Activatable bispecific anti-CD89 and anti-PD-L1 proteins and their uses

Abstract

A protein molecule is provided herein that specifically binds to PD-L1 and also exhibits specific CD89 binding that can be activated in diseased tissue. Furthermore, the use of such a protein molecule for treating cancer is provided herein. The protein molecule comprises a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain, and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD89 VL domain, and a second immunoglobulin light chain constant region.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 497,092, filed on 19 April 2023, which is incorporated herein by reference in its entirety.

[0002] Technical field This disclosure relates to activatable bispecific proteins and cancer treatment. [Background technology]

[0003] background In immuno-oncology therapy, most major drug targets are not expressed only in diseased tissue, but also in non-disease tissue. In addition, many drugs used to treat cancer utilize very potent cell-killing mechanisms. Consequently, drug engagement of targets in non-disease tissue often leads to undesirable side effects.

[0004] PD-L1 is a cell surface receptor that is a member of the immunoglobulin superfamily and is primarily expressed on myeloid cells and regulatory T (Treg) cells in non-disease tissues. However, PD-L1 has also been observed to be highly expressed on some cancer cells. PD-L1 binds to the membrane protein PD1. The interaction between PD-L1 and PD1 on T cells downregulates the inflammatory activity of T cells and promotes immune autotolerance. Therefore, PD-L1 has been described as an immune checkpoint. Consequently, antagonist anti-PD-L1 monoclonal antibodies that block interaction with PD1 have demonstrated potential as immunotherapeutic agents that exhibit good tolerability in disease environments such as cancer by freeing the anti-cancer T cell response from innate immunosuppression. Thus, PD-L1 is a drug target used to amplify the anti-cancer effects of the adaptive immune system. However, in many cases, tumors can use multiple "evasion mechanisms" to neutralize the effects of anti-PD-L1 agents.

[0005] CD89 is a receptor for IgA and binds to the heavy chain constant region of IgA antibodies. CD89 is present on the cell surface of myeloid cells, including neutrophils, monocytes, macrophages, and eosinophils. Binding and activation of CD89 by IgA leads to potent induction of antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) against IgA-opsonized cells, which can be significantly more potent than induction by similar IgGs. Despite these observations, the development of IgA antibodies as therapeutic agents has been unsuccessful overall due to the difficulty in expressing and purifying IgA antibodies and their poor pharmacokinetics (PK) in humans, which minimize exposure to diseased tissues.

[0006] Historically, antibodies generated against the CD89 extracellular domain do not compete with the binding of endogenous IgA. When binding to CD89, these antibodies can induce direct activation signals, leading to an increase in the inflammatory activity of innate immune cells. Using the binding domains from these antibodies, bispecific agents that ligate to CD89 have also been created, which can direct the killing activity of innate immune cells towards target-rich diseased tissues. Thus, an anti-PD-L1 antibody can become a more potent and broad-acting therapeutic agent by acquiring the ability to also bind to CD89, thereby strongly enhancing the killing mechanism of innate immune cells in a PD-L1 antibody-resistant disease environment. This can be combined with "synthetic IgA immunity" that can induce the function of major checkpoint inhibitors to synergistically stimulate the adaptive immune system. However, the ability to make this combination function in a single therapeutic agent structure (e.g., in a standard bispecific antibody format with fully active PD-L1 and CD89 binding domains) is limited by the relatively broad expression profiles of both PD-L1 and CD89 in many cell types such as T cells and innate immune cells, as well as others. This broad expression profile can cause not only dose-limiting toxicity to PD-L1 / CD89 binders but also serious peripheral sink / biodistribution problems that limit the ability of such agents to achieve a highly sufficient exposure in diseased tissues to utilize complex mechanisms. Therefore, there is a need for engineered forms of bispecific binding proteins with activity that specifically targets the diseased tissue environment. Summary of the Invention Means for Solving the Problems

[0007] Abstract As used herein, provided is a protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, wherein the heavy chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain, and the light chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD89 VL domain, and a second immunoglobulin light chain constant region.

[0008] In some embodiments, the heavy chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0009] In some embodiments, the protein further comprises a third polypeptide chain containing a hinge and an Fc region. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32.

[0010] In some embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the second linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0011] In some embodiments, the anti-PD-L1 VH domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14; the anti-PD-L1 VL domain includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; the anti-CD89 VH domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, and HCDR3 containing the amino acid sequence of SEQ ID NO: 20; and the anti-CD89 VL domain includes LCDR1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 23.

[0012] In some embodiments, the anti-PD-L1 VH domain includes the amino acid sequence of SEQ ID NO: 24, and the anti-PD-L1 VL domain includes the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CD89 VH domain includes the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CD89 VL domain includes the amino acid sequence of SEQ ID NO: 27.

[0013] In some embodiments, the heavy chain includes the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the light chain includes the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0014] In this specification, (a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 30, the light chain comprises the amino acid sequence of SEQ ID NO: 28, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32; or (b) A protein is provided in which the heavy chain comprises the amino acid sequence of SEQ ID NO: 31, the light chain comprises the amino acid sequence of SEQ ID NO: 29, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32.

[0015] In some embodiments, the heavy chain includes IgG, IgE, IgM, IgD, IgA, or IgY constant regions. In some embodiments, the heavy chain includes IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 constant regions. In some embodiments, the heavy chain includes an immunologically inactive constant region. In some embodiments, the heavy chain includes a wild-type human IgG1 constant region, a human IgG1 constant region with amino acid substitutions L234A, L235A, and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, or a human IgG4 constant region with amino acid substitution S228P, with numbering following an EU index such as Kabat.

[0016] This specification provides immunoconjugates comprising proteins disclosed herein, which are conjugated to therapeutic agents. In some embodiments, the therapeutic agent is a cytotoxic agent, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell proliferation inhibitory enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or an apoptosis promoter.

[0017] Pharmaceutical compositions comprising a protein or immunoconjugate disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient are provided herein.

[0018] Provided herein are nucleic acid molecules encoding (a) a heavy chain amino acid sequence; (b) a light chain amino acid sequence; or (c) both a heavy chain amino acid sequence and a light chain amino acid sequence of a protein disclosed herein.

[0019] An expression vector comprising nucleic acid molecules disclosed herein is provided herein.

[0020] Recombinant host cells comprising nucleic acid molecules or expression vectors disclosed herein are provided herein.

[0021] This specification provides a method for producing a protein, comprising: culturing recombinant host cells containing an expression vector disclosed herein under conditions in which a nucleic acid molecule is expressed, thereby producing a protein; and isolating the protein from the host cells or culture.

[0022] This specification provides a method for enhancing an anti-cancer immune response in a subject, the method comprising administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0023] This specification provides a method for treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0024] This specification provides a method for improving the symptoms of cancer in a subject, wherein the method comprises administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0025] In some embodiments of the methods provided herein, cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. [Brief explanation of the drawing]

[0026] [Figure 1]Figure 1 shows an exemplary asymmetric single-arm construct of the protein molecule disclosed herein in the conformations intact (left), activated protease cleavage (center), and inactivated protease cleavage (right). In the intact conformation, the anti-PD-L1 Fab-binding domain is exposed and can bind to its cognitive target. The anti-CD89 Fab domain is inhibited from binding by linkers in both the heavy and light chains, both of which are cleavable by proteolysis and can be sequentially cleaved by matrix metalloproteinases (MMPs) and / or cathepsins. The first cleavage event creates an intermediate active state that allows both anti-PD-L1 and anti-CD89 Fab from a single protein construct to bind to their cognitive targets, which may direct the activation of CD89+ immune cells in the PD-L1+ immunosuppressive microenvironment. The second cleavage dissociates the anti-PD-L1 and anti-CD89 Fabs, eliminating the ability of the single molecule to simultaneously bind to both PD-L1 and CD89.

[0027] [Figure 2] Figure 2 illustrates the mechanism of activity of the activatable bispecific protein molecule provided herein. Because the released activity of the CD89-binding domain is essentially monomeric, it cannot directly induce CD89 activation and relies on binding to PD-L1+ cells, resulting in the presentation of CD89 binding in trans to nearby immune cells and inducing CD89 signaling. The activated molecule thereby simultaneously blocks PD-L1 / PD1 signaling, restoring uninhibited TCR activation in T cells and providing CD89 signaling to amplify ADCC and ADCP activity in innate immune cells.

[0028] [Figure 3] Figure 3 shows the structure of an exemplary symmetrical two-arm construct of a protein molecule described herein. [Modes for carrying out the invention]

[0029] Detailed explanation In oncology, there are two major problems that limit the effectiveness of CD89 activation and tumor-targeted bispecific antibody drugs:

[0030] 1) Antibody target proteins found on cancer cells (e.g., PD-L1) can be expressed not only on tumor cells but also on many different cell classes in the body. This extratumor target expression may carry a risk of dose-limiting side effects because the CD89-binding domain in the standard bispecific molecule is constitutively active and can therefore direct the activation of CD89+ cells in the presence of any PD-L1+ cells, regardless of whether PD-L1+ cells are present in diseased tissue or not. Furthermore, extratumor target expression may also result in an antigen "sync" effect, thereby reducing the amount of drug that infiltrates the tumor.

[0031] 2) CD89-positive cells, such as neutrophils and monocytes, are found at high concentrations in the bloodstream and other tissues, creating a significant sink effect on this arm and influencing the distribution of free drugs available for in vivo distribution and tumor invasion.

[0032] Both factors described above minimize the potential safety and efficacy of bispecific antibodies that drive CD89 engagement and activation. The anti-PD-L1 and anti-CD89 proteins provided herein (see, for example, Figure 1) overcome peripheral sink and toxicity issues by minimizing CD89 binding outside disease tissue. This effect is achieved by adding a PD-L1 binding domain and linker above (i.e., at the amino-terminal) the CD89 binding domain. The use of an appropriate combination of upper domains and linkers yields a configuration that minimizes binding activity at the lower (i.e., carboxy-terminal) CD89 domain. The PD-L1 domain then drives high concentrations in the PD-L1-rich tumor microenvironment. This protein construct linker system utilizes the increased MMP and cathepsin activity common to solid tumors to cleave the linker peptide, exposing the CD89 binding domain and thereby conditionally activating CD89 activation activity within the tumor rather than peripherally. As a result, these combined biological functions give the molecule the potential to evade peripheral CD89 sinking and maximize the T-cell immune response against cancer cells, as outlined in Figure 2.

[0033] A protein that is conditionally activated in diseased human tissue is provided herein. The protein of this disclosure is fully active in specifically binding and blocking PD-L1 throughout the body; shows minimal CD89 binding in healthy tissue; and is highly activated in CD89 binding and activation in a PD-L1-positive diseased tissue environment. The protein of this disclosure comprises a CD89-binding domain that is masked by a PD-L1-binding domain in non-disease tissue. The protein also comprises two peptide linkers that are cleaved by one or more proteases expressed in diseased tissue (e.g., tumor). Cleavage of the linkers removes the mask of the CD89-binding domain in diseased tissue, thereby enabling selective binding and / or function of the protein in diseased tissue.

[0034] protein molecule A protein comprising two Fab fragments (anti-PD-L1 Fab and anti-CD89 Fab) is provided herein. This protein is monovalent in its intact structure and, when activated, can only have at most monovalent CD89 binding to minimize the risk of peripheral toxicity associated with bivalent activated anti-CD89 antibodies.

[0035] In some embodiments, the protein comprises a first polypeptide chain including a heavy chain and a second polypeptide chain including a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain, and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD89 VL domain, and a second immunoglobulin light chain constant region. In some embodiments, the heavy chain further comprises an immunoglobulin hinge region and an Fc domain at its C-terminus. In some embodiments, the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0036] In some embodiments, the protein further comprises a third polypeptide chain including a hinge and an Fc region. The third polypeptide chain is sometimes referred to as the "Fc-stump".

[0037] Figure 1 shows an example of the domain-labeled protein of this disclosure.

[0038] The first and second linkers are cleavable by matrix metalloproteinases (MMPs) and / or cathepsins found in diseased tissues such as tumors. The linkers within the protein are immunoglobulin-derived hinge sequences that are sensitive to proteolysis and can be sequentially cleaved. The first cleavage occurs in an intact structure, resulting in an intermediate active state that allows anti-PD-L1 Fab and anti-CD89 Fab from a single protein construct to bind to their cognitive targets. A second cleavage event at the second linker removes the covalent bond between anti-PD-L1 Fab and anti-CD89 Fab, resulting in the loss of the molecule's ability to recruit PD-L1+ cells to kill T cells. This secondary cleavage event constitutes a "self-destruct mechanism" that minimizes the risk of the activating molecule evading the tumor microenvironment. Severing linkers based on immunoglobulin hinge sequences may also mobilize enhanced immunoeffector functions at the cell membrane via endogenous anti-hinge antibodies (antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell phagocytosis (ADCP)), a known phenomenon in human patients with (or without) autoreactive underlying diseases.

[0039] In some embodiments, the protein comprises one or more amino acid sequences provided in Table 1 or Table 2. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] In the VH and VL domain sequences, the CDR sequence is underlined. [Table 2-1] [Table 2-2]

[0040] The design of the anti-PD-L1 / anti-CD89 protein may be based on a sequence derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector function.

[0041] The proteins disclosed herein include domains and regions of antibody molecules. The term “antibody” broadly refers to an immunoglobulin (Ig) molecule, which generally consists of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential target-binding properties of the Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0042] In full-length antibodies, each heavy chain contains a heavy chain variable domain (abbreviated herein as the VH domain) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. The constant regions of IgG, IgA, and IgD contain a flexible hinge region between the CH1 and CH2 domains. Each light chain contains a light chain variable domain (abbreviated herein as the VL domain) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and regions containing more conserved regions called framework regions (FRs). Each VH and VL domain consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Framework areas and CDRs are defined and described, for example, in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, or IMGT Lefranc MP. Unique database numbering system for immunogenetic analysis. Immunol Today. 1997 Nov;18(11):509.

[0043] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be the native Fc region or a variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The numbering of residues in the Fc region follows EU indices such as Kabat. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. The Fc region can exist in dimeric or monomeric form. The Fc region binds to various cellular receptors, such as Fc receptors, and to other immune molecules, such as complement proteins.

[0044] The protein provided herein comprises two Fab fragments. The Fab fragments are monovalent antigen-binding fragments consisting of VL, VH, CL, and CH1 domains.

[0045] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2) or subclass. IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains, as well as two antigen-binding domains, each composed of VH and VL. Generally, IgA antibodies consist of two monomers, each monomer consisting of two heavy chains and two light chains (similar to IgG, IgD, and IgE antibodies); thus, an IgA molecule has four antigen-binding domains, each again composed of VH and VL. A particular IgA antibody is monomeric in that it consists of two heavy chains and two light chains. Secreted IgM antibodies are generally composed of five monomers, each monomer consisting of two heavy chains and two light chains (similar to IgG and IgE antibodies). Therefore, the IgM molecule has 10 antigen-binding domains, each again consisting of VH and VL. The cell surface morphology of IgM has a two-heavy-chain / two-light-chain structure, similar to IgG, IgD, and IgE antibodies.

[0046] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody or an antigen-binding portion thereof), or a protein containing a binding domain derived from an immunoglobulin, and an antigen to which the immunoglobulin or protein is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (K d ) of that interaction, with a smaller K d indicating higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of an antigen-binding site / antigen complex, which rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both the "on-rate constant" (K on ) and the "off-rate constant" (K off ) can be determined by calculation of concentration and the actual association and dissociation rates. (See Malmqvist, Nature 361:186-187 (1993)) The ratio of K off / K on makes it possible to cancel out all parameters not related to affinity and is equal to the dissociation constant K d . (See Davies et al. (1990) Annual Rev Biochem 59:439-473) An antibody or antigen-binding portion provided herein is said to specifically bind to PD-L1 or CD89 when the equilibrium binding constant (K d ), measured by an assay such as a radioligand binding assay or a similar assay known to those skilled in the art, is from about 1 pM to 10 μM or less, preferably 10 nM or less, more preferably 10 nM or less, and most preferably 100 pM or less. The K dOne method for determining this is by using surface plasmon resonance (SPR), typically by using a biosensor system such as the Biacore® system.

[0047] Functionally, the binding affinity of the proteins provided herein is 10 -5 M~10 -12 It may be within the range of M. For example, the binding affinity of the protein provided herein is 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10-7 M, 10 -6 M~10 -7 M, or 10 -5 M~10 -6 It is M.

[0048] This specification provides a protein comprising a first polypeptide chain including a heavy chain and a second polypeptide chain including a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain, and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD89 VL domain, and a second immunoglobulin light chain constant region.

[0049] In some embodiments, the heavy chain includes, from N-terminus to C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0050] In some embodiments, the protein further comprises a third polypeptide chain including a hinge and an Fc region. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32.

[0051] In some embodiments, the first linker includes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the second linker includes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0052] In some embodiments, the anti-PD-L1 VH domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14; the anti-PD-L1 VL domain includes LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17; the anti-CD89 VH domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, and HCDR3 containing the amino acid sequence of SEQ ID NO: 20; and the anti-CD89 VL domain includes LCDR1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 23.

[0053] In some embodiments, the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO: 24, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 25.

[0054] In some embodiments, the anti-CD89 VH domain includes the amino acid sequence of SEQ ID NO: 26.

[0055] In some embodiments, the anti-CD89 VL domain includes the amino acid sequence of SEQ ID NO: 27.

[0056] In some embodiments, the heavy chain includes the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the heavy chain includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31.

[0057] In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0058] In some embodiments, as described herein, (a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 30, the light chain comprises the amino acid sequence of SEQ ID NO: 28, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32; or (b) A protein is provided in which the heavy chain comprises the amino acid sequence of SEQ ID NO: 31, the light chain comprises the amino acid sequence of SEQ ID NO: 29, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32.

[0059] Proteins comprising heavy and light chains are also provided herein, wherein the heavy chain comprises an amino acid sequence provided herein having one, two, or three conserved amino acid substitutions, and the light chain comprises an amino acid sequence provided herein having one, two, or three conserved amino acid substitutions. In some embodiments, the conserved amino acid substitutions are made only in the FR sequence and not in the CDR sequence. In some embodiments, the conserved amino acid substitutions are not made in the first or second linker sequence.

[0060] In some embodiments, the proteins provided herein include an immunoglobulin heavy chain constant region at the C-terminus of the heavy chain. In some embodiments, the immunoglobulin heavy chain constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin heavy chain constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin heavy chain constant region is IgG1. In some embodiments, the immunoglobulin heavy chain constant region is immunologically inactive. In some embodiments, the immunoglobulin heavy chain constant region includes one or more mutations to reduce or inhibit FcγR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the constant region of the immunoglobulin heavy chain is a wild-type human IgG1 constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S, or a human IgG4 constant region containing amino acid substitution S228P, with numbering following an EU index such as Kabat. In some embodiments, the positions of amino acid residues in the constant region of the immunoglobulin molecule are numbered according to an EU index such as Kabat (Ward et al., 1995 Therap. Immunol. 2:77-94).

[0061] In some embodiments, the proteins provided herein may include an immunoglobulin light chain constant region which is a kappa light chain. In some embodiments, the kappa light chain includes SEQ ID NO: 7.

[0062] In some embodiments, the proteins provided herein may include an immunoglobulin light chain constant region, which is a lambda light chain.

[0063] In some embodiments, the proteins provided herein may include an immunoglobulin heavy chain constant region comprising the amino acid sequences of the Fc regions of human IgG4, human IgG4(S228P), human IgG2, human IgG1, and human IgG1 effector null. For example, the Fc region of human IgG4(S228P) includes the following substitution compared to the Fc region of wild-type human IgG4: S228P. For example, the Fc region of human IgG1 effector null includes the following substitutions compared to the Fc region of wild-type human IgG1: L234A, L235A, and G237A. In some embodiments, the protein may include an immunoglobulin heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 8, SEQ ID NOs. 9, SEQ ID NOs. 10, and SEQ ID NOs. 11.

[0064] An immunoconjugate comprising a protein disclosed herein, linked to a therapeutic agent, is provided herein. In some embodiments, the therapeutic agent is a cytotoxic agent, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell proliferation inhibitory enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or an apoptosis promoter.

[0065] Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, anti-angiogenic agents, antiproliferative agents, apoptosis inducers, and cell proliferation inhibitory enzymes and cytolytic enzymes (e.g., RNAses). Further therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, anti-angiogenic agents, antiproliferative agents, or apoptosis promoters. Since the descriptions of these drugs are not mutually exclusive, therapeutic agents may be described using one or more of the above terms.

[0066] Suitable therapeutic agents for use in immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansine, CC-1065 and duocalmycin, calicheamicin and other enediins, and auristatin. Other examples include antifolic acid agents, vinca alkaloids, and anthracyclines. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers may also be used in immunoconjugates. In addition, conjugates can be prepared using secondary carriers such as liposomes or polymers as cytotoxic agents. Suitable cytotoxins include agents that inhibit or block cellular function and / or cause cellular destruction. Typical cytotoxins include antibiotics, tubulin polymerization inhibitors, alkylating agents that bind to and disrupt DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins, such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins.

[0067] Representative cytotoxins include doxorubicin, daunorubicin, idarubicin, acralubicin, zorubicin, mitoxantrone, epirubicin, carbicin, nogaramycin, menogalyl, pirarubicin, barurubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuhdine, capecitabine, cladribine, decitabine, floxuhdine, fludarabine, gugerotin, puromycin, tegafur, thiazophrene, adriamycin, and cis. Examples of platins include, but are not limited to, cisplatin and carboplatin, mitomycin, thiotepa, taxane, vincristine, mitoxantrone, bleomycin, mechloretamine, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, taxol, taxol analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxane, vincristine, daunorubicin, epirubicin, actinomycin, anthramycin, azaserin, bleomycin, tamoxifen, idarubicin, dorastatin / aulistatin, hemiasterin, esperamicin, and meitansinoids.

[0068] Suitable immunomodulatory agents include anti-hormones that block hormonal effects on tumors, and immunosuppressants that suppress cytokine production, downregulate autoantigen expression, or mask MHC antigens. [Table 3]

[0069] Pharmaceutical composition The activatable proteins provided herein (also referred to herein as “active compounds”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise a protein (or an immunoconjugate containing the protein) and a pharmaceutically acceptable carrier, diluent, or excipient. Such materials should be non-toxic and should not interfere with the efficacy of the protein. The exact nature of the carrier or other materials depends on the route of administration, which may be by injection, bolus, infusion, or any other suitable route as discussed below.

[0070] As used herein, the term “pharmaceutically acceptable” means molecular entities and compositions that, when administered using routes known in the art, do not generally cause allergic reactions or other serious adverse reactions. Molecular entities and compositions approved by U.S. federal or state regulatory authorities, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, more specifically in humans, are considered “pharmaceutically acceptable.” As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for drug administration. Suitable carriers are listed in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in the art, which is incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in compositions is intended unless any conventional media or agent is incompatible with the active compound. Auxiliary active compounds may also be incorporated into compositions. Pharmaceutically acceptable carriers, diluents, or excipients may be compounds or combinations of compounds that do not induce secondary reactions and, for example, facilitate the administration of proteins, enhance their lifespan and / or efficacy in the body, or increase their solubility in solution.

[0071] The pharmaceutical compositions disclosed herein may be formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and isotonic modifiers such as sodium chloride or dextrose. pH may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0072] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow for easy needle penetration. It must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Inhibition of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, it is preferable to include isotonic agents in the composition, such as sugars, polyhydric alcohols like mannitol and sorbitol, and sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0073] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing one or a combination of the components listed above, and subsequently sterilizing by filtration, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and any other required components not listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, where the powder of the active component plus any further desired components is obtained from a previously sterile filtered solution.

[0074] Oral compositions generally contain an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is applied orally, rinsed and spat out, or swallowed. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders, e.g., microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch or lactose; disintegrants, e.g., alginic acid, Primojel®, or corn starch; lubricants, e.g., magnesium stearate; lubricants, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose or saccharin; or flavoring agents, e.g., peppermint, methyl salicylate, or orange flavoring.

[0075] For administration by inhalation, the compound may be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or from a nebulizer.

[0076] Systemic administration may also be by mucosal or percutaneous means. With regard to mucosal or percutaneous administration, a penetrating agent suitable for the barrier to be penetrated may be used in the formulation. Such penetrating agents are generally known in the art, and examples of these, with regard to mucosal administration, include cleansing agents, bile salts, and fusidic acid derivatives. Mucosal administration may be achieved through the use of nasal sprays or suppositories. With regard to percutaneous administration, the active compound is formulated into ointments, plasters, gels, or creams, as is generally known in the art.

[0077] The pharmaceuticals may also be prepared in the form of suppositories (for example, with conventional suppository bases, such as cocoa butter and other glycerides) or in the form of retained enemas for rectal delivery.

[0078] In some embodiments, the active compound is prepared with a controlled-release formulation, including a carrier that protects the compound from rapid elimination from the body, such as an implant and a microencapsulation delivery system. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Methods for preparing such formulations are apparent to those skilled in the art. The materials are also commercially available. Liposome suspensions can also be used as pharmaceutically acceptable carriers.

[0079] Formulating oral or parenteral compositions in dosing unit forms for ease of administration and uniformity of dosage is particularly advantageous. As used herein, a dosing unit form refers to a physically distinct unit suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications for the dosing unit forms of the present invention are determined and directly depend on the specific properties of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art for formulating such active compounds for the treatment of an individual.

[0080] In some embodiments, proteins may be provided in a lyophilized form for reconstitution before administration. For example, a lyophilized antibody molecule may be reconstituted in sterile water and mixed with saline before administration to an individual.

[0081] The pharmaceutical compositions provided herein may be included in a container, pack, or dispenser, along with instructions for administration.

[0082] Methods for producing nucleic acid molecules, vectors, host cells, and proteins Nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding the amino acid sequence of a protein disclosed herein (or the amino acid sequence of (i) the VH domain, (ii) the VL domain, or (iii) both the VH domain and the VL domain of a protein) are provided herein. Furthermore, nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding the (i) heavy chain, (ii) light chain, or (iii) both the heavy chain and the light chain of a protein disclosed herein are provided herein. In some embodiments, the nucleic acid molecule encoding the VH domain, VL domain, heavy chain, or light chain includes a signal sequence (or encodes a leader peptide). In some embodiments, the nucleic acid molecule encoding the VH domain, VL domain, heavy chain, or light chain does not include a signal sequence (or does not encode a leader peptide).

[0083] Expression vectors comprising nucleic acid molecules disclosed herein are also provided herein. In a particular vector, the nucleic acid molecule is operably ligated to one or more regulatory sequences suitable for the expression of a nucleic acid segment in a host cell. In some cases, the expression vector includes a sequence that mediates replication and contains one or more select markers. As used herein, “vector” means a construct that can deliver, preferably express, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0084] Recombinant host cells comprising an expression vector or nucleic acid molecule disclosed herein are provided herein. “Host cell” includes individual cells, cell lines or cell cultures that may or may have been recipients of a vector for the incorporation of a polynucleotide insertion. Host cells include offspring of a single host cell. Offspring may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to spontaneous, accidental, or planned mutations. Expression vectors may be transfected into host cells by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. In some embodiments, the recombinant host cell comprises a single vector or single nucleic acid molecule encoding both the heavy and light chains of the protein disclosed herein. In some embodiments, the recombinant host cell comprises (i) a first vector or first nucleic acid molecule encoding the heavy chain of the protein disclosed herein and (ii) a second vector or second nucleic acid molecule encoding the light chain of the protein disclosed herein.

[0085] The protein molecule or portion thereof of the present invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthesis techniques, computational techniques, or combinations thereof, or other techniques readily known in the art.

[0086] Furthermore, a method for producing the protein disclosed herein is provided, comprising culturing recombinant host cells containing the expression vector disclosed herein under conditions in which a nucleic acid segment is expressed, thereby producing the protein. The protein can then be isolated from the host cells or culture. A method for producing the protein is provided herein, comprising culturing recombinant host cells containing the expression vector disclosed herein under conditions in which a nucleic acid molecule is expressed, thereby producing the protein, and isolating the protein from the host cells or culture.

[0087] The proteins disclosed herein may be produced by any of the various methods known to those skilled in the art. In certain embodiments, the proteins disclosed herein may be produced by recombination. For example, a nucleic acid sequence, or a portion thereof, encoding one or more of the heavy or light chains provided herein may be introduced into bacterial cells (e.g., E. coli, B. subtilis) or eukaryotic cells (e.g., yeast such as S. cerevisiae, or mammalian cells such as CHO cell lines, various Cos cell lines, HeLa cells, HEK293 cells, various myeloma cell lines, or transformed B cells or hybridomas), or into an in vitro translation system, and the translated polypeptide may be isolated. In some embodiments, the light and heavy chain proteins are produced intracellularly, along with signal sequences that are removed during the production of the mature proteins disclosed herein.

[0088] Those skilled in the art can determine whether a protein containing a given polypeptide sequence binds to the PD-L1 protein and / or CD89 protein using standard methodologies, such as Western blotting and ELISA.

[0089] Medical use of activatable proteins Methods and uses of activatable proteins, immunoconjugates, and pharmaceutical compositions disclosed herein for providing therapeutic benefits to subjects with cancer.

[0090] The activatable proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may be used in methods of treating the human or animal body, including prophylactic or preventive measures (e.g., pre-onset treatment in a subject to reduce the risk of a condition occurring in a subject; delay its onset; or reduce its severity after onset). Methods of treatment may include administering the protein, immunoconjugate, or pharmaceutical composition to a subject in need.

[0091] A method for enhancing an anti-cancer immune response in a subject is provided herein, comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein. In some embodiments, the anti-cancer immune response is a T-cell response. In some embodiments, the anti-cancer immune response is a complement response.

[0092] This specification provides a method for treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0093] This specification provides a method for improving the symptoms of cancer in a subject, wherein the method comprises administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0094] This specification provides a method for reducing the size of a tumor in a subject, the method comprising administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0095] This specification provides a method for inhibiting tumor growth in a subject, wherein the method comprises administering a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein to the subject.

[0096] In some embodiments, cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

[0097] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a blood cancer.

[0098] In some embodiments, the cancer of blood tissue is lymphoma. In some embodiments, the cancer is mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia (AML), B lymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm (BPDCN), or hairy cell leukemia.

[0099] As used herein, the terms “effective dose” or “therapeutic dose” mean an amount of a pharmaceutical product, such as a protein, immunoconjugate, or pharmaceutical composition disclosed herein, sufficient to reduce or improve the severity and / or duration of cancer, or one or more of its symptoms, to halt disease progression, cause disease regression, prevent recurrence, development, onset, or progression of one or more symptoms associated with the disease, or to enhance or improve the prophylactic or therapeutic effect of another related treatment for cancer (e.g., a prophylactic or therapeutic agent).

[0100] The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of delivery of the composition, the method of administration, the administration schedule, and other factors known to the healthcare professional. The prescription of the treatment, e.g., the determination of the dosage, is within the responsibility of the general practitioner and other physicians and may depend on the severity of the symptoms and / or progression of the disease being treated. Appropriate doses of antibody-based protein molecules are well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated and used as appropriate in this specification or in the Physician's Desk Reference (2003) in relation to the type of medicine being administered. The therapeutically effective or suitable dose of an antibody-based protein molecule can be determined by comparing its in vitro and in vivo activities in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dose will depend on several factors, including whether the antibody-based protein is for prophylaxis or treatment, the size and location of the area to be treated, the exact properties of the antibody-based protein, and the properties of any detectable labels or other molecules bound to the antibody-based protein.

[0101] Typical protein doses range from 100 μg to 1 g for systemic administration and from 1 μg to 1 mg for intradermal injection. A higher initial loading dose may be administered, followed by one or more lower doses. In some embodiments, the protein is the IgG1 or IgG4 isotype. The dose for a single treatment in adult subjects may be proportionally adjusted for children and infants. Treatment may be repeated at the physician's discretion, at intervals of daily, twice weekly, weekly, or monthly. The treatment schedule for a subject may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the treated state.

[0102] The treatment may be cyclical, and the interval between administrations may be approximately two weeks or longer, for example, approximately three weeks or longer, approximately four weeks or longer, approximately once a month or longer, approximately five weeks or longer, or approximately six weeks or longer. For example, treatment may be every two to four weeks or every four to eight weeks. Treatment may be given before and / or after surgery, and / or directly administered or applied to the anatomical site of the surgical procedure or invasive procedure. Preferred formulations and routes of administration are described above.

[0103] In some embodiments, the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may be administered by subcutaneous injection. Subcutaneous injection may be administered using an autoinjector, for example, for long-term prophylaxis / treatment.

[0104] In some embodiments, the therapeutic effects of the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may persist in a subject for one month or more, two months or more, three months or more, four months or more, five months or more, or six months or more.

[0105] In some embodiments, the subject may be treated with the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein, and additional therapeutic agents or treatments used to treat cancer or symptoms or complications of cancer. The proteins, immunoconjugates, or pharmaceutical compositions disclosed herein, and additional therapeutic agents or treatments may be administered simultaneously or sequentially.

[0106] In some embodiments, the subjects are humans, non-human primates, pigs, horses, cattle, dogs, cats, guinea pigs, mice, or rats. In some embodiments, the subjects are adults. In some embodiments, the subjects are children.

[0107] Furthermore, this specification provides for use in the treatment of diseases or disorders, proteins, immunoconjugates, or pharmaceutical compositions disclosed herein.

[0108] In this specification, proteins, immunoconjugates, or pharmaceutical compositions disclosed herein for use as pharmaceuticals are provided.

[0109] definition Unless otherwise specified, terms used herein have the definitions commonly used in the art. Some terms are defined below, and further definitions may be found in the remainder of the detailed description.

[0110] The terms “a” or “an” can refer to one or more of the entities, i.e., to multiple referents. Therefore, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, a reference to an “element” with the indefinite article “a” or “an” does not rule out the possibility of more than one element being present unless the context explicitly requires the presence of one or only one element.

[0111] Unless otherwise specified or the context makes otherwise clear, the term “about” means within 10% above or below the reported number (except when such a number exceeds 100% of the possible value or falls below 0%). When used with a range or set of values, the term “about” applies to each of the values ​​listed in that range or set of values ​​unless otherwise specified. In this application, the terms “about” and “approximately” are used as equivalent.

[0112] As used herein, the term “sequence identity” refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences remain invariant throughout the entire alignment window of residues, e.g., nucleotides or amino acids. The “identity percentage” for aligned segments of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion thereof. The “identity percentage” is 100 times the identity percentage. The identity percentage can be calculated using the Clustal Omega alignment program, available at ebi.ac.uk / Tools / msa / clustalo, with default parameters. See, for example, Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega” (2011 October 11) Molecular Systems Biology 7:539. For the purpose of calculating sequence identity, extensions such as tags are not included.

[0113] As used herein, the term "HCDR" refers to the heavy chain complementarity determination region. As used herein, the term "LCDR" refers to the light chain complementarity determination region.

[0114] The terms “amino terminus,” “N terminus,” “carboxyl terminus,” and “C terminus” are used herein to indicate locations within a polypeptide chain. Where the context permits, these terms are used to refer to specific sequences or parts of a polypeptide to indicate proximity or relative positions. For example, a particular sequence located at the carboxyl terminus relative to a reference sequence within a polypeptide is located in proximity to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the entire polypeptide.

[0115] As used herein, the term “conservative substitution” refers to the replacement of one amino acid with another amino acid that does not significantly alter the functional activity. A preferred example of a “conservative substitution” is the replacement of one amino acid with another amino acid that has a value of 0 or greater in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915–10919): [Table 4]

[0116] The term "immunoconjugate" refers to the proteins of this disclosure that are conjugated to cytotoxic agents, cell proliferation inhibitors, and / or therapeutic agents.

[0117] The term “isolated molecule” (where the molecule is, for example, a protein, nucleic acid, polynucleotide, or antibody) is defined as a molecule that, depending on its origin or source of derivatives, (1) is not associated with naturally related components that accompany it in its natural state, (2) substantially contains no other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cells from which it originates in nature is “isolated” from its naturally associated components. A molecule can also be made substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be assayed by several means well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and by staining the gel using techniques well known in the art to visualize the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0118] When used herein in relation to the bioactivity of proteins disclosed herein, the terms “inhibit,” “block,” or “neutralize” mean the ability of a protein to inhibit, but substantially counteract, prohibit, block, suppress, delay, destroy, eliminate, halt, reduce, or reverse, for example, the progression, intensity, or severity of, but not limited to, the binding of PD-L1 to PD-1 or the binding of CD89 to IgA.

[0119] As used herein, the terms “to treat,” “to treat,” or “to treat” (and their grammatical variations thereof) mean that the severity of the condition in question is reduced, at least partially improved, or stabilized, and / or some relief, reduction, decrease, or stabilization of at least one clinical symptom is achieved, and / or there is a delay in the progression of the disease or disability.

[0120] PD-L1 is also known as programmed cell death ligand 1, CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1, and hPD-L1.

[0121] CD89 is also known as differentiation cluster 89 and TP44. CD89 is a transmembrane glycoprotein that binds to both subclasses of IgA.

[0122] As used herein, the terms “prevent,” “prevent,” and “prevent” (and their grammatical variations) refer to the prevention and / or delay of the onset of a disease, disorder, and / or clinical symptom in a subject, and / or a reduction in the severity of the onset of the disease, disorder, and / or clinical symptom compared to what would occur in the absence of the compositions and / or methods described herein. Prevention may be complete, for example, the complete absence of the disease, disorder, and / or clinical symptom. Prevention may also be partial, such that the onset of the disease, disorder, and / or clinical symptom in a subject is less severe than what would occur in the absence of the compositions and / or methods described herein.

[0123] As used herein, “therapeutic dose” is the amount of the protein or pharmaceutical composition provided herein that is effective in treating or preventing a disease or disorder, or in improving its signs or symptoms, in a subject. The “therapeutic dose” may vary, for example, depending on the disease and / or symptoms of the disease, the severity of the symptoms of the disease and / or disorder, the age, weight, and / or health of the patient to be treated, and the judgment of the prescribing physician.

[0124] As used herein, and unless otherwise specified, the terms “hinge linker,” “linker,” “hinge,” “first linker,” “second linker,” “downward hinge linker” (“LHL”), “inter-Fab linker,” and their derivatives, whether in the plural or singular, refer to sequences that are distinct from any hinge sequences in an immunoglobulin hinge region, which may be derived from an immunoglobulin hinge region and may link two polypeptides, e.g., polypeptides from different Fab regions, and which may be part of the protein of the present invention.

[0125] All references, articles, publications, patents, patent gazettes, and patent applications cited herein are incorporated by reference in their entirety for the purposes of this specification. However, no reference to any reference, article, publication, patent, patent gazette, or patent application cited herein shall be understood as, or any form of, an acknowledgment or suggestion that they constitute valid prior art or form part of common knowledge in any country of the world.

[0126] The section headings used herein are for structural purposes only and should not be construed as limiting the subjects described.

[0127] This disclosure is further clarified by the following embodiments, which are merely illustrative and not intended to be limiting in any way. [Examples]

[0128] Production of therapeutic proteins that are activated under optimized conditions. Introduction In this example, the inventors succeeded in generating an active protein under optimized conditions. This conditionally active protein was successfully expressed.

[0129] material and method Protein cloning, transient expression, purification, and characterization The polypeptide-encoding DNA sequence was cloned via restriction-ligation cloning into separate expression cassettes encoding separate human IgG1 heavy and light chain constant regions in separate plasmid vectors to create activatable constructs for expression. The protein was expressed in CHO cells and purified from the culture supernatant via a combination of protein A affinity chromatography (ProA), ion exchange chromatography (IEX), and / or size exclusion chromatography (SEC). The purified protein was characterized by SEC, SDS-PAGE, and mass spectrometry.

[0130] Results and Discussion Design, cloning, expression, and characterization of protein constructs To produce proteins for functional testing, DNA cassettes were designed for each construct type using combinations of variable domains, constant domains, and linker sequences found in Table 1. These humanized variants were then combined with PD-L1 v domains, constant domains, and linkers to form full-length heavy and light chain sequences (Table 2). Using these full chains, two initial designs were synthesized and cloned into expression vectors encoding human IgG1 heavy and light chain sequences, as well as a free hinge-Fc fragment (Fc Stump, Table 2). The anti-PD-L1 variable domain sequence used in the protein constructs disclosed herein is the variable domain sequence provided in US7,943,743 B2. The anti-CD89 variable domain sequence used in the protein constructs disclosed herein is the variable domain sequence provided in US2003 / 0082643 A1. Proteins were produced by transient transfection into CHO cells and then purified by proA, IEX, and / or SEC. After the ProA step, the protein was examined for yield and homogeneity by SEC (Table 4). The fully purified protein demonstrated high purity (>95%) and homogeneity by analytical SEC, demonstrating that the best-behaving construct can be expressed and purified as an intact, stable product in a single process. [Table 5] Embodiment 1. A protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, The heavy chain comprises, from the N-terminus to the C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain. A protein in which the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD89 VL domain, and a second immunoglobulin light chain constant region. 2. The protein according to Embodiment 1, wherein the heavy chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain. 3. The protein according to Embodiment 1 or 2, further comprising a third polypeptide chain including a hinge and an Fc region. 4. The protein according to Embodiment 3, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32. 5. The protein according to any one of Embodiments 1 to 4, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 6. The protein according to any one of Embodiments 1 to 6, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 7. The anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO: 14. The anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO: 17. The anti-CD89 VH domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, and HCDR3 containing the amino acid sequence of SEQ ID NO: 20. The anti-CD89 VL domain includes LCDR1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 23. The protein according to any one of Embodiments 1 to 6. 8. The protein according to any one of Embodiments 1 to 7, wherein the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO: 24 and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 25. 9. The protein according to any one of Embodiments 1 to 8, wherein the anti-CD89 VH domain comprises the amino acid sequence of SEQ ID NO: 26. 10. The protein according to any one of Embodiments 1 to 9, wherein the anti-CD89 VL domain comprises the amino acid sequence of SEQ ID NO: 27. 11. The protein according to any one of Embodiments 1 to 10, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. 12. The protein according to any one of Embodiments 1 to 11, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29. 13. (a) The heavy chain contains the amino acid sequence of SEQ ID NO: 30, the light chain contains the amino acid sequence of SEQ ID NO: 28, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 32; or (b) The protein according to Embodiment 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 31, the light chain comprises the amino acid sequence of SEQ ID NO: 29, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32. 14. The protein according to Embodiment 2 or 3, wherein the heavy chain comprises an IgG, IgE, IgM, IgD, IgA, or IgY constant region. 15. The protein according to Embodiment 2 or 3, wherein the heavy chain comprises a constant region of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. 16. The protein according to Embodiment 2 or 3, wherein the heavy chain comprises an immunologically inactive constant region. 17. The protein according to Embodiment 2 or 3, wherein the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, or a human IgG4 constant region containing amino acid substitution S228P, and the numbering follows an EU index such as Kabat. 18. An immunoconjugate comprising a protein according to any one of Embodiments 1 to 17, linked to a therapeutic agent. 19. The immunoconjugate according to Embodiment 18, wherein the therapeutic agent is a cytotoxic agent, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell proliferation inhibitory enzyme, a cell lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or an apoptosis promoter. 20. A pharmaceutical composition comprising a protein according to any one of Embodiments 1 to 17, or an immunoconjugate according to Embodiment 18 or 19, and a pharmaceutically acceptable carrier, diluent, or excipient. 21. A protein according to any one of Embodiments 1 to 17, (a) Heavy chain amino acid sequence; (b) Light chain amino acid sequence; or (c) Both heavy chain amino acid sequence and light chain amino acid sequence A nucleic acid molecule that codes for something. 22. An expression vector comprising the nucleic acid molecule described in Embodiment 21. 23. Recombinant host cells comprising the nucleic acid molecule described in Embodiment 21 or the expression vector described in Embodiment 22. 24. A method for producing a protein, Recombinant host cells containing the expression vector described in Embodiment 22 are cultured under conditions in which nucleic acid molecules are expressed, thereby producing proteins; Isolating proteins from host cells or cultures Methods that include... 25. A method for enhancing an anti-cancer immune response in a subject, the method comprising administering a therapeutically effective amount of a protein described in any one of Embodiments 1 to 17, an immunoconjugate described in Embodiment 18 or 19, or a pharmaceutical composition described in Embodiment 20 to the subject. 26. A method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of a protein according to any one of Embodiments 1 to 17, an immunoconjugate according to Embodiment 18 or 19, or a pharmaceutical composition according to Embodiment 20 to the subject. 27. A method for improving the symptoms of cancer in a subject, the method comprising administering a therapeutically effective amount of a protein described in any one of Embodiments 1 to 17, an immunoconjugate described in Embodiment 18 or 19, or a pharmaceutical composition described in Embodiment 20 to the subject. 28. The method according to any one of Embodiments 25 to 27, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. 29. A protein comprising a first heavy chain and a first light chain, The first heavy chain is (a) a first portion (P1) of the first antigen-binding domain, comprising a first heavy chain variable domain (VH) and, optionally, a first heavy chain constant domain 1 (CH1); (b) A first linker that can be cleaved by a protease; (c) The first portion (P2) of the second antigen-binding domain, comprising a second VH and, optionally, a second CH1; (i) The N-terminus of the first linker is connected to the C-terminus of the first CH1 of P1, and the C-terminus of the first linker is connected to the N-terminus of the second VH of P2; or (ii) The first portion of the second antigen-binding domain (P2) is such that the N-terminus of the first linker is linked to the C-terminus of the first VH of P1, and the C-terminus of the first linker is linked to the N-terminus of the second VH of P2. Includes, The first light chain, (a) a second portion (P3) of the first antigen-binding domain, comprising a first light chain variable domain (VL) and, optionally, a first light chain constant domain (CL); (b) a second linker that can be cleaved by a protease; (d) A second portion (P4) of the second antigen-binding domain, comprising a second VL and, if applicable, a second CL; (i) The N-terminus of the second linker is connected to the C-terminus of the first CL of P3, and the C-terminus of the second linker is connected to the N-terminus of the second VL of P4; or (ii) The second portion of the second antigen-binding domain (P4) is such that the N-terminus of the first linker is linked to the C-terminus of the first VL of P3, and the C-terminus of the second linker is linked to the N-terminus of the second VL of P4. Includes, The first antigen-binding domain binds to PD-L1; A protein in which, when the first and second linkers are cleaved, the second antigen-binding domain binds to CD89. 30. The protein according to Embodiment 29, wherein CH1 of P1 is covalently linked to CL of P3. 31. The protein according to Embodiment 29 or 30, wherein CH1 of P2 is covalently linked to CL of P4. 32. A protein comprising a first heavy chain and a first light chain, The first heavy chain is (a) the first portion (P1) of the first antigen-binding domain, which includes the first heavy chain variable domain (VH); (b) A first linker that can be cleaved by a protease; (c) The first portion (P2) of the second antigen-binding domain, which includes the second VH; A first heavy chain in which the N-terminus of the first linker is linked to the C-terminus of P1, and the C-terminus of the first linker is linked to the N-terminus of the second VH of P2; The first light chain, (c) The second portion (P3) of the first antigen-binding domain, which includes the first light chain variable domain (VL); (d) a second linker that can be cleaved by a protease; (d) The second portion (P4) of the second antigen-binding domain, which includes the second VL; The first light chain has a second linker whose N-terminus is linked to the C-terminus of P3, and the C-terminus of the second linker is linked to the N-terminus of the second VL of P4; Includes, The first antigen-binding domain binds to PD-L1; A protein in which, when the first and second linkers are cleaved, the second antigen-binding domain binds to CD89. 33. The protein according to Embodiment 32, wherein P1 further comprises a first heavy chain constant region 1 (CH1), and the C-terminus of the first CH1 of P1 is ligated to the N-terminus of the first linker. 34. The protein according to embodiment 33, wherein the N-terminus of the first CH1 of P1 is ligated to the C-terminus of the VH of P1. 35. The protein according to Embodiment 32, wherein P3 further comprises a first CL, and the C-terminus of the first CL of P3 is ligated to the N-terminus of the second linker. 36. The protein according to embodiment 35, wherein the N-terminus of the first CL of P3 is ligated to the C-terminus of the first VL of P3. 37. The protein according to any one of embodiments 32 to 36, wherein P2 further contains a second CH1, and the N-terminus of the second CH1 of P2 is ligated to the C-terminus of the second VH of P2. 38. The protein according to any one of embodiments 32 to 37, wherein P4 further comprises a second CL, and the N-terminus of the second CL of P4 is ligated to the C-terminus of the second VL of P4. 39. The protein according to any one of embodiments 35 to 38, wherein the first CH1 of P1 is covalently linked to the first CL of P3. 40. The protein according to any one of embodiments 35 to 39, wherein the second CH1 of P2 is covalently linked to the second CL of P4. 41. The protein according to any one of embodiments 29 to 40, wherein the first antigen-binding domain comprises P1 and P3. 42. A protein according to any one of embodiments 29 to 41, wherein P1 and P3 are covalently linked to form a Fab. 43. The protein according to any one of embodiments 29 to 42, wherein the second antigen-binding domain comprises P2 and P4. 44. A protein according to any one of embodiments 29 to 43, wherein P2 and P4 are covalently linked to form a Fab. 45. The protein according to any one of Embodiments 29 to 44, wherein the first heavy chain further comprises a first dimerization domain. 46. ​​The protein according to Embodiment 45, wherein the first dimerization domain comprises a first hinge domain and a first immunoglobulin constant domain (Fc domain). 47. The protein according to Embodiment 46, wherein the Fc domain comprises a first heavy chain constant region 2 (CH2) and a first heavy chain constant region 3 (CH3). 48. The protein according to embodiments 45 to 47, wherein the C-terminus of P2 is ligated to the N-terminus of the first hinge domain. 49. The protein according to any one of embodiments 29 to 48, wherein the first linker comprises an amino acid sequence having at least 85% identity with one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 50. The protein according to Embodiment 49, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 51. The protein according to any one of embodiments 29 to 50, wherein the second linker comprises an amino acid sequence having at least 85% identity with one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 52. The protein according to Embodiment 51, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 53. The first VH of P1 is (a) The heavy chain complementarity determination region 1 (HCDR1) amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 34, or SEQ ID NO: 36; (b) The heavy chain complementarity determination region 2 (HCDR2) amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 35, or SEQ ID NO: 37; and (c) and the heavy chain complementarity determination region 3 (HCDR3) amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 38 A protein according to any one of embodiments 29 to 52, including the protein described above. 54. The first VL of P3 is (a) The light chain complementarity determination region 1 (LCDR1) amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 39; (b) Light chain variable domains containing the amino acid sequence of Sequence ID No. 16 or DA's light chain complementarity determining region 1 (LCDR2); and (c) Light chain variable domain containing the light chain complementarity determination region 3 (LCDR3) amino acid sequence of SEQ ID NO: 17 A protein according to any one of embodiments 29 to 53, including the protein described above. 55. The second VH of P2 is (a) The HCDR1 amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 40, or SEQ ID NO: 21; (b) the HCDR2 amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 41, or SEQ ID NO: 43; and (c) HCDR3 amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 44 A protein according to any one of embodiments 29 to 54, including the protein described above. 56. The second VL of P4 is (a) The LCDR1 amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 45; (b) the LCDR2 amino acid sequence of Sequence ID No. 22 or GA; and (c) Amino acid sequence of LCDR3 of SEQ ID NO. 23 A protein according to any one of embodiments 29 to 55, including the protein described above. The protein according to any one of embodiments 29 to 56, wherein the first VH of 57.P1 contains a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 24. 58. The protein according to any one of embodiments 29 to 57, wherein the first VH of P1 contains the sequence of SEQ ID NO: 24. The protein according to any one of Embodiments 29 to 58, wherein the first VL of P3 contains a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 25. 60. The protein according to any one of embodiments 29 to 59, wherein the first VL of P3 contains the sequence of SEQ ID NO: 25. 61. The protein according to any one of Embodiments 29 to 60, wherein the second VH of P2 contains a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 26. 62. The protein according to any one of embodiments 29 to 61, wherein the second VH of P2 contains the sequence of SEQ ID NO: 26. The protein according to any one of Embodiments 29 to 62, wherein the second VL of P4 contains a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 27. 64. The protein according to any one of embodiments 29 to 63, wherein the second VL of P4 contains the sequence of SEQ ID NO: 27. 65. The first heavy chain is (a) A first VH of P1 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 24; and (b) A second VH of P2 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 26. A protein according to any one of embodiments 29 to 64, including the protein described above. 66. The first heavy chain is (a) the first VH of P1 containing the sequence of sequence number 24; and (b) The second VH of P2 containing the sequence of sequence number 26 A protein according to any one of embodiments 29 to 65, including the protein described above. 67. The first light chain, (a) A first VL of P3 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 25; and (b) A second VL of P4 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO: 27. A protein according to any one of embodiments 29 to 66, including the protein described above. 68. The first light chain, (a) the first VL of P3 containing the sequence of sequence number 25; and (b) The second VL of P4 containing the sequence of sequence number 27 A protein according to any one of embodiments 29 to 67, including the protein described above. 69.(a) The first heavy chain is (i) A first VH of P1 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 24; and (ii) A second VH of P2 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 26. Includes, (b) The first light chain, (i) the first VL of P3 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of sequence number 25; and (ii) A second VL of P4 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO: 27. A protein according to any one of embodiments 29 to 68, including the protein described above. 70.(a) The first heavy chain is (i) the first VH of P1 containing the sequence of sequence number 24; and (ii) The second VH of P2 containing the sequence of sequence number 26 Includes, (b) The first light chain, (i) the first VL of P3 containing the sequence of sequence number 25; and (ii) The second VL of P4 containing the sequence of sequence number 27 A protein according to any one of embodiments 29 to 69, including the protein described above. The protein according to any one of Embodiments 29 to 70, wherein the first CH1 of 71.P1 contains a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 5. The protein according to any one of embodiments 29 to 71, wherein the first CH1 of 72.P1 contains the sequence of SEQ ID NO: 5. The protein according to any one of Embodiments 29 to 72, wherein the second CH1 of 73.P2 contains a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 5. 74. The protein according to any one of embodiments 29 to 73, wherein the second CH1 of P2 contains the sequence of SEQ ID NO: 5. The protein according to any one of embodiments 29 to 74, wherein the first CL of 75.P3 is a kappa light chain constant domain. 76. The protein according to Embodiment 75, wherein the kappa light chain constant domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO: 7. 77. The protein according to embodiment 75 or 76, wherein the kappa light chain constant domain comprises the sequence of SEQ ID NO: 7. The protein according to any one of embodiments 29 to 77, wherein the second CL of 78.P4 is a kappa light chain constant domain. 79. The protein according to Embodiment 78, wherein the kappa light chain constant domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 7. 80. The protein according to Embodiment 78 or 79, wherein the kappa light chain constant domain comprises the sequence of SEQ ID NO: 7. 81. The protein according to any one of Embodiments 29 to 80, wherein the first heavy chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 30 or Sequence ID No. 31. 82. The protein according to any one of embodiments 29 to 81, wherein the first heavy chain comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31. 83. The protein according to any one of embodiments 29 to 82, wherein the first light chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO: 28 or SEQ ID NO: 29. 84. The protein according to any one of embodiments 29 to 83, wherein the first light chain comprises the sequence of SEQ ID NO: 28 or SEQ ID NO: 29. 85. The first heavy chain and the first light chain, Sequence IDs 30 and 28, respectively; Sequence IDs 31 and 28, respectively; Sequence IDs 30 and 29, respectively; or Sequence IDs 31 and 29, respectively; A protein according to any one of embodiments 29 to 84, comprising the sequence of . 86. The protein according to any one of embodiments 46 to 85, wherein the first dimerization domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO: 32. 87. The protein according to any one of embodiments 46 to 86, wherein the first dimerization domain comprises the sequence of SEQ ID NO: 32. 88. The protein according to any one of embodiments 29 to 87, wherein the first heavy chain comprises one or more heavy chain constant regions selected from the group consisting of an IgG constant region or a functional fragment thereof, an IgE constant region or a functional fragment thereof, an IgM constant region or a functional fragment thereof, an IgD constant region or a functional fragment thereof, an IgA constant region or a functional fragment thereof, an IgY constant region or a functional fragment thereof, an IgG1 constant region or a functional fragment thereof, an IgG2 constant region or a functional fragment thereof, an IgG3 constant region or a functional fragment thereof, an IgG4 constant region or a functional fragment thereof, an IgA1 constant region or a functional fragment thereof, and an IgA2 constant region or a functional fragment thereof. 89. The protein according to any one of embodiments 29 to 88, wherein the first heavy chain comprises one or more immunologically inactive constant regions. 90. The protein according to any one of Embodiments 29 to 89, wherein the first heavy chain comprises one or more heavy chain constant regions selected from the group consisting of a wild-type human IgG1 constant region, a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, and a human IgG4 constant region containing amino acid substitution S228P, and the numbering follows an EU index such as Kabat. 91. The protein according to Embodiment 89 or 90, wherein the heavy chain constant region includes a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. 92. The protein according to any one of embodiments 29 to 91, further comprising a second heavy chain. 93. The protein according to Embodiment 92, wherein the second heavy chain further comprises a second dimerization domain. 94. The protein according to Embodiment 93, wherein the second dimerization domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Sequence ID No. 32. 95. The protein according to either embodiment 93 or 94, wherein the second dimerization domain comprises the sequence of SEQ ID NO: 32. 96. The protein according to Embodiment 93, wherein the first dimerization domain and the second dimerization domain are dimerized. 97. The second heavy chain, (a) Second P1; (b) a third linker in which the first linker is cleavable by a protease; and (c) P2 of the second page A protein according to any one of embodiments 100 to 104, including the above. 98. Further containing a second light chain, the second light chain is (a) Second P3; (b) The fourth linker; and (c) P4 of the second page The protein according to Embodiment 97, including the protein described in Embodiment 97. 99. A protein according to any of the preceding embodiments, wherein the C-terminus of the first heavy chain is ligated to the N-terminus of the first dimerization domain, and the C-terminus of the first light chain is ligated to the N-terminus of the second dimerization domain. 100. A protein containing the structure [P1]-[A]-[P2]-[P3]-[B]-[P4], (a)[P1] comprises a first heavy chain variable domain (VH) of the first antigen-binding domain and, optionally, a first heavy chain constant domain 1 (CH1); (b)[A] comprises a first linker that can be cleaved by a protease; (c)[P2] comprises a second VH of the second antigen-binding domain and, optionally, a second CH1; (d)[P3] comprises a first light chain variable domain (VL) of the first antigen-binding domain and, optionally, a first light chain constant domain (CL); (e)[B] contains a second linker that can be cleaved by a protease; (f)[P4] comprises a second VL of the second antigen-binding domain and, optionally, a second CL; (g)[P1] and [P3] are linked by a covalent bond; (h)[P2] and [P4] are linked by a covalent bond; (i) The C-terminus of [P1] is ligated to the N-terminus of [A]; (j) The C-terminus of [A] is ligated to the N-terminus of [P2]; (k)[P3]'s C-terminus is ligated to the N-terminus of [B]; (l) The C-terminus of [B] is ligated to the N-terminus of [P4]; The first antigen-binding domain is bound to PD-L1; A protein in which, when [A] and [B] are cleaved, a second antigen-binding domain binds to CD89.

Claims

1. A protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, The heavy chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain. A protein wherein the light chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD89 VL domain, and a second immunoglobulin light chain constant region.

2. The protein according to claim 1, wherein the heavy chain comprises, in order from the N-terminus to the C-terminus, the anti-PD-L1 VH domain, the first CH1 domain, the first linker, the anti-CD89 VH domain, the second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

3. The protein according to claim 1 or 2, further comprising a third polypeptide chain including a hinge and an Fc region.

4. The protein according to claim 3, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

32.

5. The protein according to any one of claims 1 to 4, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

6. The protein according to any one of claims 1 to 6, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

7. The anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 12, HCDR2 containing the amino acid sequence of SEQ ID NO: 13, and HCDR3 containing the amino acid sequence of SEQ ID NO:

14. The anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 15, LCDR2 containing the amino acid sequence of SEQ ID NO: 16, and LCDR3 containing the amino acid sequence of SEQ ID NO:

17. The anti-CD89 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, and HCDR3 containing the amino acid sequence of SEQ ID NO:

20. The anti-CD89 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO:

23. The protein according to any one of claims 1 to 6.

8. The protein according to any one of claims 1 to 7, wherein the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO: 24, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO:

25.

9. The protein according to any one of claims 1 to 8, wherein the anti-CD89 VH domain comprises the amino acid sequence of SEQ ID NO:

26.

10. The protein according to any one of claims 1 to 9, wherein the anti-CD89 VL domain comprises the amino acid sequence of SEQ ID NO:

27.

11. The protein according to any one of claims 1 to 10, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO:

31.

12. The protein according to any one of claims 1 to 11, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

29.

13. (a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 30, the light chain comprises the amino acid sequence of SEQ ID NO: 28, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32; or (b) The protein according to claim 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 31, the light chain comprises the amino acid sequence of SEQ ID NO: 29, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

32.

14. The protein according to claim 2 or 3, wherein the heavy chain comprises an IgG, IgE, IgM, IgD, IgA, or IgY constant region.

15. The protein according to claim 2 or 3, wherein the heavy chain comprises an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 constant region.

16. The protein according to claim 2 or 3, wherein the heavy chain includes an immunologically inactive constant region.

17. The protein according to claim 2 or 3, wherein the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, or a human IgG4 constant region containing amino acid substitution S228P, and the numbering follows an EU index such as Kabat.

18. An immunoconjugate comprising a protein according to any one of claims 1 to 17, linked to a therapeutic agent.

19. The immunoconjugate according to claim 18, wherein the therapeutic agent is a cytotoxic agent, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell proliferation inhibitory enzyme, a cell lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or an apoptosis promoter.

20. A pharmaceutical composition comprising a protein according to any one of claims 1 to 17, or an immunoconjugate according to claim 18 or 19, and a pharmaceutically acceptable carrier, diluent, or excipient.

21. A protein according to any one of claims 1 to 17, (a) Heavy chain amino acid sequence; (b) Light chain amino acid sequence; or (c) Both heavy chain amino acid sequence and light chain amino acid sequence A nucleic acid molecule that codes for something.

22. An expression vector comprising the nucleic acid molecule described in claim 21.

23. Recombinant host cells comprising the nucleic acid molecule described in claim 21 or the expression vector described in claim 22.

24. A method for producing protein, Recombinant host cells containing the expression vector described in claim 22 are cultured under conditions in which nucleic acid molecules are expressed, thereby producing the protein; Isolating the protein from the host cells or culture. Methods that include...

25. A method for enhancing an anti-cancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1 to 17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

26. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1 to 17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

27. A method for improving the symptoms of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1 to 17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

28. The method according to any one of claims 25 to 27, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

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