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

Abstract

Provided herein are protein molecules that specifically bind to PD-L1 and also exhibit activatable specific CD89 binding in diseased tissue. Further provided herein are uses of such protein molecules for the treatment of cancer.

Description

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 497,092, filed April 19, 2023, the contents of which are incorporated in their entirety. TECHNICAL FIELD

[0002] The present disclosure relates to activatable bispecific proteins and treatment of cancer. BACKGROUND

[0003] In immuno-oncology therapies, a small number of key drug targets are expressed only in diseased tissue, with the majority also expressed in non-diseased tissue. Furthermore, many drugs employed in cancer therapy employ potent cell-killing mechanisms of action. As a result, engagement of the drug with the target in non-diseased tissue often leads to unwanted side effects.

[0004] PD-L1 is a cell surface receptor that is a member of the immunoglobulin superfamily and is expressed primarily on myeloid cells and regulatory T (Treg) cells in non-diseased tissue. However, PD-L1 is also found to be highly expressed on some cancer cells. PD-L1 binds to the membrane protein PD1. The interaction of PD-L1 with PD1 on T cells downregulates T cell inflammatory activity, which promotes immune self-tolerance. PD-L1 has thus been described as an immune checkpoint. As a result, antagonistic anti-PD-L1 monoclonal antibodies that block the interaction with PD1 have proven to be possible to act as well-tolerated immunotherapeutic agents in disease settings such as cancer by “liberating” anti-cancer T cell responses from natural immune limitations. As such, PD-L1 is a drug target for enhancing the anti-cancer effects of the adaptive immune system. In many cases, however, tumors can use a variety of “escape mechanisms” to counteract 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 potently induces antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) against IgA-opsonized cells, which is induced much more potently than by analogous IgG. Despite these observations, the development of IgA antibodies as therapeutic agents has been generally unsuccessful because IgA antibody structures are difficult to express and purify, and have poor pharmacokinetics (PK) in humans, resulting in very low exposure in diseased tissue.

[0006] Historically, antibodies have been generated against the CD89 ectodomain that do not compete for binding with endogenous IgA. These antibodies, upon binding to CD89, can induce direct activation signals that enhance the inflammatory activity of innate immune cells. Using the binding domains from these antibodies, CD89- linked bispecific agents have also been generated that can direct innate immune cell killing activity to target-enriched diseased tissues. Thus, anti-PD-Ll antibodies can be made more potent and broadly acting by acquiring the ability to also bind CD89, thereby strongly enhancing innate immune cell killing mechanisms in the context of PD-Ll antibody resistant diseases. This would combine synergistically the functions of a key checkpoint inhibitor of the adaptive immune system and an inducible "synthetic IgA immunity." However, the ability to make this combination work in a single therapeutic agent structure (e.g., a standard bispecific antibody format with fully active PD-Ll and CD89 binding domains) is limited by the relatively broad expression profile of PD-Ll and CD89 on many cell types, such as T cells and innate immune cells. This broad expression profile can not only lead to dose-limiting toxicities of PD-Ll / CD89 binding agents, but also to severe peripheral sink / biodistribution problems that limit the ability of such agents to achieve sufficiently high exposure in diseased tissues to leverage the combined mechanisms. Thus, there is a need for engineered forms of bispecific binding proteins with activity that specifically targets the diseased tissue environment. SUMMARY

[0007] Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, an anti-PD-Ll heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD89 VH domain, and a second CH1 domain; wherein the light chain comprises, in order from N-terminus to C-terminus, an anti-PD-Ll 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 N-terminus to C-terminus, an anti-PD-Ll 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 comprising 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 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 14; the anti-PD-L1 VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 17; the anti-CD89 VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 20; and the anti-CD89 VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0012] 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. In some embodiments, the anti-CD89 VH domain comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CD89 VL domain comprises the amino acid sequence of SEQ ID NO: 27.

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

[0014] Provided herein is a protein, wherein (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 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 comprises an IgG, IgE, IgM, IgD, IgA, or IgY constant region. In some embodiments, the heavy chain comprises an IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 constant region. In some embodiments, the heavy chain comprises an immunologically inert constant region. In some embodiments, the heavy chain comprises a wild-type human IgGl constant region, a human IgGl constant region comprising the 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 comprising the amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat.

[0016] Provided herein is an immunoconjugate comprising a protein disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0017] Provided herein is a pharmaceutical composition comprising a protein or immunoconjugate disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] Provided herein is a nucleic acid molecule 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] Provided herein is an expression vector comprising a nucleic acid molecule disclosed herein.

[0020] Provided herein is a recombinant host cell comprising a nucleic acid molecule or an expression vector disclosed herein.

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

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

[0023] Provided herein is a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0024] Provided herein is a method of alleviating a symptom of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0025] In some embodiments of the methods provided herein, 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, cancer of the mouth or throat, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Schematic diagrams depicting exemplary asymmetric single-arm constructs of the protein molecules disclosed herein in the intact conformation (left), the activated protease cleavage conformation (middle), and the inactivated protease cleavage conformation (right). In the intact conformation, the anti-PD-Ll Fab binding domain is exposed and able to bind its cognate target. The binding of the anti-CD89 Fab domain is inhibited by linkers in both the heavy and light chains, which are each proteolytically cleavable and can be sequentially cleaved by matrix metalloproteases (MMPs) and / or cathepsins. The first cleavage event creates an intermediate active state, enabling both the anti-PD-Ll and anti-CD89 Fabs from a single protein construct to bind their cognate targets, potentially directing activation of CD89+ immune cells in a PD-L1+ immunosuppressive microenvironment. The second cleavage separates the anti-PD-Ll and anti-CD89 Fabs, eliminating the ability of a single molecule to simultaneously bind PD-L1 and CD89.

[0027] Figure 2 Schematic diagrams depicting the mechanism of activity of the activatable bispecific protein molecules provided herein. Due to the monomeric nature of the CD89 binding domain’s release activity, it cannot directly cause CD89 activation, but rather relies on binding to PD-L1+ cells, resulting in CD89 binding being presented to nearby immune cells in trans to induce CD89 signaling. The activated molecule thereby simultaneously blocks PD-L1 / PD1 signaling to restore unoppressed TCR activation in T cells, and provides CD89 signaling to amplify innate immune cell ADCC and ADCP activity.

[0028] Figure 3Structures of exemplary symmetric dual-arm constructs of the protein molecules described herein are depicted. DETAILED DESCRIPTION

[0029] There are two key issues that limit the efficacy of CD89-activating, tumor-targeting bispecific antibody drugs in oncology: 1) The antibody target protein found on cancer cells (e.g., PD-L1) can be expressed in many different cell classes in the body, not just on tumor cells. This off-tumor target expression can lead to a risk of dose-limiting side effects because the CD89-binding domain in a standard bispecific molecule has constitutive activity, and thus can direct CD89+ cell activation in the presence of any PD-L1+ cell, whether or not it is in diseased tissue. Off-tumor target expression can also lead to an antigen "sink" effect, reducing the amount of drug that penetrates the tumor.

[0030] 2) CD89-positive cells, such as neutrophils and monocytes, are present in the bloodstream and are highly concentrated in other tissues, creating a large sink effect for this arm, affecting biodistribution and availability of free drug for tumor penetration.

[0031] Both of these factors 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, e.g., Figure 1 ) overcome the peripheral sink and toxicity issues by minimizing CD89 binding outside of diseased tissue. This effect is achieved by adding the PD-L1 binding domain and linker above (i.e., amino-terminal) to the CD89 binding domain. Using the appropriate upper domain and linker combination creates a configuration that minimizes binding activity in the lower (i.e., carboxy-terminal) CD89 domain. The PD-L1 domain then drives high concentration in the tumor microenvironment, which is rich in PD-L1. The protein construct linker system takes advantage of the elevated MMP and cathepsin activity that is common in solid tumors to cleave the linker peptide, exposing the CD89 binding domain, and thus conditionally activating CD89 activation activity in the tumor but not in the periphery. The biological function of these combinations thus gives the molecule the potential to avoid peripheral CD89 sink and maximize T cell immune response against cancer cells, as Figure 2 outlined.

[0032] Provided herein are proteins that have conditional activity in diseased human tissue. The proteins of the present disclosure have full activity in specifically binding and blocking systemic PD-L1; exhibit minimal CD89 binding in healthy tissue; and become highly activated in CD89 binding and activation once in the environment of a PD-L1 positive diseased tissue. The proteins of the present disclosure comprise a CD89 binding domain that is masked by a PD-L1 binding domain in non-diseased tissue. The protein further comprises two peptide linkers that are cleaved by one or more proteases expressed in diseased tissue (e.g., a tumor). Linker cleavage exposes the CD89 binding domain in the diseased tissue, thus allowing the protein to selectively bind and / or function in the diseased tissue.

[0033] Protein molecules Provided herein are proteins comprising two Fab fragments (anti-PD-L1 Fab and anti-CD89 Fab). The protein is monovalent in the intact structure and can only have maximal monovalent CD89 binding upon activation to minimize the risk of peripheral toxicities associated with bivalent activating anti-CD89 antibodies.

[0034] In some embodiments, the protein comprises a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, in order 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; wherein the light chain comprises, in order 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, at its C-terminus, an immunoglobulin hinge region and an Fc domain. In some embodiments, the heavy chain comprises, in order 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.

[0035] In some embodiments, the protein further comprises a third polypeptide chain comprising a hinge and an Fc region. The third polypeptide chain can be referred to as an “Fc-stump.”

[0036] Figure 1 A schematic of an exemplary protein of the present disclosure with labeled domains is shown.

[0037] The first and second linkers can be cleaved by matrix metalloproteinases (MMPs) and / or cathepsins present in diseased tissue, such as tumors. The linkers in the protein are immunoglobulin-derived hinge sequences that are both proteolytically sensitive and can be sequentially cleaved, with the first cleavage event taking the full structure and creating an intermediate active state that enables the anti-PD-Ll Fab and anti-CD89 Fab from a single protein construct to bind their cognate targets. The second cleavage event in the second linker removes the covalent linkage between the anti-PD-Ll Fab and anti-CD89 Fab, eliminating the molecule’s ability to recruit T cells to kill PD-L1+ cells. Thus, this second cleavage event constitutes a “self-destruct mechanism” that minimizes the risk of the activated molecule escaping the tumor microenvironment. The cleavable linker based on immunoglobulin hinge sequences can also recruit increased immune effector functions (antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP)) at the cell membrane via endogenous anti-hinge antibodies, a known phenomenon in human patients with (and even without) underlying autoimmune diseases.

[0038] In some embodiments, the protein comprises one or more of the amino acid sequences provided in Table 1 or Table 2.

[0039] Table 1: Exemplary Sequences

[0040]

[0041]

[0042]

[0043] In the VH and VL domain sequences, the CDR sequences are underlined.

[0044] Table 2: Full Length Sequences of Exemplary Heavy and Light Chains

[0045]

[0046] The anti-PD-Ll / anti-CD89 protein design can be based on sequences derived from IgGl, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and can or can not have effector function capabilities.

[0047] The proteins disclosed herein comprise domains and regions of antibody molecules. The term "antibody" refers broadly to immunoglobulin (Ig) molecules, typically comprising 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 characteristics of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0048] In a full-length antibody, each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH domain) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. The IgG, IgA, and IgD constant regions comprise a flexible hinge region between the CH1 domain and the CH2 domain. Each light chain comprises a light chain variable domain (abbreviated herein as VL domain) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL domain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Framework regions and CDRs have been defined and described, for example, in Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. 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.

[0049] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus. The numbering of the residues in the Fc region is in accordance with the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. The Fc region can exist in a dimeric or monomeric form. The Fc region binds to various cellular receptors such as Fc receptors and other immunological molecules such as complement proteins.

[0050] The protein described in this article contains two Fab fragments. The Fab fragment is a monovalent antigen-binding fragment composed of the VL, VH, CL, and CH1 domains.

[0051] 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 typically contain two identical heavy chains and two identical light chains, as well as two antigen-binding domains, each consisting of a VH and a VL. Typically, IgA antibodies consist of two monomers, each consisting of two heavy chains and two light chains (as with IgG, IgD, and IgE antibodies); thus, the IgA molecule has four antigen-binding domains, each consisting of a VH and a VL. Some IgA antibodies are monomeric because they consist of two heavy chains and two light chains. Secretory IgM antibodies typically consist of five monomers, each consisting of two heavy chains and two light chains (as with IgG and IgE antibodies). Therefore, the IgM molecule has ten antigen-binding domains, each consisting of a VH and a VL. Cell surface IgM has a two-heavy-chain / two-light-chain structure, similar to IgG, IgD and IgE antibodies.

[0052] As used herein, the terms "immunobinding" and "immunobinding property" refer to a type of nonvalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody or its antigen-binding portion) or a protein containing an immunoglobulin-derived binding domain and an immunoglobulin or a protein-specific antigen. The strength or affinity of an immune-binding interaction can be expressed by the dissociation constant (K0) of the interaction. d Let K be the smaller one. d This represents a higher affinity. The immunobinding properties of a selected peptide can be quantified using methods well-known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that also affect the rates in both directions. Therefore, the "association rate constant" (Kassociation rate constant) is used. on ) and "dissociation rate constant" (K off Both can be determined by calculating the concentration and the actual association and dissociation rates. (See Malmqvist, Nature 361: 186-187 (1993)). K off / K on The ratio can cancel out all parameters unrelated to affinity and is equal to the dissociation constant K. d (See Davies et al. (1990)) Annual Rev Biochem59: 439-473). When the equilibrium binding constant (K) d When the concentration is ≤10 μM, preferably ≤10 nM, more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM, the antibody or antigen-binding moiety provided herein is considered to specifically bind to PD-L1 or CD89, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. A K-type assay for determining antibodies... d The method involves using surface plasmon resonance (SPR), typically employed with biosensor systems such as the Biacore® system.

[0053] Functionally, the protein binding affinity presented in this article can reach 10. -5 M to 10 -12 Within a certain range. For example, the binding affinity of the proteins provided in this article is from 10... -6 M to 10 -12 M, from 10 -7 M to 10 -12 M, from 10 -8 M to 10 -12 M, from 10 -9 M to 10 -12 M, from 10 -5 M to 10 -11 M, from 10 -6 M to 10 -11 M, from 10 -7 M to 10 -11 M, from 10 -8 M to 10 -11 M, from 10 -9 M to 10 -11 M, from 10 -10 M to 10 -11 M, from 10 -5 M to 10 -10 M, from 10 -6 M to 10 -10 M, from 10 -7 M to 10 -10 M, from 10 -8 M to 10 -10 M, from 10 -9 M to 10 -10 M, from 10 -5 M to 10 -9 M, from 10 -6 M to 10 -9 M, from 10 -7 M to 10 -9 M, from 10 -8M to 10 -9 M, from 10 -5 M to 10 -8 M, from 10 -6 M to 10 -8 M, from 10 -7 M to 10 -8 M, from 10 -5 M to 10 -7 M, from 10 -6 M to 10 -7 M or from 10 -5 M to 10 -6 M.

[0054] Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, in order 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; wherein the light chain comprises, in order 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.

[0055] In some embodiments, the heavy chain comprises, in order 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.

[0056] In some embodiments, the protein further comprises a third polypeptide chain comprising a hinge and an Fc region. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32.

[0057] 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.

[0058] In some embodiments, the anti-PD-L1 VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 14; the anti-PD-L1 VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 17; the anti-CD89 VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 20; and the anti-CD89 VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0059] 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.

[0060] In some embodiments, the anti-CD89 VH domain comprises the amino acid sequence of SEQ ID NO: 26.

[0061] In some embodiments, the anti-CD89 VL domain comprises the amino acid sequence of SEQ ID NO: 27.

[0062] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. 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: 30 or SEQ ID NO: 31.

[0063] 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.

[0064] In some embodiments, provided herein is a protein, wherein (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 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.

[0065] Also provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence provided herein with 1, 2, or 3 conservative amino acid substitutions; and wherein the light chain comprises an amino acid sequence provided herein with 1, 2, or 3 conservative amino acid substitutions. In some embodiments, the conservative amino acid substitutions are made only in the FR sequences, and not in the CDR sequences. In some embodiments, no conservative amino acid substitutions are made in the first or second linker sequences.

[0066] In some embodiments, the protein provided herein comprises 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 IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In some embodiments, the immunoglobulin heavy chain constant region is IgGl. In some embodiments, the immunoglobulin heavy chain constant region is immunologically inert. In some embodiments, the immunoglobulin heavy chain constant region comprises one or more mutations to reduce or prevent FcyR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the immunoglobulin heavy chain constant region is a wild-type human IgGl constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgGl constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgGl constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a human IgG4 constant region comprising amino acid substitution S228P, wherein the numbering is according to the EU index as in Kabat. In some embodiments, the positions of the amino acid residues in the immunoglobulin molecule constant region are numbered according to the EU index as in Kabat (Ward et al., 1995 Therap. Immunol. 2: 77-94).

[0067] In some embodiments, the proteins provided herein can comprise an immunoglobulin light chain constant region that is a kappa light chain. In some embodiments, the kappa light chain comprises SEQ ID NO: 7.

[0068] In some embodiments, the proteins provided herein can comprise an immunoglobulin light chain constant region that is a lambda light chain.

[0069] In some embodiments, the proteins provided herein can comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of a human IgG4, human IgG4 (S228P), human IgG2, human IgGl, human IgGl effector null Fc region. For example, a human IgG4 (S228P) Fc region comprises the following substitutions compared to a wild-type human IgG4 Fc region: S228P. For example, a human IgGl effector null Fc region comprises the following substitutions compared to a wild-type human IgGl Fc region: L234A, L235A, and G237A. In some embodiments, the proteins can comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0070] Provided herein is an immunoconjugate comprising a protein disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0071] Examples of suitable therapeutic agents include, but are not limited to, immunomodulatory agents, cytotoxins, radioisotopes, chemotherapeutic agents, anti-angiogenic agents, anti-proliferative agents, pro-apoptotic agents, and cytostatic and cytolytic enzymes (e.g., RNases). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding an immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent. These drug descriptors are not mutually exclusive, and a therapeutic agent can be described using one or more of the above terms.

[0072] Examples of suitable therapeutic agents for immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansines, CC-1065 and duocarmycins, calicheamicins and other enediyines, and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers can also be used in immunoconjugates. In addition, secondary carriers can be used to prepare conjugates, such as liposomes or polymers, as cytotoxic agents. Suitable cytotoxins include agents that inhibit or prevent the function of cells and / or cause destruction of cells. Representative cytotoxins include antibiotics, microtubuline polymerization inhibitors, alkylating agents that bind to and damage DNA, and agents that destroy the function of protein synthesis or essential cellular proteins such as protein kinases, phosphatases, topoisomerases, enzymes, and cell cycle proteins.

[0073] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carrubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, and gemcitabine. Itabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin The following are listed: tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, and methotrexate. Fluorouracil, etoposide, taxol, taxol analogs, platinum-based drugs (such as cisplatin and carboplatin), mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, autramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins / olestradiol, hemiasterlinEsperamicins and maytansinoids.

[0074] Suitable immunomodulators include anti-hormonal agents that block the action of hormones at tumor sites and immunosuppressants that inhibit cytokine production, down-regulate self-antigen expression, or mask MHC antigens.

[0075] Table 3: Chain sequence combinations for exemplary proteins

[0076] Pharmaceutical compositions 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 the protein (or immunoconjugate comprising the protein) and a pharmaceutically acceptable carrier, diluent, or excipient. Such materials should be nontoxic and should not interfere with the efficacy of the protein. The precise nature of the carrier or other material will depend on the route of administration, which can be by injection, by the oral route, by infusion, or any other suitable route, as discussed below.

[0077] As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an allergic or other untoward reaction when administered to an animal, especially a human, as appropriate for a given route of administration. Molecular entities and compositions approved by a regulatory agency of the United States Federal or state governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans, are considered “pharmaceutically acceptable.” As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington’s Pharmaceutical Sciences (a standard reference text in the field), 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. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is envisaged. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutically acceptable carrier, diluent, or excipient can be a compound or combination of compounds that do not cause a secondary reaction and allow, for example, the protein to be administered, extended its shelf life, and / or increase its efficacy in the body or increase its solubility in solution.

[0078] The pharmaceutical compositions disclosed herein can be formulated to be compatible with 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 for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solutions, non-volatile oils, polyethylene glycol, glycerol, 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 acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0079] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (in the water-soluble case) or dispersants and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersants. For intravenous administration, suitable carriers include physiological saline, antibacterial aqueous solutions, and Cremophor EL. ® (BASF, Parsipani, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to a degree of ease of injection. It must be stable under manufacturing and storage conditions; and must be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which may contain, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating (such as lecithin), maintaining the desired particle size in the case of a dispersion, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. Extended absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0080] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or more of the ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile powder, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0081] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0082] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0083] Systemic administration can also be by way of transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into a

[0084] The agents can also be formulated in suppository form (using conventional suppository binders such as cocoa butter and other glycerides) or in the form of a retention enema for rectal delivery.

[0085] In some embodiments, the active compound is prepared with an agent that protects the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained from a number of commercial suppliers. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.

[0086] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the application are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0087] In some embodiments, the protein can be provided in lyophilized form, so as to be reconstituted prior to administration. For example, a lyophilized antibody molecule can be reconstituted in sterile water prior to administration to an individual, and mixed with saline.

[0088] The pharmaceutical compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.

[0089] Nucleic acid molecules, vectors, host cells and methods of producing proteins Provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding an amino acid sequence of a protein disclosed herein (or an amino acid sequence of (i) a VH domain, (ii) a VL domain, or (iii) both a VH domain and a VL domain of a protein). Further provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding (i) a heavy chain, (ii) a light chain, or (iii) both a heavy chain and a light chain of a protein disclosed herein. In some embodiments, the nucleic acid molecule encoding the VH domain, the VL domain, the heavy chain, or the light chain comprises a signal sequence (or encodes a leader peptide). In some embodiments, the nucleic acid molecule encoding the VH domain, the VL domain, the heavy chain, or the light chain does not comprise a signal sequence (or does not encode a leader peptide).

[0090] Also provided herein are expression vectors comprising the nucleic acid molecules described herein. In certain vectors, the nucleic acid molecule is operably linked to one or more regulatory sequences suitable for expression of the nucleic acid segment in a host cell. In some cases, the expression vector comprises sequences that mediate replication and comprises one or more selectable markers. As used herein, "vector" refers to a construct that is capable of delivering and preferably expressing 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, cosmids or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0091] Provided herein is a recombinant host cell comprising an expression vector or nucleic acid molecule disclosed herein. A "host cell" includes an individual cell, cell line, or cell culture that can be or has been a recipient for a vector. Host cells include progeny of the original subject cell. The progeny can not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Expression vectors can be transfected into host cells by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. In some embodiments, the recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both the heavy chain and the light chain of a protein disclosed herein. In some embodiments, the recombinant host cell comprises (i) a first vector or a first nucleic acid molecule encoding the heavy chain of a protein disclosed herein and (ii) a second vector or a second nucleic acid molecule encoding the light chain of a protein disclosed herein.

[0092] The protein molecules of the present application, or portions thereof, can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, computational techniques, or a combination of such techniques or other techniques readily apparent to those skilled in the art.

[0093] Further provided herein is a method of producing a protein disclosed herein, the method comprising: culturing a recombinant host cell comprising an expression vector described herein under conditions in which the nucleic acid segment is expressed, thereby producing the protein. The protein can then be isolated from the host cell or culture. Provided herein is a method of producing a protein, the method comprising: culturing a recombinant host cell comprising an expression vector disclosed herein under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0094] The proteins disclosed herein can be produced by any of a variety of methods known to those of skill in the art. In certain embodiments, the proteins disclosed herein can be produced recombinantly. For example, a nucleic acid sequence encoding one or more of the heavy or light chains provided herein, or a portion thereof, can be introduced into a bacterial cell (e.g., Escherichia coli E. coli ), Bacillus subtilis B. subtilis ), or a eukaryotic cell (e.g., a yeast, such as Saccharomyces cerevisiae S. cerevisiae , or a mammalian cell, such as a CHO cell line, various Cos cell lines, a HeLa cell, a HEK293 cell, various myeloma cell lines, or a transformed B cell or hybridoma), or an in vitro translation system, and the translated polypeptides can be isolated. In some embodiments, the light chain protein and the heavy chain protein are produced in a cell with a signal sequence that is removed when the mature protein disclosed herein is produced.

[0095] One of skill in the art is able to determine whether a protein comprising a given polypeptide sequence binds to a PD-L1 protein and / or a CD89 protein using standard methods, e.g., Western blotting, ELISA, etc.

[0096] Medical uses of activatable proteins Methods and uses of the activatable proteins, immunoconjugates, and pharmaceutical compositions disclosed herein are provided herein for providing a therapeutic benefit to a subject having a cancer.

[0097] The activatable proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can be used in a method of treatment of the human or animal body by a prophylactic or preventative treatment (e.g., treatment before the onset of a condition in a subject to reduce the risk of that condition occurring in the subject; to delay its onset; or to lessen its severity after its onset). The method of treatment can comprise administering the protein, immunoconjugate, or pharmaceutical composition to a subject in need thereof.

[0098] Provided herein is 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, 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.

[0099] Provided herein is a method for treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0100] Provided herein is a method for alleviating a symptom of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0101] Provided herein is a method for reducing tumor size in a subject, comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0102] Provided herein is a method for inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0103] In some embodiments, 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, cancer of the mouth or throat, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or a cancer of hematological tissue.

[0104] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0105] In some embodiments, the cancer of hematological tissue is a lymphoma. In some embodiments, the cancer is mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, acute myeloid leukemia (AML), B-lymphocyte leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or hairy cell leukemia.

[0106] As used herein, the term “effective amount” or “therapeutically effective amount” refers to the amount of an agent (e.g., a protein, immunoconjugate, or pharmaceutical composition) disclosed herein that is sufficient to reduce or ameliorate the severity and / or duration of a cancer, or one or more symptoms thereof, prevent the development of a disease, cause the regression of a disease, prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disease, or enhance or improve the prophylactic or therapeutic effect of another relevant therapy (e.g., a prophylactic or therapeutic agent) on a cancer.

[0107] The actual amount to be administered, and the rate and time-course of administration, will depend on the nature and severity of the disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration, and other factors known to medical practitioners. Treatment protocols, e.g., dosing decisions, etc., are within the responsibility of the general practitioner and other medical doctors and can depend on the symptoms, severity and / or progression of the disease being treated. Appropriate dosages of antibody-based protein molecules are well known in the art (Ledermann JA et al., 1991, Int. J. Cancer47 : 659-664; Bagshawe KD et al., 1991, Antibody, Immunocouples and Radiopharmaceuticals 4: 915-922). Specific dosages can be found in this text or in the texts cited herein or in the Physician's Desk Reference (2003), as appropriate for the type of administered drug that can be used. A therapeutically effective amount or suitable dose of an antibody-based protein molecule can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods of extrapolating an effective dose in mice and other test animals to humans are known. The precise dose will depend on a variety of factors including whether the antibody-based protein is used for prevention or therapy, the size and location of the area to be treated, the precise nature of the antibody-based protein, and the nature of any detectable label or other molecule attached to the antibody-based protein.

[0108] For systemic application, a typical protein dose will be in the range of 100 μg to 1 g, and for intradermal injection, in the range of 1 μg to 1 mg. An initial higher loading dose can be administered, followed by one or more lower doses. In some embodiments, the protein is an IgGl or IgG4 isotype. Single therapeutic doses for adult subjects can be proportionally adjusted for children and infants. Treatment can be repeated at daily, twice-weekly, weekly, or monthly intervals, as determined by the physician. The subject's treatment schedule can depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

[0109] Treatment can be periodic, and the period between administrations can be about two weeks or more, such as about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment can be once every two to four weeks or once every four to eight weeks. Treatment can be given pre-operatively and / or post-operatively, and / or can be administered or applied directly at the anatomical site of a surgical treatment or invasive procedure. Suitable formulations and routes of administration are as described above.

[0110] In some embodiments, the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can be administered in the form of a subcutaneous injection. Subcutaneous injections can be administered using an auto-injector, such as for long-term prophylaxis / treatment.

[0111] In some embodiments, the therapeutic effect of a protein, immunoconjugate, or pharmaceutical composition disclosed herein can last for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of a protein, immunoconjugate, or pharmaceutical composition disclosed herein can last for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more in a subject.

[0112] In some embodiments, a subject can be treated with a protein, immunoconjugate, or pharmaceutical composition disclosed herein and an additional therapeutic agent or therapy for treating a cancer or a symptom or complication of cancer. The protein, immunoconjugate, or pharmaceutical composition disclosed herein and the additional therapeutic agent or therapy can be administered simultaneously or sequentially.

[0113] In some embodiments, the subject is a human, non-human primate, pig, horse, cow, dog, cat, guinea pig, mouse, or rat. In some embodiments, the subject is an adult human. In some embodiments, the subject is a child.

[0114] Further provided herein are the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein for use in treating a disease or disorder.

[0115] Provided herein are the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein for use as a medicament.

[0116] Definitions The terms used herein, unless otherwise indicated, have the definitions commonly associated with them in the art. Some terms are defined below, and other definitions can be found throughout the detailed description.

[0117] The term “a” or “an” refers to one or more of that entity, i.e., can refer to a plural as well as a singular number. Thus, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. Furthermore, the indefinite article “a” or “an” does not exclude the possibility that more than one element of the element is present, unless the context clearly indicates the presence of one and only one element.

[0118] Unless otherwise indicated or unless context clearly indicates otherwise, the term “about” refers to within 10% above or below a reported numerical value (unless such number would exceed 100% of a possible value or below 0%). When used in connection with a range or series of values, the term “about” applies to each of the range’s endpoints or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0119] The term "sequence identity," as used herein, refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of residues (e.g., nucleotides or amino acids). The "fraction of identity" for a segment of aligned sequences 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 segment of the reference sequence (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The "percent identity" is the fraction of identity multiplied by 100. Percent identity can be calculated using the alignment program Clustal Omega, available at ebi.ac.uk / Tools / msa / clustalo with default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (Mol. Sys. Biol. 7:539 (2011) doi:10.1038 / msb.2011.75) Molecular Systems Biology 7: 539). For the calculation of the identity of a sequence, extensions such as tags are not included.

[0120] The term "HCDR," as used herein, refers to a heavy chain complementarity determining region. The term "LCDR," as used herein, refers to a light chain complementarity determining region.

[0121] The terms "amino-terminal," "N-terminal," "carboxyl-terminal," and "C-terminal" as used herein refer to positions within a polypeptide chain. Where the context allows, these terms are used in reference to a particular sequence or portion of a polypeptide to indicate proximity or relative position. For example, a sequence that is located carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily located at the carboxyl terminus of the entire polypeptide.

[0122] The term "conservative substitution," as used herein, refers to the replacement of one amino acid by another amino acid without significant deleterious alteration of functional activity. Preferred examples of "conservative substitutions" are replacements of one amino acid with another that has a value of > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919): .

[0123] The term "immunoconjugate" refers to a protein of the disclosure conjugated to a cytotoxic agent, cytostatic agent, and / or therapeutic agent.

[0124] The term "isolated molecule" (in the case of a molecule that is, for example, a protein, nucleic acid, polynucleotide, or antibody) is a molecule that is (1) not associated with naturally associated components that accompany it in its natural state, (2) substantially free of other molecules from the same species (3) expressed by a cell from a different species or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from a cell of its natural source will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assayed by a variety of means well known in the art. For example, the purity of a polypeptide sample can be assayed by denaturing polyacrylamide gel electrophoresis and gel staining using techniques well known in the art to visualize the polypeptide. For some purposes, higher resolution can be provided by use of HPLC or other means of purification well known in the art.

[0125] As used herein with respect to the biological activity of the proteins disclosed herein, the terms "inhibit," "block," or "neutralize" refer to the ability of a protein to significantly antagonize, prohibit, prevent, limit, slow, disrupt, eliminate, stop, reduce, or reverse the progression, intensity, or severity of, for example, the thing being inhibited, including but not limited to the binding of PD-L1 to PD-1 or the binding of CD89 to IgA.

[0126] As used herein, the terms "treat," "treating," or "treatment" (and grammatical variations thereof) refer to the reduction in severity, at least partial amelioration, or stabilization of a subject's condition, and / or some improvement in at least one clinical symptom, and / or delay in the progression of the disease or condition.

[0127] PD-L1 is also known as programmed cell death ligand 1, CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1, and hPD-L1.

[0128] CD89 is also known as cluster of differentiation 89 and TP44. CD89 is a transmembrane glycoprotein that binds two subclasses of IgA.

[0129] As used herein, the terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to precluding and / or delaying the onset of a disease, disorder, and / or clinical symptoms in a subject and / or reducing the severity of a disease, disorder, and / or clinical symptoms that would occur in the absence of the compositions and / or methods described herein. Prevention can be complete, e.g., the total absence of a disease, disorder, and / or clinical symptoms. Prevention can also be partial, such that the occurrence and / or severity of a disease, disorder, and / or clinical symptoms in a subject is less than would occur in the absence of the compositions and / or methods described herein.

[0130] As used herein, a "therapeutically effective amount" is an amount of a protein or pharmaceutical composition provided herein effective to treat or prevent a disease or disorder in a subject or to alleviate a sign or symptom thereof. A "therapeutically effective amount" can vary depending, for example, on the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease, the age, weight, and / or health status of the patient to be treated, and the judgment of the prescribing physician.

[0131] As used herein, unless otherwise indicated, the terms "hinge linker," "linker," "hinge," "first linker," "second linker," "lower hinge linker" ("LHL"), "Fab inter-Fab linker," and derivatives thereof, in the plural and singular forms, refer to a sequence that can connect two polypeptides, e.g., polypeptides of different Fab regions, e.g., a sequence derived from an immunoglobulin hinge region, and separate from any hinge sequence in the immunoglobulin hinge region that can be part of a protein of the application.

[0132] All references, articles, publications, patents, patent publications, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. The mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or admission that they are common general knowledge of the public or constitute part of the common general knowledge in any country in the world.

[0133] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0134] The present disclosure will be further clarified by the following examples, which set forth in detail specific embodiments of this application. These examples are intended to be illustrative only and are not intended to limit the scope of the disclosure.

[0135] Examples Generation of conditionally active therapeutic proteins Introduction In this example, we successfully generated optimized conditionally active proteins. These conditionally active proteins expressed well.

[0136] Materials and Methods Cloning, transient expression, purification, and characterization of proteins DNA sequences encoding polypeptides were cloned into separate human IgGl heavy and light chain constant region encoded expression cassettes in separate plasmid vectors via restriction-ligation cloning to create activatable constructs for expression. Proteins were expressed in CHO cells and purified from culture supernatant by a combination of Protein A affinity chromatography (ProA), ion exchange chromatography (IEX), and / or size exclusion chromatography (SEC). Purified proteins were characterized by SEC, SDS-PAGE, and mass spectrometry.

[0137] Results and Discussion Protein construct design cloning, expression, and characterization To generate proteins for functional testing, DNA cassettes for each construct type were designed using combinations of variable domains, constant domains, and linker sequences 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). With these full-length chains, 2 initial designs were synthesized and cloned into expression vectors encoding human IgGl heavy and light chain sequences and a free hinge-Fc fragment (Fc residues, Table 2). The anti-PD-L1 variable domain sequences used in the protein constructs disclosed herein are the variable domain sequences provided in U.S. 7,943,743 B2. The anti-CD89 variable domain sequences used in the protein constructs disclosed herein are the variable domain sequences provided in U.S. 2003 / 0082643 Al. The proteins were produced by transient transfection of CHO cells and then purified by ProA, IEX, and / or SEC. After the ProA step was complete, the proteins were tested for yield and homogeneity by SEC (Table 4). Fully purified proteins exhibited high purity (>95%) and homogeneity by analytical SEC, indicating that the best-performing constructs could be expressed and purified as intact, stable products in a single process.

[0138] Table 4: Productivity analysis of selected proteins

[0139] HMW = high molecular weight product LMW = low molecular weight product EMBODIMENTS 1. A protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, in order from N-terminal to C-terminal, an anti-PD-L1 heavy chain variable (VH) domain, a first CHI domain, a first linker, an anti-CD89 VH domain, and a second CHI domain; wherein the light chain comprises, in order from N-terminal to C-terminal, 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.

[0140] 2. The protein of embodiment 1, wherein the heavy chain comprises, in order from N-terminal to C-terminal, the anti-PD-L1 VH domain, the first CHI domain, the first linker, the anti-CD89 VH domain, the second CHI domain, a hinge, a CH2 domain, and a CH3 domain.

[0141] 3. The protein of embodiment 1 or 2, wherein the protein further comprises a third polypeptide chain comprising a hinge and an Fc region.

[0142] 4. The protein of embodiment 3, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32.

[0143] 5. The protein of any one of embodiments 1-4, 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.

[0144] 6. The protein of any one of embodiments 1-6, 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.

[0145] 7. The protein of any one of embodiments 1-6, wherein the anti-PD-L1 VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 14; wherein the anti-PD-L1 VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 17; wherein the anti-CD89 VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 20; wherein the anti-CD89 VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0146] 8. The protein according to any one of embodiments 1-7, 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.

[0147] 9. The protein according to any one of embodiments 1-8, wherein the anti-CD89 VH domain comprises the amino acid sequence of SEQ ID NO: 26.

[0148] 10. The protein according to any one of embodiments 1-9, wherein the anti-CD89 VL domain comprises the amino acid sequence of SEQ ID NO: 27.

[0149] 11. The protein according to any one of embodiments 1-10, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31.

[0150] 12. The protein according to any one of embodiments 1-11, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0151] 13. The protein according to embodiment 3, wherein (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 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.

[0152] 14. The protein according to embodiment 2 or 3, wherein the heavy chain comprises an IgG, IgE, IgM, IgD, IgA, or IgY constant region.

[0153] 15. The protein of embodiment 2 or 3, wherein the heavy chain comprises an IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 constant region.

[0154] 16. The protein of embodiment 2 or 3, wherein the heavy chain comprises an immunologically inert constant region.

[0155] 17. The protein of embodiment 2 or 3, wherein the heavy chain comprises a wild-type human IgGl constant region, a human IgGl constant region comprising the 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 comprising the amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat.

[0156] 18. An immunoconjugate comprising the protein of any one of embodiments 1-17 linked to a therapeutic agent.

[0157] 19. The immunoconjugate of embodiment 18, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0158] 20. A pharmaceutical composition comprising the protein of any one of embodiments 1-17 or the immunoconjugate of embodiment 18 or 19, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0159] 21. A nucleic acid molecule 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 the protein of any one of embodiments 1-17.

[0160] 22. An expression vector comprising the nucleic acid molecule of embodiment 21.

[0161] 23. A recombinant host cell comprising the nucleic acid molecule of embodiment 21 or the expression vector of embodiment 22.

[0162] 24. A method of producing a protein, the method comprising: culturing a recombinant host cell comprising the expression vector of embodiment 22 under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0163] 25. A method of enhancing an anti-cancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of embodiments 1-17, the immunoconjugate of embodiment 18 or 19, or the pharmaceutical composition of embodiment 20.

[0164] 26. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of embodiments 1-17, the immunoconjugate of embodiment 18 or 19, or the pharmaceutical composition of embodiment 20.

[0165] 27. A method of alleviating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of embodiments 1-17, the immunoconjugate of embodiment 18 or 19, or the pharmaceutical composition of embodiment 20.

[0166] 28. The method of any one of embodiments 25-27, 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, cancer of the mouth or throat, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0167] 29. A protein comprising a first heavy chain and a first light chain, wherein the first heavy chain comprises: (a) a first part of a first antigen binding domain (P1) comprising a first heavy chain variable domain (VH) and optionally a first heavy chain constant domain 1 (CH1); (b) a first linker, wherein the first linker is cleavable by a protease; and (c) a first part of a second antigen binding domain (P2) comprising a second VH and optionally a second CH1; wherein: (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 N-terminus of the first linker is connected to the C-terminus of the first VH of P1 and the C-terminus of the first linker is connected to the N-terminus of the second VH of P2; and wherein the first light chain comprises: (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, wherein the second linker is cleavable by a protease; and (d) a second portion (P4) of the second antigen binding domain comprising a second VL and optionally a second CL; wherein: (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 N-terminus of the first linker is connected to the C-terminus of the first VL of P3 and the C-terminus of the second linker is connected to the N-terminus of the second VL of P4; and wherein the first antigen binding domain binds PD-L1; and wherein the second antigen binding domain binds CD89 when the first linker and the second linker are cleaved.

[0168] 30. The protein according to embodiment 29, wherein the CH1 of P1 is covalently linked to the CL of P3.

[0169] 31. The protein according to embodiment 29 or 30, wherein the CH1 of P2 is covalently linked to the CL of P4.

[0170] 32. A protein comprising a first heavy chain and a first light chain, wherein the first heavy chain comprises: (a) a first portion (P1) of a first antigen binding domain comprising a first heavy chain variable domain (VH); (b) a first linker, wherein the first linker is cleavable by a protease; and (c) a first portion (P2) of a second antigen binding domain comprising a second VH; wherein the N-terminus of the first linker is connected to the C-terminus of P1 and the C-terminus of the first linker is connected to the N-terminus of the second VH of P2; wherein the first light chain comprises: (c) a second portion (P3) of the first antigen binding domain comprising a first light chain variable domain (VL); (d) a second linker, wherein the second linker is cleavable by a protease; and (d) a second portion (P4) of the second antigen binding domain comprising a second VL; wherein the N-terminus of the second linker is connected to the C-terminus of P3 and the C-terminus of the second linker is connected to the N-terminus of the second VL of P4; wherein the first antigen binding domain binds PD-L1; and wherein the second antigen binding domain binds CD89 when the first linker and the second linker are cleaved.

[0171] 33. The protein according to embodiment 32, wherein the P1 further comprises a first heavy chain constant domain 1 (CH1), wherein the C-terminus of the first CH1 of P1 is connected to the N-terminus of the first linker.

[0172] 34. The protein according to embodiment 33, wherein the N-terminus of the first CH1 of P1 is connected to the C-terminus of the VH of P1.

[0173] 35. The protein according to embodiment 32, wherein the P3 further comprises a first CL, wherein the C-terminus of the first CL of P3 is connected to the N-terminus of the second linker.

[0174] 36. The protein according to embodiment 35, wherein the N-terminus of the first CL of P3 is connected to the C-terminus of the first VL of P3.

[0175] 37. The protein according to any one of embodiments 32-36, wherein the P2 further comprises a second CH1, wherein the N-terminus of the second CH1 of P2 is connected to the C-terminus of the second VH of P2.

[0176] 38. The protein according to any one of embodiments 32-37, wherein the P4 further comprises a second CL, wherein the N-terminus of the second CL of P4 is connected to the C-terminus of the second VL of P4.

[0177] 39. The protein according to any one of embodiments 35-38, wherein the first CH1 of P1 is covalently connected to the first CL of P3.

[0178] 40. The protein according to any one of embodiments 35-39, wherein the second CH1 of P2 is covalently connected to the second CL of P4.

[0179] 41. The protein according to any one of embodiments 29-40, wherein the first antigen binding domain comprises P1 and P3.

[0180] 42. The protein according to any one of embodiments 29-41, wherein the P1 and the P3 are covalently connected to form a Fab.

[0181] 43. The protein according to any one of embodiments 29-42, wherein the second antigen binding domain comprises P2 and P4.

[0182] 44. The protein according to any one of embodiments 29-43, wherein the P2 and the P4 are covalently linked to form a Fab.

[0183] 45. The protein according to any one of embodiments 29-44, wherein the first heavy chain further comprises a first dimerization domain.

[0184] 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).

[0185] 47. The protein according to embodiment 46, wherein the Fc domain comprises a first heavy chain constant domain 2 (CH2) and a first heavy chain constant domain 3 (CH3).

[0186] 48. The protein according to embodiments 45-47, wherein the C-terminus of the P2 is linked to the N-terminus of the first hinge domain.

[0187] 49. The protein according to any one of embodiments 29-48, wherein the first linker comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0188] 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.

[0189] 51. The protein according to any one of embodiments 29-50, wherein the second linker comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0190] 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.

[0191] 53. The protein according to any one of embodiments 29-52, wherein the first VH of P1 comprises: (a) a heavy chain complementarity determining 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 determining region 2 (HCDR2) amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 35, or SEQ ID NO: 37; and (c) the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 38.

[0192] 54. The protein according to any one of embodiments 29-53, wherein the first VL of P3 comprises: (a) the light chain complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 39; (b) the light chain complementarity determining region 1 (LCDR2) amino acid sequence of SEQ ID NO: 16 or DA; and (c) the light chain complementarity determining region 3 (LCDR3) amino acid sequence of SEQ ID NO: 17.

[0193] 55. The protein according to any one of embodiments 29-54, wherein the second VH of P2 comprises: (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) the HCDR3 amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 44.

[0194] 56. The protein according to any one of embodiments 29-55, wherein the second VL of P4 comprises: (a) the LCDR1 amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 45; (b) the LCDR2 amino acid sequence of SEQ ID NO: 22 or GA; and (c) the LCDR3 amino acid sequence of SEQ ID NO: 23.

[0195] 57. The protein according to any one of embodiments 29-56, wherein the first VH of PI comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 24.

[0196] 58. The protein according to any one of embodiments 29-57, wherein the first VH of PI comprises the sequence of SEQ ID NO: 24.

[0197] 59. The protein according to any one of embodiments 29-58, wherein the first VL of P3 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 25.

[0198] 60. The protein according to any one of embodiments 29-59, wherein the first VL of P3 comprises the sequence of SEQ ID NO: 25.

[0199] 61. The protein according to any one of embodiments 29-60, wherein the second VH of P2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 26.

[0200] 62. The protein according to any one of embodiments 29-61, wherein the second VH of P2 comprises the sequence of SEQ ID NO: 26.

[0201] 63. The protein according to any one of embodiments 29-62, wherein the second VL of P4 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 27.

[0202] 64. The protein according to any one of embodiments 29-63, wherein the second VL of P4 comprises the sequence of SEQ ID NO: 27.

[0203] 65. The protein according to any one of embodiments 29-64, wherein the first heavy chain comprises: (a) the first VH of PI having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 24; and (b) the second VH of P2 having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 26.

[0204] 66. The protein according to any one of embodiments 29-65, wherein the first heavy chain comprises: (a) the first VH of PI comprising the sequence of SEQ ID NO: 24; and (b) the second VH of P2 comprises the sequence of SEQ ID NO: 26.

[0205] 67. The protein according to any one of embodiments 29-66, wherein the first light chain comprises: (a) the first VL of P3 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 25; and (b) the second VL of P4 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 27.

[0206] 68. The protein according to any one of embodiments 29-67, wherein the first light chain comprises: (a) the first VL of P3 comprises the sequence of SEQ ID NO: 25; and (b) the second VL of P4 comprises the sequence of SEQ ID NO: 27.

[0207] 69. The protein according to any one of embodiments 29-68, (a) wherein the first heavy chain comprises: (i) the first VH of P1 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 24; and (ii) the second VH of P2 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 26; and (b) wherein the first light chain comprises: (i) the first VL of P3 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 25; and (ii) the second VL of P4 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 27.

[0208] 70. The protein according to any one of embodiments 29-69, (a) wherein the first heavy chain comprises: (i) the first VH of P1 comprises the sequence of SEQ ID NO: 24; and (ii) the second VH of P2 comprises the sequence of SEQ ID NO: 26; and (b) wherein the first light chain comprises: (i) the first VL of P3 comprising the sequence of SEQ ID NO: 25; and (ii) the second VL of P4 comprising the sequence of SEQ ID NO: 27.

[0209] 71. The protein according to any one of embodiments 29-70, wherein the first CH1 of P1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 5.

[0210] 72. The protein according to any one of embodiments 29-71, wherein the first CH1 of P1 comprises the sequence of SEQ ID NO: 5.

[0211] 73. The protein according to any one of embodiments 29-72, wherein the second CH1 of P2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 5.

[0212] 74. The protein according to any one of embodiments 29-73, wherein the second CH1 of P2 comprises the sequence of SEQ ID NO: 5.

[0213] 75. The protein according to any one of embodiments 29-74, wherein the first CL of P3 is a kappa light chain constant domain.

[0214] 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 to the sequence of SEQ ID NO: 7.

[0215] 77. The protein according to embodiment 75 or 76, wherein the kappa light chain constant domain comprises the sequence of SEQ ID NO: 7.

[0216] 78. The protein according to any one of embodiments 29-77, wherein the second CL of P4 is a kappa light chain constant domain.

[0217] 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 to the sequence of SEQ ID NO: 7.

[0218] 80. The protein of either embodiment 78 or 79, wherein the kappa light chain constant domain comprises the sequence of SEQ ID NO: 7.

[0219] 81. The protein of any one of embodiments 29-80, wherein the first heavy chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.

[0220] 82. The protein of any one of embodiments 29-81, wherein the first heavy chain comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.

[0221] 83. The protein of any one of embodiments 29-82, wherein the first light chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0222] 84. The protein of any one of embodiments 29-83, wherein the first light chain comprises the sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

[0223] 85. The protein of any one of embodiments 29-84, wherein the first heavy chain and the first light chain comprise the following sequences, respectively: SEQ ID NO: 30 and SEQ ID NO: 28; SEQ ID NO: 31 and SEQ ID NO: 28; SEQ ID NO: 30 and SEQ ID NO: 29; or SEQ ID NO: 31 and SEQ ID NO: 29.

[0224] 86. The protein of any one of embodiments 46-85, wherein the first dimerization domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the sequence of SEQ ID NO: 32.

[0225] 87. The protein of any one of embodiments 46-86, wherein the first dimerization domain comprises the sequence of SEQ ID NO: 32.

[0226] 88. The protein according to any one of embodiments 29-87, wherein the first heavy chain comprises one or more heavy chain constant regions selected from the group consisting of an IgG constant region or functional fragment thereof, an IgE constant region or functional fragment thereof, an IgM constant region or functional fragment thereof, an IgD constant region or functional fragment thereof, an IgA constant region or functional fragment thereof, an IgY constant region or functional fragment thereof, an IgG1 constant region or functional fragment thereof, an IgG2 constant region or functional fragment thereof, an IgG3 constant region or functional fragment thereof, an IgG4 constant region or functional fragment thereof, an IgA1 constant region or functional fragment thereof, and an IgA2 constant region or functional fragment thereof.

[0227] 89. The protein according to any one of embodiments 29-88, wherein the first heavy chain comprises one or more immunologically inert constant regions.

[0228] 90. The protein according to any one of embodiments 29-89, 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 comprising the 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 comprising the amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat.

[0229] 91. The protein according to embodiment 89 or 90, wherein the heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100% sequence identity to the sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.

[0230] 92. The protein according to any one of embodiments 29-91, further comprising a second heavy chain.

[0231] 93. The protein according to embodiment 92, wherein the second heavy chain further comprises a second dimerization domain.

[0232] 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 to the sequence of SEQ ID NO: 32.

[0233] 95. The protein according to any embodiment 93 or 94, wherein the second dimerization domain comprises the sequence of SEQ ID NO: 32.

[0234] 96. The protein of embodiment 93, wherein the first dimerization domain is dimerized with the second dimerization domain.

[0235] 97. The protein of any one of embodiments 100-104, wherein the second heavy chain comprises: (a) a second P1 ; (b) a third linker, wherein the first linker is cleavable by a protease; and (c) a second P2.

[0236] 98. The protein of embodiment 97, further comprising a second light chain, wherein the second light chain comprises: (a) a second P3; (b) a fourth linker; and (c) a second P4.

[0237] 99. The protein of any preceding embodiment, wherein the C-terminus of the first heavy chain is connected to the N-terminus of the first dimerization domain, and the C-terminus of the first light chain is connected to the N-terminus of the second dimerization domain.

[0238] 100. A protein comprising the structure: [P1]-[A]-[P2]-[P3]-[B]-[P4], wherein: (a) [P1] comprises a first heavy chain variable domain (VH) of a first antigen binding domain and optionally a first heavy chain constant domain 1 (CH1); (b) [A] comprises a first linker, wherein the first linker is cleavable by a protease; (c) [P2] comprises a second VH of a 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] comprises a second linker, wherein the second linker is cleavable 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 covalently linked; (h) [P2] and [P4] are covalently linked; (i) the C-terminus of [P1] is connected to the N-terminus of [A]; (j) the C-terminus of [A] is connected to the N-terminus of [P2]; (k) the C-terminus of [P3] is connected to the N-terminus of [B]; (l) the C-terminus of [B] is connected to the N-terminus of [P4]; wherein the first antigen binding domain binds PD-L1; wherein the second antigen binding domain binds CD89 when [A] and [B] are cleaved.

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 N-end to C-end, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first joint, an anti-CD89 VH domain, and a second CH1 domain; The light chain comprises, in order 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 of claim 1, wherein the heavy chain comprises, in order from N-terminus to C-terminus, the anti-PD-L1VH domain, the first CH1 domain, the first linker, the anti-CD89 VH domain, the second CH1 domain, the hinge, the CH2 domain, and the CH3 domain.

3. The protein according to claim 1 or 2, wherein the protein further comprises a third polypeptide chain, the third polypeptide chain comprising 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 1-4, 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 1-6, 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 protein according to any one of claims 1-6, 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.

8. The protein according to any one of claims 1-7, 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 1-8, 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 1-9, 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 1-10, 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 1-11, wherein the light chain comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:

29.

13. The protein according to claim 3, wherein (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 heavy chain contains the amino acid sequence of SEQ ID NO:31, the light chain contains the amino acid sequence of SEQ ID NO:29, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:

32.

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

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

16. The protein according to claim 2 or 3, wherein the heavy chain comprises an immune inert 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 comprising 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 comprising amino acid substitution S228P, wherein the numbering is based on the EU index as in Kabat.

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

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

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

21. A nucleic acid molecule encoding a protein according to any one of claims 1-17. (a) Heavy chain amino acid sequence; (b) Light chain amino acid sequence; or (c) Both the heavy chain amino acid sequence and the light chain amino acid sequence.

22. An expression vector comprising the nucleic acid molecule according to claim 21.

23. A recombinant host cell comprising the nucleic acid molecule according to claim 21 or the expression vector according to claim 22.

24. A method for producing a protein, the method comprising: The protein is produced by culturing a recombinant host cell containing the expression vector according to claim 22 under conditions in which the nucleic acid molecule is expressed. as well as The protein is isolated from the host cell or culture.

25. A method for enhancing an anticancer 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 1-17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

26. A method of 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 1-17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

27. A method for alleviating cancer symptoms in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1-17, 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 25-27, wherein the cancer is gastrointestinal stromal carcinoma (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

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