1 (w / v)% polysorbate 80. In some embodiments, the composition comprises about 75 mM to about 125 mM succinate, for example, about 100 mM succinate. In some embodiments, the composition comprises about 0.05 (w / v)% to about 0.1 (w / v)% polysorbate 80, for example, about 0.08 (w / v)% polysorbate 80. In some embodiments, the pH of the composition is about 5.0 to about 7.0, for example, about 6.0. In some embodiments, the composition comprises about 100 mM succinate in water and about 0.08 (w / v)% polysorbate 80, the pH of the composition is about 6.0, and the composition is formulated for injection.

[0097] In some embodiments, a 20X IVSS solution is provided, the approximately 20X solution containing approximately 25 mM to approximately 150 mM succinate and approximately 0.01 (w / v)% to approximately 0.1 (w / v)% polysorbate 80. In some embodiments, the 20X IVSS solution contains approximately 100 mM succinate and approximately 0.08 (w / v)% polysorbate 80. In some embodiments, the 20X IVSS solution contains approximately 100 mM succinate and approximately 0.08 (w / v)% polysorbate 80 in water, the pH of the composition is approximately 6.0, and the composition is formulated for injection.

[0098] In some embodiments, the 20X IVSS solution is diluted to a 1X concentration. In some embodiments, the 1X IVSS solution contains about 1 to about 10 mM succinate and about 0.001 (w / v)% to about 0.01 (w / v)% polysorbate 80. In some embodiments, the 1X IVSS solution contains about 5 mM succinate and about 0.004 (w / v)% polysorbate 80. In some embodiments, the 1X IVSS solution contains about 5 mM succinate and about 0.004 (w / v)% polysorbate 80 in water, the pH of the composition is about 6.0, and the composition is formulated for infusion. In some embodiments, the 1X IVSS solution further contains a therapeutic protein, such as an anti-CD123 x anti-CD3 bispecific binding protein or an anti-CD8 x monomer IL-10 binding protein.

[0099] In some embodiments, a 1X IVSS solution (with or without therapeutic protein) is used to coat drug delivery system components adapted for the delivery of at least one therapeutic protein prior to the delivery of the therapeutic protein.

[0100] In some embodiments, the composition may further include one or more additional components, such as pharmaceutically acceptable carriers or excipients.

[0101] Therapeutic proteins The compositions and methods described herein can be used in connection with the preparation, storage, and / or administration of many different types of therapeutic proteins to prevent their adsorption to one or more surfaces. Therapeutic proteins may be, for example, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, modified scaffold proteins, enzymes, growth factors, hormones, cytokines, interferons, interleukins, and thrombolytic agents. In some embodiments, the therapeutic protein is a ligand for a target receptor.

[0102] Joint domain In some embodiments, the therapeutic protein comprises at least one 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 a fusion protein in any 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 include, for example, a specific cytokine or molecule that directs the binding domain polypeptide to, for example, a particular cell type, a toxin, an additional cell receptor, or an antibody.

[0103] In some embodiments, the binding domain described herein is antibody-derived and variable heavy chain (V H ) and variable light chain (V L ) includes. For example, a single-chain variable fragment (scFv) is V H and V L The chain includes (i) an immunoglobulin heavy chain variable region (V) containing HCDR1, HCDR2, and HCDR3. H ); and (ii) immunoglobulin light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) includes.

[0104] In some embodiments, the polypeptides and proteins described herein include a binding domain which is an scFv. In such embodiments, the binding domain may also be referred to as the scFv domain. In some embodiments, the binding domain is a V specific to the target of interest. H and V L It is a single-chain Fv fragment (scFv) containing a region. In certain embodiments, V H and V L The region is human or humanized. In some modifications, the binding domain is linked by a peptide linker. L and V HThis is a single-chain Fv (scFv) containing a region.

[0105] In certain embodiments, the polypeptide binding domain described herein is (i) an immunoglobulin light chain variable region (V) comprising CDR LCDR1, LCDR2, and LCDR3. L ), and (ii) immunoglobulin heavy chain variable region (V) including CDR HCDR1, HCDR2, and HCDR3 H ) includes. In some embodiments, the amino acid sequences provided for the polypeptide construct do not include human immunoglobulin reader sequences. The indicated CDR sequences and amino acid substitution sites are defined using the IMGT criteria (Brochet et al, Nucl. Acids Res. (2008) 36, W503-508).

[0106] In certain embodiments, the combined domain V of the Disclosure L and / or V H The region is parent V L and / or V H V of the region L and / or V H It is derived from (for example, 1618 / 1619 described in International Publication No. 2016 / 185016) and is a known monoclonal antibody V L and / or V H Compared to the sequence, this may include, at will, one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or any combination of the above changes. The insertions, deletions, or substitutions include the amino or carboxyl terminus or both ends of this region, provided that each CDR contains zero changes or at most one, two, or three changes. L and / or V HThey can be located anywhere within the region. In some embodiments, the binding domains containing the modified VL and / or VH regions can still specifically bind to their target with an affinity similar to, or higher than, that of the parent binding domain.

[0107] V L and V H The use of peptide linkers for region linking is well known in the art, and numerous papers exist in this particular field. In some embodiments, the peptide linker is 15-mer and consists of three repeats of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 128) ((Gly4Ser)3) (SEQ ID NO: 59). Other linkers have been used, and phage display technology and selective infective phage technology have been used to diversify linker sequences and select appropriate linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., Protein Eng. 11, 405-410, 1998). In certain embodiments, V L and V H The region is given by the formula (Gly4Ser) n The linkers are connected by a peptide linker having an amino acid sequence including (wherein n=1~5)(SEQ ID NO: 129). For example, in one embodiment of the present invention, the linker includes (Gly4Ser)4(SEQ ID NO: 61). Other suitable linkers can be obtained by optimizing a simple linker by random mutagenesis. In some embodiments, the V of scFv described herein H The region may be located at the N-terminus of the linker sequence. In some embodiments, the V of the scFv described herein L The region may be located at the C-terminus of the linker sequence.

[0108] Hinge In addition to the binding domain, the therapeutic polypeptide may further include 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 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 additionally have prolines substituted with another amino acid (e.g., serine or alanine). For example, the modified human IgG1 hinge described above may additionally have prolines substituted with another amino acid residue located at the carboxyl terminus for 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 a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a wild-type immunoglobulin hinge region such as the wild-type human IgG1 hinge, the wild-type human IgG2 hinge, or the wild-type human IgG4 hinge.

[0109] Immunoglobulin constant domain Therapeutic proteins may also include an immunoglobulin constant (Fc) domain (hereinafter also referred to herein as the constant region, Fc domain, Fc region, etc.). In certain embodiments, the constant region includes IgG CH2 and CH3 domains, e.g., IgG1 CH2 and CH3 domains. In certain embodiments, the constant region does not include a CH1 domain. In certain embodiments, the constant domains constituting the constant region are human or derived from human sequences. In some embodiments, the Fc domain includes mutations at positions 234, 235, 237, and 322. In some embodiments, the Fc domain includes mutations at positions 234, 235, 237, 318, 320, and 322. In some embodiments, the Fc domain includes mutations L234A, L235A, G237A, and K322A. In some embodiments, the Fc domain includes mutations L234A, L235A, G237A, E318A, K320A, and K322A. In some embodiments, the Fc domain is derived from IgG1. In some embodiments, the IgG1-derived Fc domain contains two or more mutations that, when administered to a patient, prevent the polypeptide from depleting CD86 and / or IL-10R-expressing cells. In some embodiments, two or more mutations in the IgG1 Fc domain prevent or substantially reduce Fc-mediated crosslinking.

[0110] In some embodiments, the immunoglobulin constant region comprises one of the amino acid sequences of SEQ ID NOs. 32-35, or a variant thereof. Inclusion of the immunoglobulin constant region delays the elimination of the polypeptides and proteins of the present invention from circulation after administration to a subject. Mutations or other alterations allow for further relatively easy modulation of polypeptide effector functions (e.g., ADCC, ADCP, CDC, complement binding, and binding to Fc receptors), which may be increased or decreased depending on the disease being treated, as known in the art and as described herein. In certain embodiments, the polypeptides and proteins described herein include 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 the polypeptide or protein described herein compared to the corresponding wild-type immunoglobulin constant region.

[0111] The constant regions present in the polypeptides and proteins of this disclosure may include, or be derived from, some or all of, a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof. For example, an immunoglobulin constant region may include 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 parts of a CH2 domain and a CH3 domain. In certain embodiments, the polypeptides or proteins described herein do not contain a CH1 domain.

[0112] The polypeptides or proteins described herein may comprise wild-type immunoglobulin CH2 domains or modified immunoglobulin CH2 domains derived from a specific immunoglobulin class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD) and from various species (including humans, mice, rats, and other mammals). In certain embodiments, the CH2 domain of the polypeptides or proteins described herein is a wild-type immunoglobulin CH2 domain, such as the wild-type CH2 domains of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, as shown in Sequence ID No. 115, 199-201 and 195-197 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In certain embodiments, the CH2 domain is the wild-type human IgG1 CH2 domain represented by Sequence ID No. 115 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequence is incorporated herein by reference).

[0113] In certain embodiments, the modified CH2 region in the polypeptide or protein of the Disclosure includes, 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%, or 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., IGGH2c).

[0114] The modified immunoglobulin CH2 regions in the polypeptides or proteins of this disclosure can be derived from various immunoglobulin isotypes such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, and from CH2 regions of various species (including human, mouse, rat, and other mammals). In certain embodiments, the modified immunoglobulin CH2 regions in the fusion proteins of this disclosure can be derived from the CH2 regions of human IgG1, IgG2, or IgG4, or mouse IgG2a (e.g., IGGH2c), the sequences of which are shown in Sequence IDs 115, 199, 201, and 320 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In certain embodiments, the modified CH2 domains of polypeptides or proteins described herein are modified human IgG1 CH2 domains having mutations known in the art that enhance or reduce immunological activity (i.e., effector function) such as ADCC, ADCP, CDC, complement binding, Fc receptor binding, or any combination thereof.

[0115] In certain embodiments, the CH2 domain of the polypeptide or protein described herein is a modified immunoglobulin CH2 region (e.g., a modified human IgG1 CH2 domain) comprising one or more amino acid deletions or substitutions. In some embodiments, the CH2 domain comprises an asparagine amino acid substitution at position 297 (e.g., an asparagine to alanine substitution). Such an amino acid substitution reduces or eliminates glycosylation at this position, inhibiting efficient Fc binding to FcγR and C1q. The sequence of a modified human IgG1 CH2 domain having an Asn to Ala substitution at position 297 is shown in Sequence ID No. 324 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequence is incorporated herein by reference). In some embodiments, the modified CH2 domain comprises at least one substitution or deletion at positions 234–238. For example, the immunoglobulin CH2 region may include substitutions at positions 234, 235, 236, 237 or 238; positions 234 and 235; positions 234 and 236; positions 234 and 237; positions 234 and 238; positions 234-236; positions 234, 235 and 237; positions 234, 236 and 238; positions 234, 235, 237, and 238; substitutions at positions 236-238; or substitutions of any other combination of 2, 3, 4, or 5 amino acids at positions 234-238. In some embodiments, the modified CH2 region includes one or more (e.g., two, three, four, or five) amino acid deletions at positions 234-238, for example, at one of positions 236 or 237, while other positions are substituted. In certain embodiments, one or more amino acid residues at positions 234–238 are substituted with one or more alanine residues. In further embodiments, only one amino acid residue at positions 234–238 is deleted, and at the same time, one or more of the remaining amino acids at positions 234–238 may be substituted with another amino acid (e.g., alanine or serine).

[0116] In some embodiments, the above mutations reduce or eliminate the ADCC activity or Fc receptor binding ability of polypeptides containing the modified CH2 domain.

[0117] In certain embodiments, the CH2 domain of the polypeptide or protein described herein is a modified immunoglobulin CH2 domain (e.g., a modified human IgG1 CH2 domain) comprising one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331. For example, the immunoglobulin CH2 domain may comprise substitutions at positions 253, 310, 318, 320, 322, or 331, positions 318 and 320, positions 318 and 322, or any other combination of two, three, four, five, or six amino acids at positions 253, 310, 318, 320, 322, or 331. In such embodiments, the mutations reduce or eliminate the CDC activity of the polypeptide comprising the modified CH2 domain.

[0118] In certain embodiments, in addition to the amino acid substitution at position 297, the modified CH2 region of the polypeptide or protein described herein (e.g., the modified human IgG1 CH2 domain) may further include one or more (e.g., two, three, four, or five) additional substitutions at positions 234-238. For example, the immunoglobulin CH2 region may include substitutions at positions 234 and 297, positions 234, 235, and 297, positions 234, 236, and 297, positions 234-236, and 297, positions 234, 235, 237, and 297, positions 234, 236, 238, and 297, positions 236-238, and 297, or substitutions of any other combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297. In addition, or instead, the modified CH2 region may contain one or more (e.g., two, three, four, or five) amino acid deletions at positions 234–238, such as position 236 or position 237. Additional mutations reduce or eliminate the ADCC activity or Fc receptor binding ability of the polypeptide containing the modified CH2 domain. In certain embodiments, one or more amino acid residues at positions 234–238 are substituted with one or more alanine residues. In further embodiments, only one amino acid residue at positions 234–238 is deleted, while one or more of the remaining amino acids at positions 234–238 are substituted with another amino acid (e.g., alanine or serine).

[0119] In certain embodiments, in addition to one or more (e.g., two, three, four, or five) amino acid substitutions at positions 234–238, the mutant CH2 region of the polypeptide or protein described herein in the fusion protein of this disclosure (e.g., the modified human IgG1 CH2 domain) may include one or more (e.g., two, three, four, five, or six) additional amino acid substitutions (e.g., substitutions with alanine) at one or more positions (e.g., positions I253, H310, E318, K320, K322, or P331) involved in complement binding. Examples of mutant immunoglobulin CH2 regions include the human IgG1, IgG2, IgG4, and mouse IgG2a CH2 regions having alanine substitutions at positions 234, 235, 237 (if present), 318, 320, and 322. Exemplary mutant immunoglobulin CH2 regions are the mouse IGGH2c CH2 regions with alanine substitutions at L234, L235, G237, E318, K320, and K322.

[0120] In further embodiments, in addition to the amino acid substitution at position 297 and the additional deletion or substitution at positions 234-238, the modified CH2 region of the polypeptide or protein described herein (e.g., modified human IgG1 CH2 domain) may further include one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331. For example, an immunoglobulin CH2 region may include (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., two, three, four, five, or six) amino acid substitutions at positions I253, H310, E318, K320, K322, and P331, for example, one, two, or three substitutions at positions E318, K320, and K322. The amino acid at the above position may be substituted with alanine or serine.

[0121] In certain embodiments, the substituted CH2 region of the polypeptide or protein described herein includes (i) an amino acid substitution of asparagine at position 297 and one amino acid substitution at positions 234, 235, 236 or 237; (ii) an amino acid substitution of asparagine at position 297 and two amino acid substitutions at positions 234-237; (iii) an amino acid substitution of asparagine at position 297 and three amino acid substitutions at positions 234-237; (iv) an amino acid substitution of asparagine at position 297, amino acid substitutions at positions 234, 235 and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and 322.

[0122] Exemplary modified immunoglobulin CH2 regions having an asparagine amino acid substitution at position 297 include: a human IgG1 CH2 region having alanine substitutions at L234, L235, G237, and N297 and a deletion at G236 (Sequence ID 325 of U.S. Patent Application Publication 2013 / 0129723, the above sequence is incorporated herein by reference); a human IgG2 CH2 region having alanine substitutions at V234, G236, and N297 (Sequence ID 326 of U.S. Patent Application Publication 2013 / 0129723, the above sequence is incorporated herein by reference); and a human IgG4 CH2 region having alanine substitutions at F234, L235, G237, and N297 and a deletion at G236. CH2 region (Sequence ID 322 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference), human IgG4 CH2 region having alanine substitutions at F234 and N297 (Sequence ID 343 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference), human IgG4 CH2 region having alanine substitutions at L235 and N297 (Sequence ID 344 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference), human IgG4 CH2 region having alanine substitutions at G236 and N297 (Sequence ID 345 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference), and human IgG4 having alanine substitutions at G237 and N297 The CH2 region includes (Sequence ID 346 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference). These CH2 regions can be used in the polypeptides of this disclosure.

[0123] In certain embodiments, in addition to the above-described amino acid substitutions, the modified CH2 region of the polypeptide or protein described herein (e.g., the modified human IgG1 CH2 domain) may include one or more additional amino acid substitutions at one or more positions other than those described above. Such amino acid substitutions may be conserved or non-conserved. For example, in certain embodiments, in the modified IgG2 CH2 region, P233 may be changed to E233 (see, for example, Sequence ID No. 326 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference). In addition, or instead, in certain embodiments, the modified CH2 region may include one or more amino acid insertions, deletions, or both. Insertions, deletions, or substitutions may be located anywhere in the immunoglobulin CH2 region, such as at the N or C-terminal position of the wild-type immunoglobulin CH2 region obtained from a hinge-linking between the CH2 region and another region (e.g., a binding domain or an immunoglobulin heterodimerization domain).

[0124] In certain embodiments, the modified CH2 domain of the 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) (Sequence ID 595 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference), and optionally having an N297 mutation (e.g., a mutation to alanine). In certain embodiments, the modified CH2 domain is a human IgG1 CH2 domain having alanine substitutions at positions 234, 235, 237, 318, 320, and 322 (i.e., a human IgG1 CH2 domain having L234A, L235A, G237A, E318A, K320A, and K322A substitutions) (Sequence ID 596 of U.S. Patent Application Publication No. 2013 / 0129723, the above sequence is incorporated herein by reference), and optionally having an N297 mutation (e.g., a mutation to alanine).

[0125] In some embodiments, the immunoglobulin constant region of the polypeptide or protein described herein comprises a human IgG1 CH2 domain including substitutions L234A, L235A, G237A, and K322A according to the EU numbering system.

[0126] CH3 domains capable of forming the immunoglobulin constant region of polypeptides or proteins described herein may be wild-type immunoglobulin CH3 domains or modified immunoglobulin CH3 domains derived from specific immunoglobulin classes or subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM) of various species (including humans, mice, rats, and other mammals). In certain embodiments, the CH3 domains of polypeptides described herein are wild-type human immunoglobulin CH3 domains, such as the wild-type CH3 domains of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, IgE, or IgM shown in Sequence ID No. 116, 208-210, 204-207, and 212 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In certain embodiments, the CH3 domain is the wild-type human IgG1 CH3 domain represented by Sequence ID No. 116 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequence is incorporated herein by reference).

[0127] In certain embodiments, the CH3 domain of the polypeptide described herein is a modified human immunoglobulin CH3 domain, such as a modified CH3 domain based on or derived from the wild-type CH3 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, IgE, or IgM. For example, the modified CH3 domain may be a human IgG1 CH3 domain having one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering). Mutations at such positions can be involved in complement binding. In certain other embodiments, the modified CH3 domain of the polypeptide described herein may be a human IgG1 CH3 domain except having one or two amino acid substitutions at position F405 or Y407. Amino acids at such positions are involved in interaction with another CH3 domain. In certain other embodiments, the modified CH3 domain of the polypeptide described herein may be a modified human IgG1 CH3 domain in which its last lysine is deleted. The sequence of this modified CH3 domain is shown in Sequence ID No. 761 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequence is incorporated herein by reference).

[0128] In certain embodiments, the polypeptides or proteins described herein include CH3 domains containing so-called "knobs-into-holes" mutations (see Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al., Protein Engineering 9:617-21, 1966). More specifically, mutations can be introduced into each of the CH3 domains of each polypeptide chain so that these two CH3 domains pair with each other for the steric complementarity required for CH3 / CH3 bonding. For example, a CH3 domain in one single-chain polypeptide of a polypeptide heterodimer may contain a T366W mutation ("knob" mutation, which substitutes a smaller amino acid for a larger amino acid), and a CH3 domain in the other single-chain polypeptide of the polypeptide heterodimer may contain a Y407A mutation ("hole" mutation, which substitutes a larger amino acid for a smaller amino acid). Other exemplary knob-into-hole mutations include (1) the T366Y mutation in one CH3 domain and the Y407T mutation in another CH3 domain, and (2) the T366W mutation in one CH3 domain and the T366S, L368A, and Y407V mutations in other CH3 domains.

[0129] The CH4 domains capable of forming the immunoglobulin constant region of the polypeptides or proteins described herein may be wild-type immunoglobulin CH4 domains or modified immunoglobulin CH4 domains derived from IgE or IgM molecules. In certain embodiments, the CH4 domain of the polypeptides described herein is a wild-type human immunoglobulin CH4 domain, such as the wild-type human IgE and IgM CH4 domains shown in Sequence ID Nos. 213 and 214 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In certain embodiments, the CH4 domain of the polypeptides described herein is a modified human immunoglobulin CH4 domain, such as a modified CH4 domain based on or derived from a human IgE or IgM CH4 domain, which has mutations that increase or decrease immunological activity known to bind to the IgE or IgM Fc region.

[0130] In certain embodiments, the immunoglobulin constant region of the 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 may comprise CH2 and CH3 domains or CH3 and CH4 domains. In certain other embodiments, the immunoglobulin constant region may comprise two CH3 domains and omit any CH2 or CH4 domains (i.e., only two or more CH3s). The multiple constant region domains forming the immunoglobulin constant region of the polypeptide described herein may be based on or derived from the same immunoglobulin molecule, or from an immunoglobulin molecule of the same class or subclass. In certain embodiments, the immunoglobulin constant region is IgG CH2-CH3 (e.g., IgG1 CH2-CH3, IgG2 CH2-CH3, and IgG4 CH2-CH3) and may be human (e.g., human IgG1, IgG2, and IgG4) CH2CH3. For example, in certain embodiments, the immunoglobulin constant region of the polypeptide described herein includes (1) wild-type human IgG1 CH2 and CH3 domains, (2) human IgG1 CH2 with the N297A mutation (i.e., CH2(N297A)) and wild-type human IgG1 CH3, or (3) human IgG1 CH2(N297A) and modified human IgG1 CH3 with a deletion of the last lysine. Alternatively, the multiple constant region domains of the polypeptide or protein described herein may 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 includes both the human IgM CH3 domain and the human IgG1 CH3 domain. The multiple constant region domains forming the immunoglobulin constant region of the polypeptide described herein may be linked together directly or linked to each other via one or more (e.g., about 2 to 10) amino acids.

[0131] Exemplary immunoglobulin constant regions usable in polypeptides or proteins described herein are shown in SEQ ID NOs: 305-309, 321, 323, 341, 342, and 762 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). Further exemplary immunoglobulin constant regions usable in polypeptides or proteins described herein are provided in the table below. [Table 1]

[0132] In certain embodiments, the immunoglobulin constant regions of each polypeptide chain of the homodimer or heterodimer protein described herein are identical to each other. In certain other embodiments, the immunoglobulin constant region of one polypeptide chain of the heterodimer is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer. For example, one immunoglobulin constant region of the heterodimer protein may contain a CH3 domain with a “knob” mutation, while the other immunoglobulin constant region of the heterodimer protein may contain a CH3 domain with a “hole” mutation.

[0133] Fc-joint domain linker In some embodiments, the polypeptide may further include an Fc-binding domain linker that ligates a binding domain (e.g., an scFv domain). In some embodiments, the Fc-binding domain linker is a Gly4Ser linker (SEQ ID NO: 128). In some embodiments, the Fc-binding domain linker is a 20-mer and consists of four repeats ((Gly4Ser)4) (SEQ ID NO: 61) of the Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 128) amino acid sequence. In some embodiments, the Fc-binding domain linker includes an amino acid sequence selected from any one of SEQ ID NOs: 50-70. Other linkers have been used, and phage display technology and selective infective phage technology have been used to diversify linker sequences and select appropriate linker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., Protein Eng. 11, 405-410, 1998). In certain embodiments, V L and V H The region is given by the formula (Gly4Ser) n The linkers are connected by a peptide linker having an amino acid sequence containing (n=1~5 in the formula) (SEQ ID NO: 129). Other suitable linkers can be obtained by optimizing a simple linker by random mutagenesis. In some embodiments, the bispecific molecule does not contain a hinge region or a constant region.

[0134] In certain embodiments, the Fc-binding domain linker is a flexible linker sequence containing a glycine-serine (e.g., Gly4Ser, SEQ ID NO: 128) repeat. In certain embodiments, the linker contains three Gly4Ser repeats (SEQ ID NO: 61) 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 contains or consists of a sequence selected from SEQ ID NOs: 50-70.

[0135] Tables 2 and 3 below show some exemplary hinge and Fc-binding domain linker sequences suitable for use in this disclosure. Additional exemplary hinge and linker regions are shown in Sequence IDs 241–244, 601, 78, 763–791, 228, 379–434, and 618–749 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). [Table 2] JPEG2026062861000003.jpg67158 [Table 3] JPEG2026062861000005.jpg68158

[0136] In addition to the domains described above, therapeutic polypeptides may further include immunoglobulin dimerization / heterodimization domains, conjugation amino acids, tags, additional binding domains, and the like. In some embodiments, the polypeptides and proteins described herein are conjugated to a drug or toxic moiety.

[0137] Bispecific / Multispecific proteins In some embodiments, the therapeutic protein may be a bispecific or multispecific protein. Non-limiting examples of bispecific molecules include the scFv-Fc-scFv molecule, the scFv-Ig molecule, and the scFv-scFv molecule. In some embodiments, the bispecific molecules described herein include, or consist of, a first binding domain scFv bound 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 order from the amino terminus to the carboxyl terminus, or from the carboxyl terminus to the amino terminus, (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.

[0138] Homodimer / Heterodimer In some embodiments, the therapeutic protein may be a homodimer or a heterodimer. In some embodiments, the therapeutic protein is a dimer of two identical polypeptides, each polypeptide comprising, in order from the amino terminus to the carboxyl terminus or from the carboxyl terminus to the amino terminus, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) (optionally) an Fc-binding domain linker, and (v) a second binding domain. In some embodiments, the bispecific or multiplespecific protein is a dimer of two identical polypeptides, each polypeptide comprising, in order from the amino terminus to the carboxyl terminus or from the carboxyl terminus to the amino terminus, (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, the bispecific protein described herein is a diabody.

[0139] In certain embodiments, the hinge present in a polypeptide that forms a heterodimer with another polypeptide chain may be an immunoglobulin hinge, such as a wild-type immunoglobulin hinge region or a modified immunoglobulin hinge region. In certain other embodiments, the hinge of one polypeptide chain of the heterodimer protein is identical to the corresponding hinge of the other polypeptide chain of the heterodimer. In certain other embodiments, the hinge of one chain is different from the hinge of the other chain (in terms of their length or sequence). Different hinges in different chains allow for manipulation of various binding affinities of the binding domains to which the hinges are linked, thereby enabling the heterodimer to selectively bind to the target of one binding domain rather than the target of the other binding domain.

[0140] In other embodiments, the polypeptides and proteins described herein include heterodimerizing domains that can heterodimerize with different heterodimerizing domains in a second non-identical polypeptide chain. In certain modifications, the second polypeptide chain for heterodimerization includes a second binding domain. Thus, in certain embodiments of this disclosure, two non-identical polypeptide chains, one containing a first binding domain and the second optionally containing a second binding domain, dimerize to form a heterodimer-binding protein. Dimerizing / heterodimerizing domains can be used when it is desirable that one or both polypeptide chains contain binding domains and to form a heterodimer from two non-identical polypeptide chains. In certain embodiments, one polypeptide chain member of a particular heterodimer described herein does not contain a binding domain. Examples of heterodimer types include those described in U.S. Patent Application Publications 2013 / 0095097 and 2013 / 0129723, and International Publication 2016 / 094873.

[0141] In certain embodiments, the first and second polypeptide chains are dimerized via the inclusion of an “immunoglobulin dimerizing domain” or “immunoglobulin heterodimerizing domain.” Herein, “immunoglobulin dimerizing domain” or “immunoglobulin heterodimerizing domain” refers to an immunoglobulin domain of the first polypeptide chain that selectively interacts with or binds to a different immunoglobulin domain of the second polypeptide chain, the interaction of which substantially contributes to or efficiently promotes the heterodimerization of the first and second polypeptide chains (i.e., the formation of a dimer between two different polypeptide chains, also referred to as a “heterodimer”). The immunoglobulin heterodimerizing domains in the polypeptide chains of the heterodimer are distinct from each other and can therefore be differentially modified to promote heterodimerization of both chains and minimize homodimerization of either chain. The immunoglobulin heterodimerizing domains provided herein enable efficient heterodimerization between different polypeptides and facilitate the purification of the resulting heterodimer protein.

[0142] As provided herein, immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different polypeptide chains by 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 the human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domains shown, for example, in Sequence ID No. 114, 186-192, and 194 of U.S. Patent Application Publication No. 2013 / 0129723, or Sequence ID No. 114 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In further embodiments, the cysteine ​​residues of the wild-type CH1 domain (e.g., human CH1) involved in the formation of a disulfide bond with the wild-type immunoglobulin CL domain (e.g., human CL) are deleted or substituted in the modified immunoglobulin CH1 domain, thereby preventing the formation of a disulfide bond between the modified CH1 domain and the wild-type CL domain.

[0143] In certain embodiments, the immunoglobulin heterodimer domain is a wild-type CL domain, such as a wild-type Cκ domain or a wild-type Cλ domain, as shown in, for example, SEQ ID NOs. 112 and 113 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In further embodiments, the immunoglobulin heterodimer domain is a modified immunoglobulin CL domain, such as a modified Cκ or Cλ domain, for example, a modified human Cκ or human Cλ domain. In certain embodiments, a cysteine ​​residue of the wild-type CL domain involved in the formation of a disulfide bond with the wild-type immunoglobulin CH1 domain is deleted or substituted in the modified immunoglobulin CL domain, for example, Cκ as shown in SEQ ID NOs. 141 of U.S. Patent Application Publication No. 2013 / 0129723 or Cλ as shown in SEQ ID NOs. 140 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequences are incorporated herein by reference). In certain embodiments, only the last cysteine ​​of the wild-type human Cκ domain is deleted in the modified Cκ domain. This is because the first arginine deleted from the wild-type human Cκ domain can be provided by a linker that has arginine at its carboxyl terminus and connects the amino terminus of the modified Cκ domain to another domain (e.g., an immunoglobulin subregion such as a subregion containing immunoglobulin CH2 and CH3 domains).

[0144] In further embodiments, the immunoglobulin heterodimer domain is a modified Cκ domain that, compared to a wild-type Cκ domain, includes one or more amino acid substitutions at positions that can be involved in the formation of an interchain hydrogen bond network at the Cκ-Cκ boundary. For example, in certain embodiments, the immunoglobulin heterodimer domain is a modified human Cκ domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68, or T70, which are substituted with different amino acids. The amino acid numbering is based on their positions in the modified human Cκ shown in Sequence ID No. 141 of U.S. Patent Application Publication No. 2013 / 0129723 (the above sequence is incorporated herein by reference). In certain embodiments, the immunoglobulin heterodimer domain is a modified human Cκ domain having one, two, three, or four amino acid substitutions at positions N29, N30, V55, or T70. The amino acids used as substituents at the above positions may be alanine, or amino acid residues having a bulk side chain such as arginine, tryptophan, tyrosine, glutamic acid, glutamine, lysine, aspartic acid, methionine, serine, or phenylalanine. The modified human Cκ domain promotes heterodimerization with the CH1 domain but minimizes homodimerization with other Cκ domains.Representative modified human Cκ domains are sequence numbers 142-178 in U.S. Patent Application Publication 2013 / 0129723; sequence numbers 160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A) in U.S. Patent Application Publication 2013 / 0129723. The sequences are shown as T70E), 178(N30Y V55A T70E), 838(N30D V55A T70E), 839(N30M V55A T70E), 840(N30S V55A T70E), and 841(N30F V55A T70E) (the above sequences are incorporated herein by reference).

[0145] In certain embodiments, in addition to or instead of the mutations in the Cκ domain described herein, both immunoglobulin heterodimer domains of the polypeptide heterodimer (i.e., immunoglobulin CH1 and CL domains) have mutations such that the resulting immunoglobulin heterodimer domains form salt bridges (i.e., ionic interactions) between amino acid residues at the mutation sites. For example, the immunoglobulin heterodimer domain of the polypeptide heterodimer may be a mutant CH1 domain combined with a mutant Cκ domain. In the mutant CH1 domain, valine (V68) at position 68 of the wild-type human CH1 domain is replaced by a negatively charged amino acid residue (e.g., aspartic acid or glutamic acid), while leucine (L29) at position 29 of the mutant human Cκ domain, where the first arginine and last cysteine ​​are deleted, is replaced by a positively charged amino acid residue (e.g., lysine, arginine, or histidine). The charge-charge interaction between the negatively charged amino acid residue of the resulting mutant CH1 domain and the positively charged amino acid residue of the resulting mutant Cκ domain forms a salt bridge, which stabilizes the heterodimer boundary between the mutant CH1 and the Cκ domain. Alternatively, V68 of wild-type CH1 can be substituted with a positively charged amino acid residue, while L29 of the mutant human Cκ domain, in which the first arginine and last cysteine ​​are deleted, can be substituted with a negatively charged amino acid residue. Exemplary mutant CH1 sequences in which V68 is substituted with a negatively or positively charged amino acid are shown in SEQ ID NOs. 844 and 845 of U.S. Patent Application Publication 2013 / 0129723 (the above sequences are incorporated herein by reference). Exemplary mutant Cκ sequences in which L29 is substituted with a negatively or positively charged amino acid are shown in SEQ ID NOs. 842 and 843 of U.S. Patent Application Publication 2013 / 0129723 (the above sequences are incorporated herein by reference).

[0146] Positions other than V68 in the human CH1 domain and L29 in the human Cκ domain can be substituted with amino acids with opposite charges to generate amino acid-amino acid ionic interactions in addition to, or instead of, mutations at V68 in the CH1 domain and L29 in the Cκ domain. Such positions can be identified by any preferred method, including random mutagenesis, analysis of the crystal structure of CH1-Cκ pairs to identify amino acid residues at the CH1-Cκ boundary, and further preferred positions among amino acid residues at the CH1-Cκ boundary using a set of criteria (e.g., tendency to participate in ionic interactions, degree of similarity to potential partner residues, etc.).

[0147] In certain embodiments, the polypeptide heterodimers of the Disclosure contain only one pair of immunoglobulin heterodimerizing domains. For example, the first chain of the polypeptide heterodimer may contain a CH1 domain as the immunoglobulin heterodimerizing domain, while the second chain may contain a CL domain (e.g., Cκ or Cλ) as the immunoglobulin heterodimerizing domain. Alternatively, the first chain may contain a CL domain (e.g., Cκ or Cλ) as the immunoglobulin heterodimerizing domain, while the second chain may contain a domain domain as the immunoglobulin heterodimerizing domain. As shown herein, the immunoglobulin heterodimerizing domains of the first and second chains can be bound to form the heterodimer protein of the Disclosure.

[0148] In certain embodiments, the heterodimer protein of this disclosure may have two pairs of immunoglobulin heterodimerization domains. For example, the first chain of the heterodimer may contain two CH1 domains, while the second chain may have two CL domains that bind to the two CH1 domains in the first chain. Alternatively, the first chain may contain two CL domains, while the second chain may have two CH1 domains that bind to the two CL domains in the first chain. In certain embodiments, the first polypeptide chain contains a CH1 domain and a CL domain, while the second polypeptide chain contains a CL domain and a CH1 domain that bind to the CH1 domain and CL of the first polypeptide chain, respectively.

[0149] In some embodiments, where the heterodimer protein contains only one heterodimerization pair (i.e., one immunoglobulin heterodimerization domain in each chain), the immunoglobulin heterodimerization domain of each chain may be located at the amino terminus relative to the immunoglobulin constant region of that chain. Alternatively, the immunoglobulin heterodimerization domain of each chain may be located at the carboxyl terminus relative to the immunoglobulin constant region of that chain.

[0150] In some embodiments, the heterodimer protein contains two heterodimerization pairs (i.e., two immunoglobulin heterodimerization domains in each chain), both immunoglobulin heterodimerization domains in each chain may be located at the amino terminus relative to the immunoglobulin constant region of that chain. Alternatively, both immunoglobulin heterodimerization domains in each chain may be located at the carboxyl terminus relative to the immunoglobulin constant region of that chain. In further embodiments, one immunoglobulin heterodimerization domain in each chain may be located at the amino terminus relative to the immunoglobulin constant region of that chain, while the other immunoglobulin heterodimerization domain in each chain may be located at the carboxyl terminus relative to the immunoglobulin constant region of that chain. In other words, in these embodiments, the immunoglobulin constant region is inserted between the two immunoglobulin heterodimerization domains in each chain.

[0151] The polypeptides and proteins described herein may be prepared using scaffolds as commonly disclosed in U.S. Patent Publications 2013 / 0129723 and 2013 / 0095097, which are incorporated herein by reference in their entirety. The polypeptides described herein may comprise two non-identical polypeptide chains, each containing an immunoglobulin heterodimerization domain. The interacting immunoglobulin heterodimerization domains are different. In one embodiment, the immunoglobulin heterodimerization domain comprises a CH1 domain or a derivative thereof. In another embodiment, the immunoglobulin heterodimerization domain comprises a CL domain or a derivative thereof. In one embodiment, the CL domain is a Cκ or Cλ isotype or a derivative thereof.

[0152] Exemplary therapeutic proteins: anti-CD86 x mono-IL-10 polypeptide and its dimer In some embodiments, the therapeutic protein contained in the composition described herein may be an IL-10 delivery polypeptide comprising a CD86-binding domain and a monomer IL-10 domain. In some embodiments, the therapeutic protein may be an IL-10 delivery polypeptide comprising a CD86-binding domain, an immunoglobulin Fc domain, and a monomer IL-10 domain. In some embodiments, the protein therapeutic may be an IL-10 delivery polypeptide comprising a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, (optionally) an Fc-binding domain linker, and a monomer IL-10 domain.

[0153] Accordingly, in some embodiments, the IL-10 delivery polypeptide may comprise, or consist of, a CD86-binding domain and a monomeric IL-10 domain. The IL-10 delivery polypeptide of this disclosure may be described as a fusion protein. Also provided are dimers of such IL-10 delivery polypeptides, e.g., homodimers and heterodimers.

[0154] The CD86 surface molecule belongs to the B7 receptor subfamily and functions as a T cell costimulator (Lu et al. 1997; Vicenti et al. 2008). It is typically expressed on antigen-presenting cells (APCs) such as dendritic cells, monocytes, and activated but non-quiescent B cells (Lu et al. 1997; Vicenti et al. 2008). It is highly expressed by naive human monocytes and DCs, and its expression is further upregulated under certain activation conditions (Hathcock et al. 1994; Sansom et al. 2003). CD86 expression on naive monocytes is estimated to range from 2,000 to 5,000 copies per cell (Wolk et al. 2007). High levels of CD86 expression are associated with inflammatory tissues in certain pathological conditions (Vuckovic et al. 2001; Nakazawa et al. 1999), and CD86 and CD80 (the latter being the second member of the B7 family) promote T cell activation through interaction with the T cell coreceptor CD28.

[0155] The CD86-binding domain specifically binds to CD86. In some embodiments, the CD86-binding domain binds to an epitope located in the extracellular domain of CD86 (e.g., human CD86). In certain embodiments, this epitope is discontinuous and / or structurally distinct. In some embodiments, the CD86-binding domain binds to CD86 but not to CD80. In some embodiments, the CD86-binding domain binds to human CD86. In some embodiments, the CD86-binding domain binds to non-human primate CD86. In some embodiments, the CD86-binding domain binds to human CD86 and also cross-reacts with cynomolgus monkey CD86. In some embodiments, the CD86-binding domain binds to cynomolgus monkey monocytes and lineage-negative populations (DCs). In some embodiments, the CD86-binding domain is humanized.

[0156] In some cases, the CD86-binding domain of an IL-10 delivery polypeptide may be a humanized CD86-binding domain derived from a FUN-1 antibody (see, e.g., Nozawa et al., J. Pathol. 1993; 169(3): 309-315). For example, the CD86-binding domain polypeptide may (i) include the immunoglobulin heavy chain variable region (V) containing HCDR1, HCDR2, and HCDR3. H ); and (2) immunoglobulin light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) may include. In some embodiments, at least one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 is derived from the FUN1 antibody. In some embodiments, HCDR1 includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, HCDR2 includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, HCDR3 includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, LCDR1 includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, LCDR2 includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, LCDR3 includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, HCDR1, HCDR2, and HCDR3 each include SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, LCDR1, LCDR2, and LCDR3 each include SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO: 6.

[0157] In certain embodiments, the CD86 binding domain is the light chain variable region (V) of sequence number 8. LThe amino acid sequence of ) contains a sequence that is at least about 85%, 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. In some embodiments, the CD86-binding domain polypeptide contains the heavy chain variable region (V) of SEQ ID NO: 7. H The amino acid sequence of ) is included. In certain embodiments, the CD86 binding domain includes a variable heavy chain having the amino acid sequence of SEQ ID NO: 7 and a variable light chain having the amino acid sequence of SEQ ID NO: 8.

[0158] CD86-binding domains suitable for use in the polypeptides of this disclosure may include or be derived from scFv. In some embodiments, scFv is V H -V L Direction or V L -V H It can be a direction. In some embodiments, scFv is V H and V L It may include linkers between regions. For several reasons, the linker is (Gly-Ser4) n This may include (n = integers from 1 to 5) (sequence number 129). In a particular embodiment, n = 4 (sequence number 61).

[0159] In some embodiments, the CD86-binding domain contains an anti-CD86 scFv that is at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD86-binding domain contains an amino acid sequence that is at least about 95% or 100% identical to SEQ ID NO: 9.

[0160] The cytokine IL-10 is a key player in suppressing inflammation. The crucial role of IL-10 in limiting inflammatory processes has been widely demonstrated in preclinical and human trials since its discovery 20 years ago (Moore et al., 2001). However, several attempts to develop IL-10 as a therapy for various inflammatory diseases have shown only limited success in clinical settings. Growing clinical evidence suggests that while IL-10 suppresses antigen presentation and promotes antigen-specific resistance, it also stimulates effector functions in various lymphocyte populations. This is best demonstrated by recent clinical results of IL-10 in enhancing the antitumor response in cancer patients by stimulating cytotoxic T cells (Chan et al., 2015). Therefore, the multifaceted effects of IL-10 and the widespread expression of IL-10R, combined with its short half-life, may have hindered its ability to suppress local inflammation in clinical trials.

[0161] IL-10 is a cytokine that exerts both repressive and stimulating functions. IL-10 is typically expressed by T cells as well as monocyte macrophages, macrophages, and dendritic cells. One of IL-10's primary functions is to inhibit T cell activation by suppressing antigen presentation by dendritic cells (DCs) and macrophages (Moore et al., 2001). In addition to inducing antigen presentation, IL-10 also induces the differentiation of regulatory DCs (Amodio et al., 2012). Unlike normal DCs, regulatory DCs induce the differentiation of antigen-dependent regulatory T cells (Tr1) (Gregori et al., 2010, Pacciani et al., 2010, Gregori et al., 2011). The crucial role of IL-10 in suppressing inflammatory processes in multiple animal models and human diseases has been widely demonstrated (Kuhn et al. 1993; Steidler et al. 2000; Lindsay et al. 2003). In addition to its clearly characterized immunosuppressive function, IL-10 also stimulates the functions of other cell types. Its stimulating functions include, in particular, the enhancement of immunoglobulin secretion by B cells (Rousset et al. 1992; Fluckiger et al. 1993; Bachereau et al. 1994) and the enhancement of cytotoxic effects by T cells (Mumm et al. 2011; Chan et al. 2015). Several attempts have been made to develop IL-10 as a therapy for treating autoimmune conditions in patients (Colombel et al. 2001; Fedorak et al. 2000; Schreiber et al. 2000; Kimball et al. 2002). However, the multifaceted effects of IL-10, its short half-life, and the widespread expression of IL-10R are very likely reasons for the lack of effectiveness of using IL-10 as an anti-inflammatory agent (Herfarth et al. 2002).

[0162] IL-10 binds to the IL-10 receptor (IL-10R). IL-10R is expressed at a very low copy number on the surface of most hematopoietic cells, estimated to be only a few hundred receptors per cell (Carson et al. 1995; Jurlander et al. 1997). IL-10R is a double-stranded molecule: the IL-10R1 chain, which binds to IL-10 with affinity, and the IL-10R2 chain, which has low-affinity interactions with IL-10 and is involved in receptor complexes with other class 2 cytokine family members (Walter 2014). Both chains contribute to signal transduction, but all IL-10-specific functions appear to reside in the IL-10R1 chain. IL-10 is a non-covalent homodimer of two combined polypeptide chains, expressed by T cells and monocytes / macrophages. IL-10 primarily induces dimerization of two IL-10R complexes, which trigger signaling and activation of STAT3 transcription factors via phosphorylation, but STAT1 can also be activated (Walter 2014; Donnelly et al. 1999). As previously mentioned, IL-10 can mediate repressive or stimulative functions depending on the cell type. It suppresses inflammatory cytokine activation and inhibits the secretion of inflammatory cytokines by myeloid cells, e.g., DCs, as well as monocytes and macrophages (Sabat et al. 2010; Mosser et al. 2008; Ouyang et al. 2011). It induces the differentiation of regulatory DCs, which in turn induce the differentiation of regulatory T cells (Tr1) (Roncarolo). However, it also promotes B cell growth and differentiation (Rousset et al. 1992; Fluckiger et al. 1993; Bachereau et al. 1994) and the effector function of cytotoxic CD8+ T cells (Mumm et al. 2011; Chan et al. 2015). The crucial role of IL-10 as a key negative regulator of inflammation has been revealed by the consequences of IL-10 deficiency in various animal models (Kuhn et al. 1993; Steidler et al. 2000; Lindsay et al. 2003).

[0163] Described herein are modified versions of IL-10 (monomer IL-10, mono-IL10, or mono-IL10) that maintain their inhibitory function while simultaneously reducing their stimulative properties. While the monomeric form of IL-10 can still interact with IL-10R, it no longer induces downstream events in human lymphocytes and simultaneously exhibits a slightly attenuated function in myeloid cells. More specifically, monomeric IL-10 interacts with and signals via IL-10R, but exhibits lower affinity for IL-10R (Josephson et al. 2000), and it interacts with the receptor in a different configuration than 1:1 mono-IL10 / soluble IL-10R versus 1:2 wild-type IL-10:wild-type IL-10 dimer / soluble IL-10R1. Despite the reduced affinity, monomeric IL-10 retains its biological activity against cells, but its potency is reduced. From a manufacturing perspective, it is noteworthy that monomer IL-10 exhibits greater thermal stability than wild-type IL-10 (Josephson et al., 2000; Westerhof et al., 2012).

[0164] In some embodiments, the IL-10 delivery polypeptide comprises a monomer IL-10 domain containing an amino acid insertion in a DE loop between IL-10 subdomains that enables intramolecular folding of the subdomain. In some embodiments, the amino acid insertion is 4 to 8 amino acids long. In some embodiments, the amino acid insertion is 5 to 10 amino acids long. In some embodiments, the amino acid insertion is 6 amino acids long. An example of monomer IL-10 described herein was modified by introducing 6 amino acids (GGGSGG, SEQ ID NO: 130) into the DE loop of wild-type IL-10 resulting in intramolecular folding of the monomer (Josephson et al. 2000). Thus, in some embodiments, the monomer IL-10 contains 6 amino acid insertions in the DE loop between IL-10 subdomains that enables intramolecular folding of the subdomain. In certain embodiments, monomer IL-10 comprises an amino acid sequence that is at least about 85%, 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 sequence number 28.

[0165] In some embodiments, the IL-10 delivery polypeptide, comprising or consisting of a CD86-binding domain and a monomer IL-10 domain, may further comprise an immunoglobulin Fc domain. 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 or derived from human sequences. In some embodiments, the Fc domain includes mutations at positions 234, 235, 237, and 322. In some embodiments, the Fc domain includes mutations at positions 234, 235, 237, 318, 320, and 322. In some embodiments, the Fc domain includes mutations L234A, L235A, G237A, and K322A. In some embodiments, the Fc domain includes mutations L234A, L235A, G237A, E318A, K320A, and K322A. In some embodiments, the Fc domain is derived from IgG1. In some embodiments, the IgG1-derived Fc domain contains two or more mutations that, when administered to a patient, prevent the polypeptide from depleting CD86 and / or IL-10R-expressing cells. In some embodiments, two or more mutations in the IgG1 Fc domain prevent or substantially reduce Fc-mediated crosslinking.

[0166] In some embodiments, the IL-10 delivery peptide may further include an Fc-binding domain linker. The Fc-binding domain linker may contain 1 to 100 amino acids, for example, 8 to 15 amino acids. In some embodiments, the Fc-binding domain linker contains an amino acid sequence derived from a type II C lectin protein, which may be NKG2A. In some embodiments, the Fc-binding domain linker contains any one of SEQ ID NOs. 50 to 70. In some embodiments, the Fc-binding domain linker is (Gly4Ser) nThe amino acid sequence includes (n=1~5) (SEQ ID NO: 129). In certain embodiments, n=4 (SEQ ID NO: 61). In some embodiments, the Fc-binding domain linker does not contain a protease cleavage site.

[0167] The IL-10 delivery peptide may further include a hinge region, such as an IgG-derived hinge region. In some embodiments, the hinge region has one or more mutant cysteine ​​residues. In some embodiments, the hinge region includes one of SEQ ID NOs. 71-109.

[0168] In some embodiments, the IL-10 delivery polypeptide comprises, from amino-terminus to carboxy-terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, an Fc-binding domain linker, and a monomeric IL-10 domain. In some embodiments, the CD86-binding domain comprises an amino acid sequence including SEQ ID NO: 9, or having at least about 95% to 100% identity with SEQ ID NO: 9, and the monomeric IL-10 domain comprises an amino acid sequence including SEQ ID NO: 28, or having at least about 95% to 100% identity with SEQ ID NO: 28. In some embodiments, the IL-10 delivery polypeptide comprises, from amino-terminus to carboxy-terminus, the CD86-binding domain of SEQ ID NO: 9, and the monomeric IL-10 domain of SEQ ID NO: 28.

[0169] In some embodiments, the IL-10 delivery polypeptide comprises, from amino-terminus to carboxyl-terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, (optionally) an Fc-binding domain linker, and a monomer IL-10 domain, wherein the CD86-binding domain comprises a variable heavy chain and a variable light chain that specifically binds CD86; the immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to Fc receptors FcγR, FcγRIIa, FcγRIIb, and / or FcγRIIIb; the Fc-binding domain linker comprises a flexible linker of 8-20 amino acids in length that does not contain a glycosylation site; the monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker; and the IL-10 delivery polypeptide forms a dimer protein with the same IL-10 delivery polypeptide.

[0170] In some embodiments, the IL-10 delivery polypeptide comprises an amino acid sequence of SEQ ID NO: 30, or a sequence that is at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to SEQ ID NO: 30. In some embodiments, the IL-10 delivery polypeptide is essentially derived from or consists of SEQ ID NO: 30. In some embodiments, the IL-10 delivery polypeptide is encoded by a nucleic acid having the sequence of SEQ ID NO: 29, or a sequence that is at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to SEQ ID NO: 29. Q0128 is an example of an IL-10 delivery polypeptide (or fusion protein) having the amino acid sequence of SEQ ID NO: 30.

[0171] In some embodiments, the IL-10 delivery peptide specifically binds to cells expressing IL-10R and CD86. In some embodiments, the IL-10 delivery polypeptide is a dimer, such as a homodimer or heterodimer. In some embodiments, the IL-10 delivery polypeptide is a monomer.

[0172] In some embodiments, when the polypeptide is dimerized to the same IL-10 delivery polypeptide, it induces STAT3 phosphorylation in monocytes and dendritic cells. The dendritic cells may be immunotolerogenic dendritic cells.

[0173] In some embodiments, when dimerized with the same IL-10 delivery polypeptide, the IL-10 delivery polypeptide does not induce phosphorylation in B, T, and NK lymphocytes, or induces minimal phosphorylation in B, T, and NK lymphocytes compared to that with IL-10. In some embodiments, the anti-CD86 domain enhances the signaling of the monomer IL-10 domain in vivo compared to Fc monomer IL-10 or Fc-IL-10 molecules that do not contain the CD86 binding domain.

[0174] In some embodiments, when an IL-10 delivery polypeptide is dimerized with another IL-10 delivery polypeptide, it exhibits enhanced potency compared to when dimerized with IL-10.

[0175] In some embodiments, when an IL-10 delivery polypeptide is dimerized with another IL-10 delivery polypeptide, it does not stimulate activated T cells.

[0176] In some embodiments, when an IL-10 delivery polypeptide is dimerized with another IL-10 delivery polypeptide, it either does not stimulate B cells or stimulates activated B cells minimally compared to when it is dimerized with IL-10.

[0177] In some embodiments, when an IL-10 delivery polypeptide is dimerized with another IL-10 delivery polypeptide, it does not induce IgM secretion, or induces minimal IgM secretion, compared to when it is dimerized with IL-10.

[0178] In some embodiments, when an IL-10 delivery polypeptide is dimerized with another IL-10 delivery polypeptide, it inhibits T cell proliferation.

[0179] In some embodiments, when an IL-10 delivery polypeptide is dimerized with another IL-10 delivery polypeptide, it suppresses antigen-presenting cell function.

[0180] In some embodiments, a CD86 receptor occupancy rate of less than 20% on monocytes is required to achieve maximum suppression of antigen presentation when the IL-10 delivery polypeptide is dimerized with the same IL-10 delivery polypeptide and administered to humans or non-human primates.

[0181] The anti-CD86 x mono-IL10 molecules described herein are designed to treat inflammatory conditions such as psoriasis by delivering a modified version of IL-10 (monomer IL-10) to antigen-presenting cells. These molecules function as improved versions of IL-10, maintaining their inhibitory function while simultaneously reducing their stimulative properties. They achieve this dual objective through a combination of two mechanisms. Firstly, the monomeric form of IL-10 present in these molecules can still interact with IL-10R but no longer induces downstream events in human lymphocytes, while simultaneously exhibiting a slightly attenuated function in myeloid cells. Secondly, binding of monomer IL-10 to the anti-CD86 targeting arm specifically enhances the signaling of monomer IL-10 in CD86-expressing cells. The inclusion of the Fc moiety in the molecule extends its half-life compared to that of wild-type IL-10, which is less than 4 hours (Huhn et al. 1996). The resulting molecules suppress antigen presentation function and T cell activation, and induce regulatory DCs, but do not stimulate the function of naive or activated B or T cells. The minimum concentrations at which these molecules induce optimal function in vitro and in vivo are below the levels required for CD86 receptor saturation. Therefore, these molecules function via mono-IL10 delivery and without CD86 blockade.

[0182] The therapeutic protein for use in the composition of the present invention may be selected from any of the above-described therapeutic proteins. For example, the therapeutic binding protein may comprise a first binding domain and a second binding domain, which are optionally separated by at least an immunoglobulin constant region. In some embodiments, the first binding domain and / or the second binding domain bind to a drug or toxin.

[0183] In some embodiments, the first or second binding domain includes (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, VH or VL, or both VH and VL, are humanized. The amino acid sequence of HCDR1 may be SEQ ID NO: 1, the amino acid sequence of HCDR2 may be SEQ ID NO: 2, the amino acid sequence of HCDR3 may be SEQ ID NO: 3, the amino acid sequence of LCDR1 may be SEQ ID NO: 4, the amino acid sequence of LCDR2 may be SEQ ID NO: 5, and the amino acid sequence of LCDR3 may be SEQ ID NO: 6. VH comprises an amino acid sequence that is at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 7. In some embodiments, VL includes an amino acid sequence that is at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 8. In some embodiments, VH includes SEQ ID NO: 7, and VL includes SEQ ID NO: 8.

[0184] In some embodiments, the first or second binding domain is a single-chain variable fragment (scFv). The light-chain variable region of the scFv may be carboxy-terminal or amino-terminal relative to the heavy-chain variable region of the scFv. In some embodiments, the scFv includes a linker polypeptide, which may be located between the light-chain and heavy-chain variable regions of the scFv. The linker polypeptide is of the formula (Gly4Ser)n The formula may include n=1 to 5 (sequence number 129).

[0185] In some embodiments, the first or second binding domain specifically binds to antigen-presenting cells. In some embodiments, the first or second binding domain binds to the IL-10 receptor.

[0186] In some embodiments, the first or second binding domain specifically binds to CD86.

[0187] In some embodiments, the binding domain includes an immunoglobulin heavy chain variable region (VH) comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and an immunoglobulin light chain variable region (VL) comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0188] In some embodiments, the binding domain includes an immunoglobulin heavy chain variable region (VH) containing sequence number 7 or at least 95% identical thereto; and an immunoglobulin light chain variable region (VL) containing sequence number 8 or at least 95% identical thereto.

[0189] In some embodiments, the binding domain includes sequence number 9, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0190] In some embodiments, the first or second binding domain specifically binds to a cytokine receptor. The cytokine receptor may be, for example, the IL-10 receptor (IL-10R).

[0191] In some embodiments, the first or second binding domain includes a cytokine or a recombinant variant of a cytokine. The cytokine or recombinant variant may be monomer IL-10. In some embodiments, monomer IL-10 specifically binds to the IL-10 receptor (IL-10R). In some embodiments, monomer IL-10 includes amino acid insertions in the DE loop between IL-10 subdomains, which allows for intramolecular folding of the subdomains. The amino acid insertions may consist of 4 to 8 amino acids or 5 to 10 amino acids. In some embodiments, monomer IL-10 includes SEQ ID NO: 28.

[0192] In some embodiments, the therapeutic protein includes an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is a human Fc domain. In some embodiments, the immunoglobulin constant region includes immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the immunoglobulin constant region includes a human IgG1 CH2 domain with substitutions L234A, L235A, G237A, and K322A according to the EU numbering system. In some embodiments, the immunoglobulin constant region includes a human IgG1 CH2 domain with substitutions L234A, L235A, G237A, E318A, K320A, and K322A according to the EU numbering system. In some embodiments, the immunoglobulin constant region includes SEQ ID NO: 131.

[0193] In some embodiments, the therapeutic protein includes a hinge region, for example, a hinge region derived from an immunoglobulin hinge region. In some embodiments, the hinge region includes SEQ ID NO: 47.

[0194] In some embodiments, the therapeutic protein includes an Fc-binding domain linker. In some embodiments, the Fc-binding domain linker is a Gly4Ser (SEQ ID NO: 128) sequence, for example, (Gly4Ser) nThis includes n=1 to 5 in the formula (sequence number 129).

[0195] In some embodiments, the Fc-binding domain linker includes a sequence derived from the stalk region of a type II C lectin protein. The type II C lectin protein may be CD69, CD72, CD94, NKG2A, or NKG2D. In some embodiments, the Fc-binding domain linker includes SEQ ID NO: 132.

[0196] In some embodiments, the therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain, wherein the CD86-binding domain comprises a variable heavy chain and a variable light chain that specifically bind CD86, the immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIb, and the monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker, and the therapeutic protein is a homodimer. In some embodiments, the CD86-binding domain comprises an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3. In some embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO: 6. In some embodiments, the CD86-binding domain includes a variable heavy chain having an amino acid sequence at least 95% identical to SEQ ID NO: 7, and a variable light chain having an amino acid sequence at least 95% identical to SEQ ID NO: 8. In some embodiments, the CD86-binding domain includes an amino acid sequence at least about 95% or 100% identical to SEQ ID NO: 9. In some embodiments, the monomer IL-10 domain includes an amino acid sequence at least 95% or 100% identical to SEQ ID NO: 28. In some embodiments, the therapeutic protein includes the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to SEQ ID NO: 30.

[0197] In some embodiments, the first or second binding domain specifically binds to antigen-presenting cells, such as monocytes or dendritic cells. Antigen-presenting cells can be monocytes or dendritic cells, such as CD86-expressing monocytes or CD86-expressing dendritic cells. In some embodiments, the first or second binding domain of the therapeutic protein specifically binds to CD86.

[0198] In some embodiments, the therapeutic protein exhibits no antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity, or only minimal activity.

[0199] In some embodiments, about 80% or more by weight, about 85% or more by weight, or about 90% or more by weight, or about 95% or more by weight of the therapeutic protein in the composition are not present as aggregates. The aggregate percentage can be measured by size exclusion high-performance liquid chromatography.

[0200] In some embodiments, the therapeutic protein does not aggregate, or aggregates minimally, after at least one freeze event and a subsequent thawing event. In some embodiments, the composition of the Disclosure comprising a glutamate buffer has a lower relative amount of multispecific protein compared to the relative amount in a composition comprising a non-glutamate buffer and the same multispecific protein, as measured by size exclusion high-performance liquid chromatography, where high molecular weight species are present after at least one freeze event and a subsequent thawing event. The freeze event may be performed, for example, at -80°C or -20°C.

[0201] In some embodiments, the compositions described herein contain 1 to 20 mg / ml, 1 to 12 mg / ml, or 5 to 10 mg / ml of therapeutic protein. In some embodiments, the compositions contain about 1 mg / ml to about 12 mg / ml, or about 5 mg / ml to about 10 mg / ml of therapeutic protein. In further embodiments, the compositions contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 mg / ml of therapeutic protein. In certain embodiments, the compositions contain about 2 mg / ml of therapeutic protein.

[0202] Exemplary protein-based therapeutic agents: anti-CD123 x anti-CD3 polypeptides and their dimers Exemplary protein-based therapeutics can bind both CD123-expressing cells and T cell receptor complexes on T cells, thereby inducing target-dependent T cell cytotoxicity, activation, and proliferation.

[0203] Accordingly, in certain embodiments, the therapeutic proteins used in connection with the methods and compositions described herein are bispecific single-chain molecules comprising a CD123-binding domain and a CD3-binding domain. In some embodiments, the CD123 and / or CD3-binding domains are antibody-derived and comprise a variable heavy chain (VH) and a variable light chain (VL). For example, the CD123 and / or CD3-binding domain may be an scFv comprising VH and VL. These binding domains and variable chains may be arranged in any order that still maintains some degree of binding to the target. For example, the variable domains are (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 CD3)-(VL CD3)-(VH CD123)-(VH CD123), (VL CD3)-(VH CD3)-(VL CD123)-(VH CD123), (VH CD3)-(VL CD3)-(VL CD123)-(VH CD123), or (VL CD3)-(VH The domains may be arranged in an order such as CD3)-(VH CD123)-(VL CD123). The pair of VH and VL regions in the CD3-binding domain may be in the format of a single-chain antibody (scFv). The VH and VL regions may be arranged in the order VH-VL or VL-VH. In some embodiments, scFv may bind to CD123 with higher efficiency than antibodies containing the same VH and VL region sequences in the same direction. In certain embodiments, scFv may bind to CD123 with higher efficiency in the VL-VH direction than in the VH-VL direction. The VH region may be located at the N-terminus relative to the linker sequence. The VL region may be located at the C-terminus relative to the linker sequence. The domain arrangement in the CD3-binding domain of a bispecific single-chain molecule may be VH-VL, with the CD3-binding domain located at the C-terminus relative to the CD123-binding domain. A bispecific molecule may include a CD3-binding scFv linked to a CD123-binding scFv. These scFvs are linked by short-chain peptides.In some embodiments, the bispecific single-chain molecules do not include a hinge region or a constant region (see, for example, U.S. Patent Application Publication 2013 / 0295121, International Publication 2010 / 037836, 2004 / 106381, and 2011 / 121110; each is incorporated herein by reference in its entirety).

[0204] CD123-double specific binding constructs may include one or more sequences shown in Tables 4, 5, and / or 6. [Table 4] JPEG2026062861000007.jpg223158JPEG2026062861000008.jpg221158JPEG2026062861000009.jpg220158JPEG2026062861000010.jpg215158JPEG2026062861000011.jpg224158JPEG2026062861000012.jpg221158JPEG2026062861000013.jpg216158JPEG2026062861000014.jpg179158JPEG2026062861000015.jpg223158JPEG2026062861000016.jpg43158JPEG2026062861000017.jpg224158JPEG2026062861000018.jpg44158JPEG2026062861000019.jpg225158JPEG2026062861000020.jpg39158JPEG2026062861000021.jpg224158JPEG2026062861000022.jpg39158JPEG2026062861000023.jpg224158JPEG2026062861000024.jpg40158JPEG2026062861000025.jpg224158JPEG2026062861000026.jpg39158JPEG2026062861000027.jpg224158JPEG2026062861000028.jpg39158JPEG2026062861000029.jpg224158JPEG2026062861000030.jpg38158JPEG2026062861000031.jpg223158JPEG2026062861000032.jpg43158JPEG2026062861000033.jpg224158JPEG2026062861000034.jpg39158JPEG2026062861000035.jpg223158JPEG2026062861000036.jpg39158JPEG2026062861000037.jpg224158JPEG2026062861000038.jpg39158JPEG2026062861000039.jpg223158JPEG2026062861000040.jpg38158JPEG2026062861000041.jpg223158JPEG2026062861000042.jpg219158JPEG2026062861000043.jpg219158JPEG2026062 861000044.jpg226158JPEG2026062861000045.jpg189158JPEG2026062861000046.jpg224158JPEG2026062861000047.jpg97158. Table 5 Table 6 JPEG2026062861000050.jpg190158

[0205] In certain embodiments, the CD123-binding domain comprises (i) an immunoglobulin light chain variable region (VL) including CDR LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) including CDR HCDR1, HCDR2, and HCDR3, where HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 144, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 146, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 148. In certain embodiments, the CD123-binding domain comprises (i) an immunoglobulin light chain variable region (VL) including CDR LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) including CDR HCDR1, HCDR2, and HCDR3. In some such embodiments, (i) LCDR1 has a sequence different from SEQ ID NO: 138 by the amino acid sequence shown in SEQ ID NO: 138 or by at least one amino acid substitution; (ii) LCDR2 has a sequence different from SEQ ID NO: 140 by the amino acid sequence shown in SEQ ID NO: 140 or by at least one amino acid substitution; (iii) LCDR3 has a sequence different from SEQ ID NO: 142 by the amino acid sequence shown in SEQ ID NO: 142 or by at least one amino acid substitution; (iv) HCDR1 has a sequence different from SEQ ID NO: 144 by the amino acid sequence shown in SEQ ID NO: 144 or by at least one amino acid substitution; (v) HCDR2 has a sequence different from SEQ ID NO: 146 by the amino acid sequence shown in SEQ ID NO: 146 or by at least one amino acid substitution; and (vi) HCDR3 has a sequence different from SEQ ID NO: 148 by the amino acid sequence shown in SEQ ID NO: 148 or by at least one amino acid substitution. The above amino acid substitutions may be conserved or non-conserved amino acid substitutions. In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 differ from sequences that detail only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.In certain embodiments, the CDR of the Disclosure includes, compared to the CDR sequence of a known monoclonal antibody, approximately one or more (e.g., approximately 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, approximately one or more (e.g., approximately 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, approximately one or more (e.g., approximately 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or a combination of the above changes. For example, the present invention includes a recombinant polypeptide comprising: (i) LCDR1 having an amino acid sequence shown in SEQ ID NO: 138 or a sequence different from SEQ ID NO: 138 by one or two amino acid substitutions; (ii) LCDR2 having an amino acid sequence shown in SEQ ID NO: 140 or a sequence different from SEQ ID NO: 140 by one or two amino acid substitutions; (iii) LCDR3 having an amino acid sequence shown in SEQ ID NO: 142 or a sequence different from SEQ ID NO: 142 by one or two amino acid substitutions; (iv) HCDR1 having an amino acid sequence shown in SEQ ID NO: 144 or a sequence different from SEQ ID NO: 144 by one or two amino acid substitutions; (v) HCDR2 having an amino acid sequence shown in SEQ ID NO: 146 or a sequence different from SEQ ID NO: 146 by one or two amino acid substitutions; and (vi) HCDR3 having an amino acid sequence shown in SEQ ID NO: 148 or a sequence different from SEQ ID NO: 148 by one or two amino acid substitutions. The above amino acid substitutions may be conserved or non-conserved amino acid substitutions.

[0206] In related embodiments, the recombinant polypeptide of the present invention has a light chain variable region (V L The amino acid sequence of (e.g., SEQ ID NO: 134), or the heavy chain variable region (V HThe recombinant polypeptide comprises, or is a sequence that is 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 (e.g., SEQ ID NO: 136), or both. In some embodiments, the recombinant polypeptide comprises a sequence that is at least about 80%, at least about 85%, at least about 88%, at least about 98%, 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 (e.g., SEQ ID NO: 136), or both. The present invention includes a recombinant polypeptide that is identical to the amino acid sequence of SEQ ID NO: 337 by 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%.

[0207] In some embodiments, the CD123 binding domain is (i) an immunoglobulin light chain variable region (V) comprising CDR LCDR1, LCDR2, and LCDR3. L ), and (ii) immunoglobulin heavy chain variable region (V) including CDR HCDR1, HCDR2, and HCDR3 H) and (vi) HCDR3 has an amino acid sequence different from SEQ ID NO: 154 by the amino acid sequence shown in SEQ ID NO: 154 or by at least one amino acid substitution; (ii) LCDR2 has an amino acid sequence different from SEQ ID NO: 156 by the amino acid sequence shown in SEQ ID NO: 156 or by at least one amino acid substitution; (iii) LCDR3 has an amino acid sequence different from SEQ ID NO: 158 by the amino acid sequence shown in SEQ ID NO: 158 or by at least one amino acid substitution; (iv) HCDR1 has an amino acid sequence different from SEQ ID NO: 160 by the amino acid sequence shown in SEQ ID NO: 160 or by at least one amino acid substitution; (v) HCDR2 has an amino acid sequence different from SEQ ID NO: 162 by the amino acid sequence shown in SEQ ID NO: 162 or by at least one amino acid substitution; and (vi) HCDR3 has an amino acid sequence different from SEQ ID NO: 164 by the amino acid sequence shown in SEQ ID NO: 164 or by at least one amino acid substitution. The above amino acid substitutions may be conserved or non-conserved amino acid substitutions. In some embodiments, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 differ from sequences detailed by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the CDRs of the Disclosure include, compared to the CDR sequences of known monoclonal antibodies, 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., conserved or non-conserved amino acid substitutions), or a combination of the above variations.

[0208] In some embodiments, the CD123-binding domain includes, or is a sequence that is 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 the light chain variable region (VL) (e.g., SEQ ID NO: 150), or the amino acid sequence of the heavy chain variable region (VH) (e.g., SEQ ID NO: 152), or both.

[0209] In some embodiments, the CD123-binding domain is humanized immunoglobulin V L and / or V H Includes the region. Immunoglobulin V L and V H Techniques for humanizing domains are known in the art and are discussed, for example, in U.S. Patent Application Publication No. 2006 / 0153837. In some embodiments, the CD123-binding domain is human immunoglobulin V L and / or V H Includes the region.

[0210] Essentially, humanization by CDR grafting involves recombining only the CDR of a non-human antibody with a human variable region framework and human constant region. Theoretically, this should substantially reduce or eliminate immunogenicity (except in cases where allotype or idiotype differences exist). However, it has also been reported that some framework residues of the original antibody must also be conserved (Reichmann et al., Nature, 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10, 029 (1989)).

[0211] Framework residues that need to be conserved can be identified through computer modeling. Alternatively, essential framework residues may be identified by comparing them with known antigen-binding site structures (Padlan, Molec. Immunol., 31(3):169-217(1994), incorporated herein by reference).

[0212] Residues that may affect antigen binding can be classified into several groups. The first group includes residues adjacent to the antigen site surface, which can therefore come into direct contact with the antigen. These residues include amino-terminal residues and residues adjacent to the CDR. The second group includes residues that, by contacting the CDR or another peptide chain in the antibody, can alter the structure of the CDR or its relative alignment. The third group includes amino acids with accepted side chains that can affect the structural integrity of the variable domain. Residues in these groups are usually found in the same position (Padlan, 1994, cited above), but their identified positions may differ depending on the numbering system (see Kabat et al., "Sequences of proteins of immunological interest," 5th ed., Pub. No. 91-3242, USDept. Health & Human Services, NIH, Bethesda, Md., 1991).

[0213] Knowledge of humanized antibodies in the relevant art is applicable to polypeptides provided in this disclosure, even if they are not antibodies.

[0214] In some embodiments, the disclosure relates to the CD123 binding domain, (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%, and at least 99% identical to the amino acid sequence shown 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 shown in SEQ ID NO: 136. (ii) The immunoglobulin light chain variable region contains an amino acid sequence that is 99% identical to the amino acid sequence shown in SEQ ID NO: 150, and the immunoglobulin heavy chain variable region contains 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 shown in SEQ ID NO: 152, and the 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 shown in SEQ ID NO: 152. (iii) The immunoglobulin light chain variable region includes an amino acid sequence that is at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequence shown in SEQ ID NO: 150, and the immunoglobulin heavy chain variable region includes 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 shown in SEQ ID NO: 168, and (iv) The immunoglobulin light chain variable region includes an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequence shown in SEQ ID NO: 150, and the immunoglobulin heavy chain variable region includes 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 shown in SEQ ID NO: 184, and (v) the immunoglobulin light chain variable region,(vi) The immunoglobulin heavy chain variable region includes 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 shown in SEQ ID NO: 198, and (vi) the immunoglobulin light chain variable region includes 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 shown in SEQ ID NO: 200, and (vi) the immunoglobulin light chain variable region includes 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 shown in SEQ ID NO: 214 (vii) 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 shown in SEQ ID NO 216, and (vii) the immunoglobulin light 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 shown in SEQ ID NO 230 (viii) The immunoglobulin heavy chain variable region includes an amino acid sequence that is 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 shown in SEQ ID NO: 232, and (viii) the immunoglobulin light chain variable region includes 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 shown in SEQ ID NO: 166, and (viii) the immunoglobulin light chain variable region includes an amino acid sequence that is at least 85%, at least 90%, at least 99% identical to the amino acid sequence shown in SEQ ID NO: 166. (ix) The immunoglobulin heavy chain variable region contains an amino acid sequence that is 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 296, and (ix) the immunoglobulin light chain variable region contains 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 shown in SEQ ID NO: 166,(x) The immunoglobulin heavy chain variable region contains an amino acid sequence that is at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO: 248, and (x) the immunoglobulin light chain variable region contains 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 shown in SEQ ID NO: 166, and the immunoglobulin heavy chain variable region contains 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 shown in SEQ ID NO: 264 (xi) The immunoglobulin light chain variable region contains 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 shown in SEQ ID NO: 166, and the immunoglobulin heavy chain variable region contains 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 shown in SEQ ID NO: 280.

[0215] In some embodiments, each CDR contains one, two, or three or fewer substitutions, insertions, or deletions compared to those derived from a monoclonal antibody or its fragment or derivative that specifically bind to the target of interest (e.g., CD123).

[0216] In some embodiments, the CD123-binding domain does not inhibit IL-3 binding to CD123.

[0217] In some embodiments, the CD123 binding molecule or protein may include a cell-binding domain for the recruitment of T cells to target cells expressing CD123. In some embodiments, the CD123 binding proteins described herein may include (i) a binding domain that specifically binds to the TCR complex or a component thereof (e.g., TCRα, TCRβ, CD3γ, CD3δ, and CD3ε), and (ii) another binding domain that specifically binds to CD123. The CD123 binding protein can essentially utilize any binding domain that binds to T cells, such as 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, E.L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986); SEQ ID NO: 341 (QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITR) and SEQ ID NO: 342 (QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWGQGTLVTVSA)) with the VL and VH amino acid sequences shown respectively; HuM291 (Chau et al. (2001) Transplantation 71:941-950; SEQ ID NO: 343 (diqmtqspsslsasvgdrvtitcsasssvsymnwyqqkpgkapkrliydtsklasgvpsrfsgsgsgtdftltisslqpedfatyycqqwssnpptfgggtkveik) and SEQ ID NO: 344 (qvqlvqsgaevkkpgasvkvsckasgytfisytmhwvrqapgqglewmgyinprsgythynqklkdkatltadksastaymelsslrsedtavyycarsayydydgfaywgqgtlvtvss)) with the V 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 variant with alanine substitutions at positions 234 and 235 (Herold et al. (2003) J. Clin. Invest. 11:409); vizilizumab (Carpenter et al. (2002) Blood 99:2712), G19-4 monoclonal antibody (Ledbetter et al., 1986, J. Immunol. 136:3945), 145-2C11 monoclonal antibody (Hirsch et al. Examples include al. (1988) J. Immunol. 140:3766) and I2C monoclonal antibodies (see, for example, U.S. Patent Application Publication Nos. 2011 / 0293619 and 20120244162). For example, a CD3-binding domain may include a CD3-binding domain disclosed in U.S. Patent Application Publication 2012 / 0244162, which includes a CD3-binding domain containing a VL region selected from Sequence IDs 17, 21, 35, 39, 53, 57, 71, 75, 89, 83, 107, 111, 125, 129, 143, 147, 161, 165, 179, and 183 of U.S. Patent Application Publication 2012 / 0244162 and / or a VH region selected from Sequence IDs 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177, and 181 of U.S. Patent Application Publication 2012 / 0244162. In some embodiments, the CD3-binding domain comprises amino acid sequences selected from SEQ ID NOs. 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, and 187 of U.S. Patent Application Publication No. 2012 / 0244162. In some embodiments, the CD3-binding domain is described in International Publication Nos. 2004 / 106380, 2005 / 040220A1, U.S. Patent Application Publication No. 2014 / 0099318, or derived from its CD3-binding domain. 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 either the antibody or sequence described in International Publication No. 2013 / 158856 (which is incorporated herein by reference in its entirety).

[0218] In some embodiments, the second binding domain of the 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 each have the amino acid sequences shown in SEQ ID NOs. 348, 349, and 350, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NOs. 345, 346, and 347, respectively; or (b) LCDR1, LCDR2, and LCDR3 each have the amino acid sequences shown in SEQ ID NOs. 354, 355, and 356, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NOs. 351, 352, and 353, respectively. In some embodiments, the second binding domain of the 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 each have the amino acid sequences shown in SEQ ID NOs. 351, 352, and 353, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NOs. 357, 359, and 359, respectively; or (b) LCDR1, LCDR2, and LCDR3 each have the amino acid sequences shown in SEQ ID NOs. 359, 367, and 368, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NOs. 363, 364, and 365, respectively.In some embodiments, the second binding domain of the 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 amino acid sequences shown in SEQ ID NOs. 372, 373, and 374, respectively, and HCDR1, HCDR2, and HCDR3 have amino acid sequences shown in SEQ ID NOs. 369, 370, and 371, respectively; or (b) LCDR1, LCDR2, and LCDR3 have amino acid sequences shown in SEQ ID NOs. 378, 379, and 380, respectively, and HCDR1, HCDR2, and HCDR3 have amino acid sequences shown in SEQ ID NOs. 375, 376, and 377, respectively. In some embodiments, the second binding domain comprising the CDR sequences detailed in this paragraph is humanized.

[0219] In some embodiments of CD123-binding proteins containing two binding domains that specifically bind to CD3ε, the second binding domain competes with CRIS-7, HuM291, or I2C monoclonal antibodies for binding to CD3ε. In some embodiments, the CD3-binding domain is located in the immunoglobulin light chain variable region (V) derived from CRIS-7, HuM291, or I2C monoclonal antibodies. L ) and the variable region of immunoglobulin heavy chain (V H ) including (for example, V of the second binding domain) L and V H(These may be humanized variable regions containing the light chain CDR and heavy chain CDR of a monoclonal antibody, respectively.) The second binding domain may contain the light chain variable region, the heavy chain variable region, or both of the DRA222, TSC455, or TSC456 CD3 binding domain. The amino acid sequences of DRA222, TSC455, and TSC456 are provided in Table 4. The DRA222 binding domain is also described in International Publication No. 2013 / 158856. TSC455 is also known as TSC394 F87Y. TSC455 is also known as TSC394 E86D F87Y or TSC394 DY. In some embodiments, the second binding domain specifically binds CD3 and includes an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; the immunoglobulin light chain variable region includes an amino acid sequence that is at least 93% identical, at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 384; or an amino acid sequence that is at least 94% identical, at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 385; the immunoglobulin heavy chain variable region includes an amino acid sequence that is at least 82% identical, at least 85% identical, at least 87% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 383. In some embodiments, a CD123-binding polypeptide or protein further comprising a CD3-binding domain may have low levels of high molecular weight aggregates generated during recombinant expression of the polypeptide or protein. CD123-binding polypeptides or proteins containing additional CD3-binding domains may exhibit relatively long-term stability in human serum, depending on the CD3-binding domains present in the polypeptide or protein.

[0220] In certain modifications, the CD3-binding domain includes one or more CD3-binding sequences (e.g., CDRs or variable regions) disclosed in U.S. Patent Publication Nos. 2013 / 0129730, 2011 / 0293619, U.S. Patent No. 7,635,472, International Publication Nos. 2010 / 037836, 2004 / 106381, or 2011 / 121110. Each of these patent documents is incorporated herein by reference in its entirety. In some embodiments, the CD3-binding domain includes one or more sequences shown in Table 7. [Table 7]

[0221] In various embodiments, the CD3-binding domain includes one or more sequences shown in Table 8. [Table 8]

[0222] In some embodiments, the therapeutic protein includes, in order from the amino terminus to the 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 includes the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In some embodiments, the first binding domain includes an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 includes SEQ ID NO: 10, HCDR2 includes SEQ ID NO: 11, and HCDR3 includes SEQ ID NO: 12. In some embodiments, LCDR1 includes SEQ ID NO: 13, LCDR2 includes SEQ ID NO: 14, and LCDR3 includes SEQ ID NO: 15. In some embodiments, HCDR1 includes sequence number 10, HCDR2 includes sequence number 11, HCDR3 includes sequence number 12; and LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15. In some embodiments, VH includes the sequence of sequence number 16, or a sequence that is at least 90% to at least 95% identical thereto. In some embodiments, VL includes the sequence of sequence number 17, or a sequence that is at least 90% to at least 95% identical thereto. In some embodiments, the first binding domain includes a sequence that is at least 95% identical to sequence number 18. In some embodiments, the second binding domain includes (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 includes sequence number 19, HCDR2 includes sequence number 20, and HCDR3 includes sequence number 21. In some embodiments, LCDR1 includes sequence number 22, LCDR2 includes sequence number 23, and LCDR3 includes sequence number 24.In some embodiments, HCDR1 includes SEQ ID NO: 19, HCDR2 includes SEQ ID NO: 20, HCDR3 includes SEQ ID NO: 21; and LCDR1 includes SEQ ID NO: 22, LCDR2 includes SEQ ID NO: 23, and LCDR3 includes SEQ ID NO: 24. In some embodiments, VH includes the sequence of SEQ ID NO: 25, or a sequence that is at least 90% to at least 95% identical thereto. In some embodiments, VL includes the sequence of SEQ ID NO: 26, or a sequence that is at least 90% to at least 95% identical thereto. In some embodiments, the second binding domain includes a sequence that is at least 95% or 100% identical to SEQ ID NO: 27. In some embodiments, the therapeutic protein includes the sequence of SEQ ID NO: 31.

[0223] Drug delivery system The compositions for preventing protein adsorption described herein can be used in many different types of drug delivery systems known to those skilled in the art.

[0224] The drug delivery system according to this disclosure may include one or more components configured to hold a liquid, such as an IV bag. In some embodiments, a therapeutic protein is suspended in the liquid inside the IV bag. The components configured to hold the liquid may have a volume of about 50, about 100, about 150, about 200, about 250, about 350, about 450, or about 500 ml. The components may be made from, for example, polyvinyl chloride (PVC), ethylene vinyl acetate, polypropylene, or copolyester ether.

[0225] The drug delivery system may further include one or more tubes. The tubes may be attached to components configured to hold liquids.

[0226] The drug delivery system of this disclosure may further include a needle for insertion into a patient.

[0227] A drug delivery system for delivering a therapeutic protein to a patient comprises at least one component adapted to deliver the therapeutic protein, the component being selected from the group consisting of a container, tube, and needle configured to hold a liquid, the inner surface of at least one component being in contact with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80, and then being in contact with a composition containing the therapeutic protein.

[0228] In some embodiments, a drug delivery system for delivering a therapeutic protein to a patient includes at least one container adapted to hold the therapeutic protein, the inner surface of at least one container being in contact with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80, and then being in contact with the composition containing the therapeutic protein.

[0229] Containers adapted to hold therapeutic proteins are also provided. In some embodiments, the inner surface of the container is first brought into contact with the composition of the present disclosure, and then it is brought into contact with the composition containing the therapeutic protein. In some embodiments, the container is substantially latex-free. In some embodiments, the container is substantially bis(2-ethylhexyl)phthalate (DEHP)-free. In some embodiments, the container is selected from the group consisting of IV bags, injectors, and tubing.

[0230] In some embodiments, a method for constructing an intravenous drug delivery system for delivering a therapeutic protein includes preparing at least one container adapted to hold the therapeutic protein, and contacting the inner surface of at least one container with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80 before the therapeutic protein is added to at least one container. In some embodiments, the composition coats the inner surface of at least one container to prevent the therapeutic protein from binding to the inner surface of the container.

[0231] Treatment methods This disclosure also provides a method for treating a subject by intravenous administration (e.g., intravenous infusion) of a therapeutic protein. The subject may be, for example, a mammal. In some embodiments, the subject is a human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, horse, or cow. In some embodiments, the subject is a human.

[0232] In some embodiments, the method includes preparing at least one container adapted to hold a therapeutic protein, contacting the inner surface of the container with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80, contacting the inner surface of the container with the composition comprising the therapeutic protein, and intravenously administering the therapeutic protein to the target. In some embodiments, the composition coats the inner surface of at least one container to prevent the therapeutic protein from binding to the inner surface of the container.

[0233] Examples The present invention will be described in more detail by reference to the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Accordingly, the present invention should not be construed as being limited to the following embodiments, but rather as encompassing any and all modifications that become apparent as a result of the teachings provided herein.

[0234] Even without further description, those skilled in the art will likely be able to prepare and utilize the compounds of the present invention and implement the claimed methods using the preceding description and the following examples. The following examples, therefore, specifically illustrate preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0235] Example 1: Preparation of IVSS solution On day T=1, a 20X IVSS composition containing 10 mM succinate and 0.08% polysorbate 80 was prepared at pH 6.0. The composition was placed in a 10 mL clear glass vial, the surface was covered with nitrogen, and the vial was sealed with a 20 mm stopper / flip-off overseal. This composition will be referred to as the "succinate formulation" throughout the examples.

[0236] A comparative 20X IVSS composition was prepared containing 333 mM histidine and 0.067% polysorbate 80 at pH 6.0. This composition was similarly placed in a 10 mL clear glass vial, the surface covered with nitrogen, and sealed using a 20 mm stopper / flip-off overseal. This composition will be referred to as the "histidine composition" throughout the examples.

[0237] Example 2: Stability of polysorbate in succinate and histidine formulations The stability of polysorbate 80 in succinate and histidine formulations was measured by quantitative analysis of polysorbate 80 by HPLC and / or qualitative evaluation of UV spectral scan profiles. For HPLC, the polysorbate concentration of both formulations was measured using an Agilent® HPLC system equipped with an ELSD detector. For UV spectral scanning, the absorbance of both formulations from 100 to 600 nm was scanned using a spectrophotometer.

[0238] The stability data are shown in Table 9. Polysorbate 80 in the succinate formulation is stable at 40°C for at least 270 days, while polysorbate 80 in the histidine formulation is stable for less than 2 months at 25°C. This result demonstrates that polysorbate 80 is more stable in the succinate formulation compared to the histidine formulation. [Table 9]

[0239] Table 10 shows the quantification of polysorbate 80 in the succinate formulation by HPLC method. The sample was held at 40°C for 270 days. Polysorbate 80 in the succinate buffer was still within the specification limits even after 270 days, and polysorbate 80 was quantified at 0.07% (the specification limit for polysorbate 80 is set at 0.06% - 0.1% polysorbate 80). [Table 10]

[0240] Stability was also evaluated using the UV scan method. The UV scan data corroborated the data obtained by the HPLC method (Figures 1A - D). At T = 41 days (Figure 1B), the histidine formulation showed a change in the UV spectrum scan profile, indicating the decomposition of polysorbate 80. At T = 77 days (Figure 1C) and T = 144 days (Figure 1D), the histidine formulation showed further decomposition of polysorbate 80 compared to T = 41 days. During the period of this experiment, there was no change in the spectrum scan profile of the succinate formulation.

[0241] The continuous stability of the succinate formulation was also observed over six months in samples maintained at 2 - 8°C and 25°C. The succinate formulation was stable at all times and temperatures tested. Table 11(a) below shows appearance data, pH, osmolality, polysorbate 80 concentration, spectral scan, and microflow imaging (MFI) at the initial, 1-month, 2-month, 3-month, and 6-month time points. Values are rounded to the nearest integer (e.g., 1.2 is rounded to 1.0). [Table 11] JPEG2026062861000056.jpg137158

[0242] Table 11(b) below shows appearance data, pH, osmolality, spectral scan information, polysorbate 80 concentration, spectral scan, and microflow imaging (MFI) data at various temperatures (2 - 8°C, 25°C) and conditions (inverted, upright) at the initial (T0), 1-month (T1), 2-month (T2), 3-month (T3), 6-month (T6), 9-month (T9), and 12-month (T12) time points. In Table 11(b), the data show the number of particles per milliliter observed, and the particles had diameters of ≥2μm, ≥5μm, ≥10μm, or ≥25μm. Values are rounded up to the nearest integer (e.g., 1.2 is rounded up to 2). [Table 12] JPEG2026062861000058.jpg236158JPEG2026062861000059.jpg238158JPEG2026062861000060.jpg^{236158}

[0243] Taken together, this data suggests that polysorbate 80 is far more stable in the succinate-based formulation than in the histidine-based formulation.

[0244] Example 3: Method using IVSS solution with TRI130 IVSS will be supplied in clinical trials along with TRI130 and CD123 x CD3 bispecific proteins.

[0245] IVSS is transported to clinical trial sites refrigerated in disposable 10 mL vials. Until use, IVSS is stored at 2-8°C in a pharmacy or designated locked location.

[0246] Before administering TRI130 to the patient, TRI130 is diluted to prepare the final dose. The TRI130 dilution is prepared in an empty IV bag, which is called a drug dilution bag.

[0247] To prepare the drug dilution bag, gently stir the TRI130 vial 5-6 times without inverting it (do not shake) to ensure that the product is properly mixed for use in the dosage formulation. Add 190 mL of saline to the empty drug dilution bag. Add 9.7 mL of IVSS to the drug dilution bag and gently mix by inverting the bag 5-6 times. Add 0.3 mL of TRI130 to the drug dilution bag and gently mix by inverting the bag 5-6 times.

[0248] To prepare the injectors for patient administration, follow these steps: Label a 50-60 mL injector with the patient's name, study number, drug name, dosage, and date and time of preparation. This is called the patient administration injector. Add the amount of saline ("A" mL) to this labeled empty 60 mL injector. The amount of saline added, "A", depends on the dosage cohort; refer to Table 13.

[0249] Next, add a certain volume of IVSS ("B" mL) from one IVSS vial using the injector and needle. The amount of IVSS added, "B" depends on the dosage cohort; refer to Table 13. Next, remove the needle and transfer the volume ("B" mL) to a 60 mL patient-administered injector using a Baxter RAPIDFILL connector (luer lock-to-luer lock). Push the contents of the injector containing the IVSS into the 60 mL patient-administered injector. Next, slightly loosen the patient-administered injector from the connector and pull the plunger back by another 1 mL to ensure all IVSS is transferred from the injector and connector. Then, tighten the injector back into the connector and mix the contents of the patient-administered injector by gently inverting it 5 or 6 times. Then, detach and discard the IVSS injector and connector.

[0250] The volume of TRI130 ("C" mL) is taken from the drug dilution bag (prepared as described above) using an injector and needle. The amount of drug added from the drug dilution bag, "C" is dose cohort dependent; refer to Table 13. The needle is then removed, and the TRI130 (volume "C") is transferred to the patient injector using a Baxter RAPIDFILL connector.

[0251] Push the contents of the injector containing TRI130 ("C" mL) into a 60 mL patient-administered injector. Slightly loosen the patient-administered injector from the connector and pull the plunger back another 1 mL to ensure all TRI130 is transferred from the injector and connector. Then tighten the injector back into the connector and mix the contents of the patient-administered injector by gently inverting it 5 or 6 times. Disconnect and discard the TRI130 injector and connector.

[0252] Subsequently, attach the filtered IV extension tubing to the patient-administered syringe and remove the end cap from the IV tubing. Extrude 1 mL of solution from the patient-administered syringe through the IV extension tubing and filter to prime the tubing. Since the IV extension tubing and filter use approximately 0.84 mL, approximately 0.16 mL exits the IV tubing and needs to be properly discarded. The end cap of the IV tubing is reattached. The patient-administered syringe and IV tubing and filter are then sent to the hospital floor or infusion center for patient administration.

[0253] Further details regarding the preparation of TRI130 for administration to patients in various cohorts are shown in Tables 12(a), 12(b), 13(a), and 13(b). Tables 12(a) and 12(b) give the volumes of saline, IVSS, and TRI130 formulation for the preparation of the drug dilution bag. Tables 13(a) and 13(b) give the volumes of saline, IVSS, and TRI130 drug solution (from the drug dilution bag) for the preparation of the patient-administered syringe. [Table 13] [Table 14] [Table 15] [Table 16]

[0254] References 1.Cleland,J.,Powell M.,et al;Crit Rev Ther Drug Carrier Syst 10(4):307-377;The development of stable protein formulations:a close look at protein aggregation,deamidation,and oxidation 2.Shire,S.,et al;J Pharma Science 93(6):1390-1402(2004);Challenges in the development of high protein concentration formulations 3.Bruce Kerwin;Journal of Pharm Sciences 97(8):2924-2935(2008);Polysorbate 20 and 80 used in the formulation of protein biotherapeutics:Structure and degradation pathways 4.Nema,S and Brendel R;Journal of Pharmaceutical Science and Technology Vol 65,No 3,May-June 2011;Excipients and their role in approved injectable products:current usage and future directions

Claims

1. A composition for reducing the adsorption of therapeutic proteins to one or more intravenous drug delivery system components, comprising succinate and polysorbate 80.

2. succinate at approximately 1 to 10 mM, and Polysorbate 80 in a concentration of approximately 0.001 (w / v)% to approximately 0.01 (w / v)% The composition according to claim 1, comprising:

3. The composition according to claim 2, comprising approximately 4 mM to approximately 6 mM succinate.

4. The composition according to claim 3, comprising approximately 5 mM succinate.

5. The composition according to any one of claims 2 to 4, comprising approximately 0.002 (w / v)% to approximately 0.008 (w / v)% of polysorbate 80.

6. The composition according to claim 5, comprising approximately 0.004 (w / v)% of polysorbate 80.

7. The composition according to any one of claims 2 to 6, wherein the pH of the composition is about 5.0 to about 7.

0.

8. The composition according to claim 7, wherein the pH of the composition is about 6.

0.

9. A composition according to any one of claims 1 to 8, comprising a therapeutic protein.

10. The composition according to any one of claims 1 to 9, wherein the therapeutic protein comprises at least a first binding domain.

11. The composition according to claim 10, wherein the first binding domain is a single-chain variable fragment (scFv).

12. The composition according to any one of claims 1 to 9, wherein the therapeutic protein comprises at least a first binding domain and a second binding domain.

13. The composition according to claim 12, wherein the first binding domain is a single-chain variable fragment (scFv) and the second binding domain is an scFv.

14. The composition according to claim 12 or 13, wherein the first binding domain specifically binds to CD123.

15. The composition according to any one of claims 12 to 14, wherein the second binding domain specifically binds to CD3ε.

16. The aforementioned therapeutic protein is arranged in order from the amino terminus to the carboxyl terminus. (a) The first binding domain, (b) Hinge region, (c) constant immunoglobulin region, and (d) The second binding domain, A composition according to any one of claims 12 to 15, comprising the above.

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

18. The first binding domain is (i) the immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3; and (ii) The variable region (VL) of the immunoglobulin light chain, including LCDR1, LCDR2, and LCDR3. A composition according to any one of claims 12 to 17, comprising:

19. The composition according to claim 18, wherein HCDR1 includes sequence number 10, HCDR2 includes sequence number 11, and HCDR3 includes sequence number 12.

20. The composition according to claim 18, wherein LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15.

21. The HCDR1 includes sequence number 10, the HCDR2 includes sequence number 11, and the HCDR3 includes sequence number 12, and LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15. The composition according to claim 18.

22. The composition according to any one of claims 12 to 21, wherein the first binding domain comprises a sequence that is at least 95% identical to sequence number 18.

23. The second binding domain described above is (i) the immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3; and (ii) The variable region (VL) of the immunoglobulin light chain, including LCDR1, LCDR2, and LCDR3. A composition according to any one of claims 12 to 22, comprising:

24. The composition according to claim 23, wherein HCDR1 includes sequence number 19, HCDR2 includes sequence number 20, and HCDR3 includes sequence number 21.

25. The composition according to claim 23, wherein LCDR1 includes sequence number 22, LCDR2 includes sequence number 23, and LCDR3 includes sequence number 24.

26. The HCDR1 includes sequence number 19, the HCDR2 includes sequence number 20, and the HCDR3 includes sequence number 21, and The LCDR1 includes sequence number 22, the LCDR2 includes sequence number 23, and the LCDR3 includes sequence number 24. The composition according to claim 23.

27. The composition according to any one of claims 12 to 26, wherein the second binding domain comprises a sequence that is at least 95% or 100% identical to sequence number 27.

28. The composition according to any one of claims 12 to 27, wherein the therapeutic protein comprises the sequence of SEQ ID NO:

31.

29. The therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain. The CD86-binding domain includes a variable heavy chain and a variable light chain that specifically bind CD86, The immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIIb. The monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker, and The aforementioned therapeutic protein is a homodimer. The composition according to claim 12 or 13.

30. The composition according to claim 29, wherein the CD86 binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

31. The composition according to claim 30, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO:

6.

32. The composition according to any one of claims 29 to 31, wherein the CD86 binding domain comprises a variable heavy chain having an amino acid sequence identical to at least 95% of SEQ ID NO: 7, and a variable light chain having an amino acid sequence identical to at least 95% of SEQ ID NO:

8.

33. The composition according to any one of claims 29 to 32, wherein the CD86 binding domain comprises an amino acid sequence that is at least about 95% or 100% identical to that of SEQ ID NO:

9.

34. The composition according to any one of claims 29 to 33, wherein the monomer IL-10 domain contains an amino acid sequence that is at least 95% or 100% identical to that of SEQ ID NO:

28.

35. The composition according to any one of claims 29 to 34, wherein the therapeutic protein comprises an amino acid sequence that is identical to SEQ ID NO: 30, or at least about 90%, at least about 95%, at least about 98%, or at least about 99% of SEQ ID NO:

30.

36. The composition according to any one of claims 9 to 36, wherein the concentration of the therapeutic protein is about 0.01 μg / mL to about 2.0 μg / mL.

37. The composition according to claim 36, wherein the concentration of the therapeutic protein is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, or about 0.09 μg / mL.

38. The composition according to claim 36, wherein the concentration of the therapeutic protein is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 μg / mL.

39. The composition according to claim 36, wherein the concentration of the therapeutic protein is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 μg / mL.

40. A composition according to any one of claims 1 to 39, comprising about 5 mM succinate in water and about 0.0004 (w / v)% polysorbate 80, wherein the pH of the composition is about 6.0 and it is formulated for injection.

41. The composition according to claim 1, comprising approximately 25 to approximately 150 mM succinate and approximately 0.01 (w / v)% to approximately 0.1 (w / v)% polysorbate 80.

42. The composition according to claim 41, having a concentration of 10X to 50X.

43. The composition according to claim 42, having a concentration of 20X.

44. A composition according to any one of claims 41 to 43, comprising approximately 75 mM to approximately 125 mM succinate.

45. The composition according to claim 44, comprising approximately 100 mM succinate.

46. The composition according to any one of claims 41 to 45, comprising approximately 0.05 (w / v)% to approximately 0.1 (w / v)% of polysorbate 80.

47. The composition according to claim 46, comprising approximately 0.08 (w / v)% of polysorbate 80.

48. The composition according to any one of claims 41 to 47, wherein the pH of the composition is about 5.0 to about 7.

0.

49. The composition according to claim 48, wherein the pH of the composition is approximately 6.

0.

50. A composition according to any one of claims 41 to 49, comprising about 100 mM succinate in water and about 0.08 (w / v)% polysorbate 80, wherein the pH of the composition is about 6.0 and it is formulated for injection.

51. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, Approximately 100 mM succinate, Approximately 0.08 (w / v)% polysorbate 80, and Approximately a therapeutically effective amount of therapeutic protein, A composition containing the following:

52. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The aforementioned therapeutic protein is arranged in order from the amino terminus to the carboxyl terminus. (a) A first binding domain that specifically binds to the first target, (b) Hinge region, (c) constant immunoglobulin region, and (d) A second binding domain that specifically binds to a second target, A composition containing the following:

53. The composition according to claim 52, wherein the first target is CD86.

54. The composition according to claim 52, wherein the first target is CD123.

55. The composition according to claim 52, wherein the second target is an IL-10 receptor.

56. The composition according to claim 52, wherein the second target is CD3ε.

57. The composition according to claim 52, wherein the first target is CD86 and the second target is an IL-10 receptor.

58. The composition according to claim 52, wherein the first target is CD123 and the second target is CD3ε.

59. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The aforementioned therapeutic protein is arranged in order from the amino terminus to the carboxyl terminus. (a) First binding domain, (b) Hinge region, (c) constant immunoglobulin region, and (d) Second binding domain, Includes, The first binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3, wherein HCDR1 includes SEQ ID NO: 10, HCDR2 includes SEQ ID NO: 11, HCDR3 includes SEQ ID NO: 12; and LCDR1 includes SEQ ID NO: 13, LCDR2 includes SEQ ID NO: 14, and LCDR3 includes SEQ ID NO:

15. The second binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3, wherein HCDR1 includes SEQ ID NO: 19, HCDR2 includes SEQ ID NO: 20, HCDR3 includes SEQ ID NO: 21; and LCDR1 includes SEQ ID NO: 22, LCDR2 includes SEQ ID NO: 23, and LCDR3 includes SEQ ID NO:

24. composition.

60. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The therapeutic protein comprises the sequence of SEQ ID NO: 31, composition.

61. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain. The CD86-binding domain includes a variable heavy chain and a variable light chain that specifically bind CD86, The immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to the Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIIb. The monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker, and The aforementioned therapeutic protein is a homodimer. composition.

62. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain. The CD86-binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and (2) an immunoglobulin light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO:

6. The monomer IL-10 domain has the amino acid sequence of SEQ ID NO:

28. composition.

63. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain. The CD86-binding domain comprises the amino acid sequence of SEQ ID NO: 9, and The monomer IL-10 domain comprises the amino acid sequence of SEQ ID NO:

28. composition.

64. A composition for reducing the adsorption of proteins to one or more intravenous drug delivery system components, succinate of approximately 1 to 10 mM, Polysorbate 80 in an amount of approximately 0.001 (w / v)% to approximately 0.01 (w / v)%, and Therapeutic proteins ranging from approximately 0.01 μg / mL to approximately 2.0 μg / mL, Includes, The therapeutic protein comprises the sequence of SEQ ID NO: 30, composition.

65. A container adapted for holding a therapeutic protein, wherein the inner surface of the container is first in contact with the composition according to any one of claims 1 to 64, and then in contact with the composition containing the therapeutic protein.

66. The container according to claim 65, which is substantially latex-free.

67. A container according to claim 65 or 66 that is substantially free of bis(2-ethylhexyl) phthalate (DEHP).

68. A container according to any one of claims 65 to 67, selected from the group consisting of an IV bag, an injector, and a tube.

69. A method for constructing an intravenous drug delivery system for the delivery of therapeutic proteins, To provide at least one container adapted to hold the therapeutic protein, and Before adding the therapeutic protein to the at least one container, the inner surface of the at least one container is brought into contact with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80. Methods that include...

70. The method according to claim 69, wherein the composition coats the inner surface of at least one container and prevents the therapeutic protein from binding to the inner surface of the container.

71. The method according to claim 69 or 70, wherein the at least one container is substantially free of latex.

72. The method according to any one of claims 69 to 71, wherein the at least one container is substantially free of bis(2-ethylhexyl) phthalate (DEHP).

73. The method according to any one of claims 69 to 72, wherein the at least one container is selected from the group consisting of an IV bag, an injector, and a tube.

74. A method of treating a subject by intravenous administration of a therapeutic protein, To provide at least one container adapted to hold the therapeutic protein, The composition comprising approximately 1 to approximately 10 mM succinate and approximately 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80 is brought into contact with the inner surface of the container. Bringing the composition containing the therapeutic protein into contact with the inner surface of the container, and The therapeutic protein is administered intravenously to the patient. Methods that include...

75. The method according to claim 74, wherein the therapeutic protein comprises at least a first binding domain.

76. The method according to claim 75, wherein the first binding domain is a single-chain variable fragment (scFv).

77. The method according to claim 74, wherein the therapeutic protein comprises at least a first binding domain and a second binding domain.

78. The method according to claim 77, wherein the first binding domain is a single-chain variable fragment (scFv) and the second binding domain is an scFv.

79. The method according to claim 77 or 78, wherein the first binding domain specifically binds to CD123.

80. The method according to any one of claims 77 to 79, wherein the second binding domain specifically binds CD3ε.

81. The aforementioned therapeutic protein is arranged in order from the amino terminus to the carboxyl terminus. (a) The first binding domain, (b) Hinge region, (c) constant immunoglobulin region, and (d) The second binding domain, The method according to any one of claims 77 to 80, including the method described in any one of claims 77 to 80.

82. The method according to claim 81, wherein the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD.

83. The first binding domain is (i) the immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3; and (ii) The variable region (VL) of the immunoglobulin light chain, including LCDR1, LCDR2, and LCDR3. The method according to any one of claims 77 to 82, including the method described in any one of claims 77 to 82.

84. The method according to claim 83, wherein HCDR1 includes sequence number 10, HCDR2 includes sequence number 11, and HCDR3 includes sequence number 12.

85. The method according to claim 83, wherein LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15.

86. The HCDR1 includes sequence number 10, the HCDR2 includes sequence number 11, and the HCDR3 includes sequence number 12, LCDR1 includes sequence number 13, LCDR2 includes sequence number 14, and LCDR3 includes sequence number 15. The method according to claim 83.

87. The method according to any one of claims 77 to 86, wherein the first binding domain comprises a sequence that is at least 95% or 100% identical to sequence number 18.

88. The second binding domain described above is (i) the immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3, and (ii) The variable region (VL) of the immunoglobulin light chain, including LCDR1, LCDR2, and LCDR3. The method according to claim 77, including the method described in claim 77.

89. The method according to claim 88, wherein HCDR1 includes sequence number 19, HCDR2 includes sequence number 20, and HCDR3 includes sequence number 21.

90. The method according to claim 88, wherein LCDR1 includes sequence number 22, LCDR2 includes sequence number 23, and LCDR3 includes sequence number 24.

91. The HCDR1 includes sequence number 19, the HCDR2 includes sequence number 20, and the HCDR3 includes sequence number 21, The LCDR1 includes sequence number 22, the LCDR2 includes sequence number 23, and the LCDR3 includes sequence number 24. The method according to claim 88.

92. The method according to any one of claims 77 to 91, wherein the second binding domain includes a sequence that is at least 95% identical to sequence number 27.

93. The method according to any one of claims 77 to 91, wherein the therapeutic protein comprises the sequence of SEQ ID NO:

31.

94. The therapeutic protein comprises, in order from the amino terminus to the carboxyl terminus, a CD86-binding domain, an immunoglobulin hinge domain, an immunoglobulin Fc domain, and a monomer IL-10 domain. The CD86-binding domain includes a variable heavy chain and a variable light chain that specifically bind to CD86. The immunoglobulin Fc domain is an IgG1 Fc domain containing two or more mutations that prevent or significantly reduce binding to the Fc receptors FcγR, FcγRIIa, FcγRIIb, and FcγRIIIIb. The monomer IL-10 domain comprises two human IL-10 subunits separated by a short linker, and The aforementioned therapeutic protein is a homodimer. The method according to any one of claims 77 to 80.

95. The method according to claim 94, wherein the CD86-binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

96. The method according to claim 95, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO:

6.

97. The method according to any one of claims 94 to 96, wherein the CD86 binding domain comprises a variable heavy chain having an amino acid sequence identical to at least 95% or 100% of SEQ ID NO: 7, and a variable light chain having an amino acid sequence identical to at least 95% or 100% of SEQ ID NO:

8.

98. The method according to any one of claims 94 to 97, wherein the CD86-binding domain comprises an amino acid sequence that is at least about 95% or 100% identical to that of SEQ ID NO:

9.

99. The method according to any one of claims 94 to 98, wherein the monomer IL-10 domain contains an amino acid sequence that is at least 95% or 100% identical to that of SEQ ID NO:

28.

100. The method according to any one of claims 94 to 99, wherein the therapeutic protein comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to SEQ ID NO: 30 or SEQ ID NO:

30.

101. The method according to any one of claims 74 to 100, wherein the therapeutic protein is administered by intravenous infusion.

102. The method according to any one of claims 74 to 101, wherein the composition coats the inner surface of at least one container and prevents the therapeutic protein from binding to the inner surface of the container.

103. The method according to any one of claims 74 to 102, wherein the subject is a mammal.

104. The method according to claim 103, wherein the subject is a human being.

105. A drug delivery system for delivering therapeutic proteins to patients, It includes at least one container adapted to hold the therapeutic protein, A system wherein the inner surface of at least one container is in contact with a composition comprising about 1 to about 10 mM succinate and about 0.001 (w / v)% to 0.01 (w / v)% polysorbate 80, and then the container is in contact with the composition comprising the therapeutic protein.

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