Activatable bispecific anti-CD47 and anti-PD-L1 proteins and uses thereof

Abstract

Provided herein is a protein molecule that specifically binds PD-L1 and exhibits activatable specific CD47 binding in diseased tissue.Further provided herein is the use of such protein molecule to treat cancer.Provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 348,842, filed June 3, 2022, U.S. Provisional Patent Application No. 63 / 342,283, filed May 16, 2022, U.S. Provisional Patent Application No. 63 / 309,759, filed February 14, 2022, and U.S. Provisional Patent Application No. 63 / 305,003, filed January 31, 2022, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (ULSL_003_04WO_SeqList_ST26.xml; size: 38,176 bytes; and creation date: Jan. 29, 2023) are incorporated by reference herein in their entirety.

[0003] Technical Field The present disclosure relates to activatable bispecific proteins and treatments for cancer. [Background technology]

[0004] background In immune oncology therapy, most of the important drug targets are not exclusively expressed in diseased tissues, and the major ones are also expressed in non-diseased tissues.In addition, many drugs used in cancer treatment use very powerful cell killing mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).As a result, the binding of targets by drugs in non-diseased tissues often causes undesirable side effects.

[0005] 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 tissues. However, PD-L1 has also been observed to be highly expressed on some cancer cells. PD-L1 binds to the membrane protein PD1. The interaction of PD-L1 with PD1 on T cells downregulates T cell inflammatory activity, which promotes immune self-tolerance. PD-L1 is therefore described as an immune checkpoint. Thus, antagonistic anti-PD-L1 monoclonal antibodies that block the interaction with PD1 have demonstrated the potential to act as well-tolerated immunotherapeutic agents in disease settings such as cancer by "liberating" T cell responses from the constraints of innate immunity. Thus, PD-L1 is a drug target used to amplify the anti-cancer effects of the adaptive immune system.

[0006] CD47 is also a cell surface receptor that acts as an immune checkpoint, but its effect is primarily to limit the activity of the innate immune system, particularly in the case of monocytes, macrophages, natural killer (NK) cells, and neutrophils. This ability to minimize innate immune cell activity is multifactorial, but primarily involves activation of the SIRPα receptor when bound by CD47. Activation of the SIRPα receptor minimizes ADCC and ADCP by innate immune cells. CD47 blocking agents that also provide strong Fc effector functions may maximize the therapeutic potential of the innate immune system by simultaneously blocking CD47 action while promoting FcγR-mediated cell killing.

[0007] Anti-PD-L1 antibodies could be made more potent therapeutics by acquiring the ability to also block CD47 and tightly bind Fcγ receptors (e.g., in a bispecific antibody format with IgG1 Fc). This combines two important checkpoint inhibitor functions that can synergistically stimulate both the innate and adaptive immune systems. However, the ability to make this combination work in a single therapeutic structure is severely limited by the extremely broad expression of CD47 on many cell types (e.g., red and white blood cells, and endothelial cells), as well as other cells. This broad expression profile for CD47 not only results in dose-limiting toxicity of CD47-binding agents with active effector functions, but also poses significant peripheral sink / biodistribution problems that limit the ability of such agents to achieve high enough exposure in diseased tissues to take advantage of their combined mechanisms. Thus, there is a need for engineered forms of bispecific binding proteins with activity that is specifically targeted to the diseased tissue environment. Summary of the Invention [Means for solving the problem]

[0008] Abstract a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; wherein the anti-PD-L1 VH Domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the anti-PD-L1 VL Domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; Provided herein is a protein, wherein the VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein the anti-CD47 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:10, an LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:12; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0009] Provided herein is a protein comprising a heavy chain and a light chain, where the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; and where the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; where the anti-PD-L1 VH Domain comprises the amino acid sequence of SEQ ID NO:21 and the anti-PD-L1 VL Domain comprises the amino acid sequence of SEQ ID NO:22; and where the anti-CD47 VH Domain comprises the amino acid sequence of SEQ ID NO:23 and the anti-CD47 VL Domain comprises the amino acid sequence of SEQ ID NO:24.

[0010] Provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:25; and wherein the light chain comprises the amino acid sequence of SEQ ID NO:26.

[0011] In some embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the second linker comprises the amino acid sequence of SEQ ID NO: 14.

[0012] In some embodiments, the immunoglobulin heavy chain constant region of the protein provided herein is an IgG, IgE, IgM, IgD, IgA, or IgY constant region. In some embodiments, the immunoglobulin heavy chain constant region of the protein provided herein is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 constant region. In some embodiments, the immunoglobulin heavy chain constant region of the protein provided herein is immunologically inert. In some embodiments, the immunoglobulin heavy chain constant region of the protein provided herein is 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, where the numbering is according to the EU index as in Kabat.

[0013] Provided herein are multimeric proteins that are dimers of two identical proteins, where each protein is a protein disclosed herein.

[0014] Provided herein are immunoconjugates 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 antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0015] Provided herein are pharmaceutical compositions comprising a protein, multimeric protein, or immunoconjugate disclosed herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0016] 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 the heavy and light chain amino acid sequences of the proteins disclosed herein. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:28, or both the nucleotide sequences of SEQ ID NO:27 and SEQ ID NO:28.

[0017] Provided herein is an expression vector comprising the nucleic acid molecule disclosed herein. Provided herein is a recombinant host cell comprising the nucleic acid molecule disclosed herein or the expression vector disclosed herein.

[0018] Provided herein is a method of producing a protein, the method comprising culturing a recombinant host cell containing 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.

[0019] 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, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0020] Provided herein is a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0021] Provided herein is a method for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0022] Provided herein are methods for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein, wherein the cancer is gastrointestinal stromal cancer (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, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0023] Provided herein is a method for reducing the size of a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0024] Provided herein is a method for inhibiting tumor growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0025] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in enhancing an anti-cancer immune response in a subject.

[0026] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in treating cancer in a subject.

[0027] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in ameliorating a symptom of cancer in a subject.

[0028] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in ameliorating a symptom of cancer in a subject, wherein the cancer is gastrointestinal stromal cancer (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, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0029] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in reducing the size of a tumor in a subject.

[0030] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in inhibiting the growth of a tumor in a subject. [Brief description of the drawings]

[0031] [Figure 1] Figure 1 shows a schematic of a protein molecule (LB101) disclosed herein in intact (left) and protease-cleaved (right) conformations. In the intact conformation, only the PD-L1 binding domain is exposed and can bind their cognate targets. The linkers in both the heavy and light chains in this design are proteolytically cleavable and can be sequentially cleaved by matrix metalloproteases (MMPs) and / or cathepsins. An initial "fast" cleavage creates an intact conformation and an intermediate protease-cleaved active state, which allows both PD-L1 Fab and CD47 Fab derived from a single protein construct to bind their cognate targets.

[0032] [Diagram 2] FIG. 2 shows a schematic representation of the mechanism of activity of the activatable bispecific protein molecules provided herein, such as LB101 (designated "CD47 LockBody").

[0033] [Figure 3A] Figure 3A shows SDS-PAGE analysis of purified LB101 proteins. Lane 1 - molecular weight standards. Lane 2 - LB101 (reducing conditions). Lane 3 - blank. Lane 4 - LB101 (non-reducing conditions).

[0034] [Figure 3B] FIG. 3B shows size-exclusion chromatography (SEC) analysis of purified LB101 protein, clearly demonstrating structural homogeneity.

[0035] [Figure 4] Figure 4 shows ELISA analysis of LB101 binding to immobilized human PD-L1 or immobilized human CD47. A time course analysis was performed for hinge linker cleavage with or without human MMP12 from 0 to 24 hours.

[0036] [Figure 5A] FIG. 5A shows phagocytosis of A549 cancer cells by human CD11b+ cells with IgG1 isotype and vehicle (no phagocytosis), LB101 without MMP12 treatment (weak phagocytosis), LB101 MMP12 treatment (strong phagocytosis) and CD47 IgG4 (strong phagocytosis).

[0037] [Figure 5B] FIG. 5B shows phagocytosis of HEL cancer cells by human CD11b+ cells with IgG1 isotype and vehicle (no phagocytosis), LB101 without MMP12 treatment (weak phagocytosis), LB101 MMP12 treatment (strong phagocytosis) and CD47 IgG4 (strong phagocytosis).

[0038] [Figure 6A] Figure 6A shows hPD-L1+ MC38 tumor growth in transgenic hPD-L1 / hPD1 C57B6 mice. Mice were treated with either IgG1 isotype (5 mg / kg), atezolizumab (anti-PD-L1 antibody) (5 mg / kg) or LB101 (8.5 mg / kg, molar equivalent to 15 mg / kg IgG).

[0039] [Figure 6B] Figure 6B shows Kaplan-Meier survival plots for hPD-L1+ MC38 tumor growth in transgenic hPD-L1 / hPD1 C57B6 mice. A total of five groups were treated with either IgG1 isotype (5 mg / kg), atezolizumab (anti-PD-L1 antibody) (5 mg / kg or 10 mg / kg), or LB101 (8.5 mg / kg or 17 mg / kg, molar equivalent to 5 mg / kg or 10 mg / kg of IgG1).

[0040] [Figure 6C] Figure 6C shows hPD-L1+ MC38 tumor growth in transgenic hPD-L1 / hPD1 C57B6 mice. Mice were treated with either IgG1 isotype (5 mg / kg), atezolizumab (anti-PD-L1 antibody) (10 mg / kg) or LB101 (17 mg / kg, molar equivalent to 10 mg / kg of IgG1).

[0041] [Figure 6D] Figure 6D shows hPD-L1+ MC38 tumor growth in individual transgenic hPD-L1 / hPD1 C57B6 mice. Mice were treated with either atezolizumab (anti-PD-L1 antibody) (5 mg / kg) or LB101 (8.5 mg / kg, molar equivalent to 15 mg / kg IgG).

[0042] [Figure 6E]Figure 6E shows hPD-L1+ MC38 tumor growth in transgenic hPD-L1 / hPD1 C57B6 mice. Mice were treated with either an IgG1 isotype control (5 mg / kg), or a dose titration of LB101 (0.3 mg / kg, 1.5 mg / kg, 4.5 mg / kg, and 8.5 mg / kg).

[0043] [Figure 7] Figure 7 shows the activity of IgG1 isotype, atezolizumab (anti-PD-L1 antibody) or LB101 (before or after treatment with MMP12 for 1 or 2 hours) in a cell-based assay that measures the ability of an agent to disrupt hPD-L1 / hPD1 signaling (measured by fold activation).

[0044] [Figure 8] Figure 8 shows hPD-L1+ MC38 tumor growth during a "re-challenge" experiment in transgenic hPD-L1 / hPD1 C57B6 mice. Naive mice (n=10) and non-naive mice that had previously shown complete tumor regression when treated with LB101 (8.5 mg / kg, n=13) or LB101 (17 mg / kg, n=9) were studied.

[0045] [Figure 9] Figure 9 shows hPD-L1+ MC38 tumor growth during a "re-challenge" experiment in transgenic hPD-L1 / hPD1 C57B6 mice over a 30 day period. Naive mice (n=10) and non-naive mice that had previously shown complete tumor regression when treated with LB101 (8.5 mg / kg, n=13) or LB101 (17 mg / kg, n=9) were studied.

[0046] [Figure 10]Figure 10 shows immune cell infiltration in hPD-L1+ MC38 tumors as measured by flow cytometry. Tumors (n=8 / group) were isolated from mice treated with human IgG1 isotype, atezolizumab (anti-PD-L1 antibody) or LB101. Numbers in each analysis are reported as % of total tumor CD45+ (immune) cells. Significant values ​​(p<0.05) for differences between groups were calculated and are shown as lines and asterisks.

[0047] [Figure 11-1] Figures 11A-11F show results from a dose-ranging (DRF) study of LB101 in cynomolgus monkeys. Male (m) and female (f) monkeys were dosed with LB101 at 5 mg / kg, 20 mg / kg or 50 mg / kg once every 7 days for four doses. Blood samples were collected and measurements were made of LB101 pharmacokinetics (Figure 11A), body weight (Figure 11B), red blood cell levels (RBC, Figure 11C), hemoglobin levels (HBG, Figure 11D), platelet levels (Figure 11E) and neutrophil levels (Figure 11F). The range of normal levels is shown as a grey box. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description Provided herein is a protein that is conditionally active in diseased human tissue. The protein of the present disclosure is fully active in specifically binding and blocking PD-L1 throughout the body; shows minimized binding of CD47 in healthy tissue; and is highly activated in binding and blocking CD47 once in diseased tissue environment. The protein of the present disclosure comprises a CD47 binding domain that is masked by the PD-L1 binding domain in non-diseased tissue. The protein also comprises two peptide linkers that are cleaved by one or more proteases expressed in diseased tissue (e.g., tumor). The linker cleavage unmasks the CD47 binding domain in diseased tissue, thus selectively allowing the protein to bind and / or function in the diseased tissue.

[0049] Protein molecules Provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region. The first linker and the second linker are cleavable by matrix metalloproteases (MMPs) and / or cathepsins found in diseased tissues (e.g., tumors). In some embodiments, the immunoglobulin heavy chain constant region comprises, from N-terminus to C-terminus, a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A schematic representation of the proteins of the present disclosure with domains labeled is shown in FIG. 1.

[0050] The anti-PD-L1 / anti-CD47 protein structure shown in FIG. 1 minimizes binding to CD47 outside of diseased tissue. This effect is achieved by adding PD-L1 and a linker on top (N-terminal) of the CD47 binding domain. Use of the appropriate upper domain / linker combination results in a configuration that completely blocks binding activity at the lower CD47 binding domain. The PD-L1 domain then drives enrichment in the PD-L1-enriched tumor microenvironment. 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 and expose the CD47 binding domain, thereby conditionally activating CD47 binding activity in the tumor rather than the periphery. The combined biological functions of the proteins provided herein thereby give the molecule the potential to enhance both innate and adaptive immune responses against cancer cells, as outlined in FIG. 2.

[0051] The protein designs disclosed herein may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, with or without effector function capabilities. In the construct shown in FIG. 1, four polypeptide chains encode four Fab domains (2×Fab A, 2×Fab B), four linker sequences, with or without an immunoglobulin hinge region and an Fc domain. The linkers in the protein designs are proteolytically cleavable and can be sequentially cleaved. An initial "fast" cleavage results in an intact structure, creating an intermediate active state that allows Fab A and Fab B from a single protein construct to bind their cognate targets. The cleaved linkers derived from immunoglobulin hinge sequences may also recruit increased immune effector functions (ADCC, CDC, and / or ADCP) at the cell membrane via endogenous anti-hinge antibodies.

[0052] The proteins disclosed herein include antibody molecule domains and regions. The term "antibody" generally refers to an immunoglobulin (Ig) molecule that comprises 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.

[0053] In full-length antibodies, 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. 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 hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL domain is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0054] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The numbering of residues in the Fc region is according to the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. The Fc region may exist in a dimeric or monomeric form. The Fc region binds to various cellular receptors (e.g., Fc receptors) and other immune molecules (e.g., complement proteins).

[0055] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or subclass. IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen-binding domains, each composed of a VH and a VL. Generally, IgA antibodies are composed of two monomers, each composed of two heavy chains and two light chains (for IgG, IgD, and IgE antibodies); thus, IgA molecules have four antigen-binding domains, each also composed of a VH and a VL. Certain IgA antibodies are monomeric antibodies in that they are composed of two heavy chains and two light chains. Secretory IgM antibodies are generally composed of five monomers, each composed of two heavy chains and two light chains (as for IgG and IgE antibodies). Thus, the IgM molecule has 10 antigen-binding domains, each also composed of a VH and a VL. The cell surface form of IgM has a two heavy chain / two light chain structure similar to IgG, IgD and IgE antibodies.

[0056] As used herein, the terms "immunological binding" and "immunological binding properties" refer to non-covalent interactions of the type that occur between an immunoglobulin molecule (e.g., an antibody or antigen-binding portion thereof), or a protein that contains an immunoglobulin-derived binding domain(s), and an antigen for which the immunoglobulin or protein is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ), where K dA smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where the rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the "on rate constant" (K on ) and "off rate constant" (K off Both K and K can be determined by calculation of the concentrations and the actual rates of association and dissociation (see Malmqvist, Nature 361:186-187 (1993)). off / K on The ratio of d (See Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies or antigen-binding portions provided herein have an equilibrium binding constant (K d An antibody is said to specifically bind PD-L1 or CD47 when its K is ≦10 μM, preferably ≦10 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM. d One method for determining is by using surface plasmon resonance (SPR), typically by using a biosensor system such as a Biacore® system.

[0057] Functionally, the binding affinity of the proteins provided herein is greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of the proteins provided herein can be in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8M~10 -12 M、10 -9 M~10 -12 M、10 -5 M~10 -11 M、10 -6 M~10 -11 M、10 -7 M~10 -11 M、10 -8 M~10 -11 M、10 -9 M~10 -11 M、10 -10 M~10 -11 M、10 -5 M~10 -10 M、10 -6 M~10 -10 M 、 10 -7 M~10 -10 M、10 -8 M~10 -10 M、10 -9 M~10 -10 M、10 -5 M~10 -9 M、10 -6 M~10 -9 M、10 -7 M~10 -9 M、10 -8 M~10 -9 M、10 -5 M~10 -8 M、10 -6 M~10 -8 M、10 -7 M~10 -8 M、10 -5 M~10 -7 M、10 -6 M~10 -7 Mまたは10 -5 M~10 -6 Mである。

[0058] Provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region. In some embodiments, the first linker is the same as the second linker. In some embodiments, the first linker is not the same as the second linker. In some embodiments, the first linker comprises an amino acid sequence selected from any one of SEQ ID NOs: 13-20. In some embodiments, the second linker comprises an amino acid sequence selected from any one of SEQ ID NOs: 13-20. In some embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the second linker comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 13; and the second linker comprises the amino acid sequence of SEQ ID NO: 14.

[0059] a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; wherein the anti-PD-L1 VH Domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO:1, HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and HCDR3 comprising the amino acid sequence of SEQ ID NO:3; Provided herein is a protein, wherein the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:4, an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:6; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0060] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; and Provided herein is a protein, wherein the VH domain comprises at least one HCDR sequence selected from HCDR1 comprising the amino acid sequence of SEQ ID NO:1, HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and HCDR3 comprising the amino acid sequence of SEQ ID NO:3; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0061] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; and Provided herein is a protein, wherein the VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:3; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0062] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; and Provided herein is a protein, wherein the VL domain comprises at least one LCDR sequence selected from LCDR1 comprising the amino acid sequence of SEQ ID NO:4, LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and LCDR3 comprising the amino acid sequence of SEQ ID NO:6; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0063] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; and Provided herein is a protein, wherein the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:4, an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:6; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0064] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region; wherein the anti-CD47 VH domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO:7, HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and HCDR3 comprising the amino acid sequence of SEQ ID NO:9; Provided herein is a protein, wherein the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:10, an LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:12; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0065] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region; Provided herein is a protein, wherein the VH domain comprises at least one HCDR sequence selected from HCDR1 comprising the amino acid sequence of SEQ ID NO:7, HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0066] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region; Provided herein is a protein, wherein the VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0067] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region; Provided herein is a protein, wherein the VL domain comprises at least one LCDR sequence selected from LCDR1 comprising the amino acid sequence of SEQ ID NO:10, LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and LCDR3 comprising the amino acid sequence of SEQ ID NO:12; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0068] A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; and wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region; Provided herein is a protein, wherein the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:10, an LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:12; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0069] a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; wherein the anti-PD-L1 VH Domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the anti-PD-L1 VL Domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; Provided herein is a protein, wherein the VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein the anti-CD47 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:10, an LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:12; wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0070] a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; wherein the anti-PD-L1 VH Domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the anti-PD-L1 VL Domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; Provided herein are proteins, wherein the VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein the anti-CD47 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:10, an LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:12; wherein the first linker comprises the amino acid sequence of SEQ ID NO:13; and wherein the second linker comprises the amino acid sequence of SEQ ID NO:14.

[0071] Further provided herein is a protein comprising a heavy chain and a light chain, where the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain VH Domain, a CH domain, a first linker, an anti-CD47 VH Domain, and an immunoglobulin heavy chain constant region; and where the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL Domain, and a second immunoglobulin light chain constant region; where the anti-PD-L1 VH Domain comprises the amino acid sequence of SEQ ID NO:21 and the anti-PD-L1 VL Domain comprises the amino acid sequence of SEQ ID NO:22; and where the anti-CD47 VH Domain comprises the amino acid sequence of SEQ ID NO:23 and the anti-CD47 VL Domain comprises the amino acid sequence of SEQ ID NO:24.

[0072] Also provided herein are proteins comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 25; and wherein the light chain comprises the amino acid sequence of SEQ ID NO: 26. Proteins comprising these heavy and light chain sequences are referred to as LB101 or LB-101. In some embodiments, the proteins comprise one or more of the amino acid sequences provided in Table 1.

[0073] Further provided herein is a protein comprising a heavy chain and a light chain, wherein 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:25; and wherein the light 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:26.

[0074] Also provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:25 with 1, 2 or 3 conservative amino acid substitutions; and the light chain comprises the amino acid sequence of SEQ ID NO:26 with 1, 2 or 3 conservative amino acid substitutions.In some embodiments, conservative amino acid substitutions are only made in FR sequences, and not in CDR sequences.In some embodiments, conservative amino acid substitutions are not made in the first linker sequence or the second linker sequence. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] In the VH and VL domain sequences, the CDR sequences are underlined. In the heavy and light chain amino acid sequences, the anti-PD-L1 VH and VL domain sequences are italicized and underlined; the cleavable linker sequence is underlined, and the anti-CD47 VH and VL domain sequences are bolded.

[0075] In some embodiments, the proteins provided herein comprise at least one Fab fragment, which is a monovalent antigen-binding fragment consisting of the VL, VH, CL and CH1 domains.

[0076] In some embodiments, the proteins provided herein comprise an immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin heavy chain constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin heavy chain constant region is IgG1. In some embodiments, the immunoglobulin heavy chain constant region is immunologically inert. In some embodiments, the immunoglobulin heavy chain constant region comprises one or more mutations to reduce or prevent FcγR 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 IgG1 constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a human IgG4 constant region comprising the amino acid substitution S228P, where the numbering is according to the EU index as in Kabat. In some embodiments, the amino acid residue positions in the constant region of an immunoglobulin molecule are numbered according to the EU index as in Kabat (Ward et al., 1995 Therap. Immunol. 2:77-94).

[0077] 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:31.

[0078] In some embodiments, the proteins provided herein can include an immunoglobulin light chain constant region that is a lambda light chain.

[0079] In some embodiments, the proteins provided herein may comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of a human IgG4, human IgG4(S228P), human IgG2, human IgG1, or human IgG1 effector null Fc region. For example, the human IgG4(S228P) Fc region comprises the following substitutions compared to a wild-type human IgG4 Fc region: S228P. For example, the human IgG1 effector null Fc region comprises the following substitutions compared to a wild-type human IgG1 Fc region: L234A, L235A, and G237A. In some embodiments, the proteins may comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.

[0080] Further provided herein is a multimeric protein that is a dimer of two identical proteins, where each protein is a protein disclosed herein. Figure 1 shows an example of such a dimer. The dimer comprises two identical heavy chains and two identical light chains. In some embodiments, each heavy chain comprises the amino acid sequence of SEQ ID NO:25, and each light chain comprises the amino acid sequence of SEQ ID NO:26.

[0081] Provided herein are immunoconjugates 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 antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0082] Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g., RNAses).Additional therapeutic agents include therapeutic nucleic acids (e.g., genes that code for immunomodulators, antiangiogenic agents, antiproliferative agents, or proapoptotic agents).These drug descriptors are not mutually exclusive, so therapeutic agents can be described using one or more of the above terms.

[0083] Examples of therapeutic agents suitable for use in immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansines, CC-1065 and duocarmycins, calicheamicins and other enediynes, and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers may also be used in immunoconjugates. In addition, conjugates may be made using a secondary carrier (e.g., liposomes or polymers) as the cytotoxic agent. Suitable cytotoxins include agents that inhibit or prevent the function of cells and / or cause the destruction of cells. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins (e.g., protein kinases, phosphatases, topoisomerases, enzymes, and cyclins).

[0084] Representative cytotoxins include doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pirarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacytidine, doxifluridine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, cisplatin ... These include, but are not limited to, platins, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, fluorouracils, etoposide, taxol, taxol analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramicin, azaserine, bleomycin, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterlin, esperamicin, and maytansinoids.

[0085] Suitable immunomodulatory agents include anti-hormonal agents that block hormone action on the tumor and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0086] 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 include the protein (or an immunoconjugate comprising said protein, or a multimeric protein comprising said protein) and a pharma- ceutically acceptable carrier, diluent or excipient. Such substances should be non-toxic and should not interfere with the efficacy of the protein. The exact nature of the carrier or other substances depends on the route of administration, which may be by injection, bolus, infusion, or any other suitable route, as discussed below. Compositions are provided herein that include the proteins disclosed herein (or an immunoconjugate comprising said protein, or a multimeric protein comprising said protein) and a pharma- ceutically acceptable carrier.

[0087] As used herein, the term "pharmaceutical acceptable" refers to molecular entities and compositions that generally do not produce allergic reactions or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that have been approved by a regulatory agency of the U.S. Federal or state government or are listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans, are considered to be "pharmaceutical acceptable". As used herein, the term "pharmaceutical 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 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 (e.g., fixed oils) may be used. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition. A pharma-ceutically acceptable carrier, diluent or excipient may be a compound or combination of compounds that does not cause secondary reactions and can, for example, facilitate the administration of the protein, increase its life span and / or its effectiveness in the body, or increase its solubility in solution.

[0088] In all embodiments of the pharmaceutical compositions provided herein, the protein may comprise a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:25; and wherein the light chain comprises the amino acid sequence of SEQ ID NO:26.

[0089] The pharmaceutical composition disclosed herein can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration). The solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent (e.g., water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); an antibacterial agent (e.g., benzyl alcohol or methylparaben); an antioxidant (e.g., ascorbic acid or sodium hydrogen sulfate); a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)); a buffer (e.g., acetate, citrate, or phosphate), and an agent for adjusting tonicity (such as sodium chloride or dextrose). The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0090] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (登録商標)(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The composition may be sterile; fluid to the extent that easy needle passage exists; stable under conditions of manufacture and storage; and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent in the composition (for example, sugar, polyalcohol such as mannitol, sorbitol, sodium chloride). Prolonged absorption of an injectable composition may be brought about by including an agent that delays absorption in the composition (for example, aluminum monostearate and gelatin).

[0091] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the ingredients listed above, followed by filtration sterilization if necessary. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients other than those listed above. In the case of sterile powders and powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying to obtain a powder of the active ingredient plus any additional desired ingredients from its solution that has previously been sterile-filtered.

[0092] Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or auxiliary substances may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients, or compounds of a similar nature: binders (e.g., microcrystalline cellulose, gum tragacanth, or gelatin); excipients (e.g., starch or lactose), disintegrants (e.g., alginic acid, Primojel, etc.), and / or other suitable agents. (登録商標) , or corn starch); a lubricant (e.g., magnesium stearate); a glidant (e.g., colloidal silicon dioxide); a sweetening agent (e.g., sucrose or saccharin); or a flavoring agent (e.g., peppermint, methyl salicylate, or orange flavoring).

[0093] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0094] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated can be used in the formulation.Such penetrants are generally known in the art, and for example, for transmucosal administration, include detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal spray or suppository.For transdermal administration, the active compound can be formulated into ointments, salves, gels, or creams as generally known in the art.

[0095] Medicaments can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0096] In some embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as controlled release formulations, 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 of skill in the art. The materials may also be obtained commercially. Liposomal suspensions may also be used as pharma-ceutically acceptable carriers.

[0097] It is particularly 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 a physically separate unit suitable as a unitary dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification for dosage unit form of the present invention is determined by and directly depends on the specific characteristics of said active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of compounding such active compound for individual treatment.

[0098] In some embodiments, the protein may be provided in lyophilized form for reconstitution prior to administration, for example, lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline solution prior to administration to an individual.

[0099] The pharmaceutical compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.

[0100] Nucleic acid molecules, vectors, host cells and methods for producing antibodies Provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding the amino acid sequences of the proteins disclosed herein (or the amino acid sequences of (i) the VH domain, (ii) the VL domain, or (iii) both the VH and VL domains of the proteins). Further provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding (i) the heavy chain, (ii) the light chain, or (iii) both the heavy and light chains of the proteins disclosed herein. In some embodiments, the nucleic acid molecules encoding the VH domain, VL domain, heavy chain, or light chain comprise a signal sequence (or encode a leader peptide). In some embodiments, the nucleic acid molecules encoding the VH domain, VL domain, heavy chain, or light chain do not comprise a signal sequence (or encode a leader peptide).

[0101] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:28, or both SEQ ID NO:27 and SEQ ID NO:28, with or without a sequence encoding a leader peptide.

[0102] Also provided herein is an expression vector comprising the nucleic acid molecule described herein. In certain vectors, the nucleic acid molecule is operably linked to one or more regulatory sequences suitable for the expression of the nucleic acid segment in a host cell. In some cases, the expression vector comprises a sequence that mediates replication and comprises one or more selectable markers. As used herein, "vector" refers to a construct that can deliver, and preferably express, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., producer cells).

[0103] 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(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. An expression vector can be transfected into a host cell by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. In some embodiments, a recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both the heavy and light chains of a protein disclosed herein. In some embodiments, a 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.

[0104] The antibody molecules of the present invention, or antigen-binding portions thereof, may be produced using techniques well known in the art (e.g., recombinant, phage display, synthetic, computational or a combination of such techniques, or other techniques readily known in the art).

[0105] Further provided herein is a method for producing a protein disclosed herein, comprising culturing a recombinant host cell comprising an expression vector as described herein under conditions in which its nucleic acid segment is expressed, thereby producing said protein.The protein can then be isolated from said host cell or culture.Provided herein is a method for producing a protein, comprising culturing a recombinant host cell comprising an expression vector as described herein under conditions in which its nucleic acid molecule is expressed, thereby producing protein; and isolating the protein from the host cell or culture.

[0106] The proteins disclosed herein can be produced by any of a variety of methods known to those skilled in the art. In certain embodiments, the proteins disclosed herein can be produced recombinantly. For example, the nucleic acid sequence encoding one or more of SEQ ID NO:25 and SEQ ID NO:26, or a portion thereof, can be introduced into bacterial cells (e.g., E. coli, B. subtilis) or eukaryotic cells (e.g., yeast such as S. cerevisiae, or mammalian cells such as CHO cell lines, various Cos cell lines, HeLa cells, HEK293 cells, various myeloma cell lines, or transformed B cells or hybridomas), or into an in vitro translation system, and the translated polypeptide can be isolated. In some embodiments, light and heavy chain proteins are produced in cells with signal sequences that are removed during the production of the mature proteins disclosed herein.

[0107] One of skill in the art can determine whether a protein comprising a given polypeptide sequence binds to PD-L1 protein and / or CD47 protein using standard methodologies (e.g., Western blot, ELISA, etc.).

[0108] Medical Uses of Activatable Proteins Provided herein are methods and uses of the activatable proteins, multimeric proteins, immunoconjugates, and pharmaceutical compositions disclosed herein to provide a therapeutic benefit to a subject with cancer.

[0109] The activatable proteins, multimeric proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may be used in methods of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., treatment prior to the onset of a condition in a subject, to reduce the risk of the condition occurring in the subject; delay its onset; or reduce its severity after onset). Methods of treatment may include administering the protein, multimeric protein, immunoconjugate, or pharmaceutical composition to a subject in need thereof.

[0110] Provided herein is a method for enhancing anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of the protein, multimeric 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.

[0111] Provided herein is a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein. Provided herein is a method for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0112] Provided herein are methods for reducing tumor size in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0113] Provided herein is a method for inhibiting tumor growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0114] 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, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0115] In some embodiments, the cancer is a solid tumor. In some embodiments, the tumor is a gastrointestinal tumor, a pancreatic tumor, a breast tumor, a lung tumor, a bronchial tumor, a colorectal tumor, a prostate tumor, a stomach tumor, an ovarian tumor, a bladder tumor, a brain tumor, a central nervous system tumor, a peripheral nervous system tumor, an esophageal tumor, a cervical tumor, a uterine tumor, an endometrial tumor, a tumor of the oral cavity or pharynx, a liver tumor, a kidney tumor, a renal tumor, a testicular tumor, a biliary tumor, a small intestine tumor, a appendix tumor, a salivary gland tumor, a thyroid tumor, or an adrenal tumor.

[0116] In some embodiments, the cancer is a hematological cancer.

[0117] In some embodiments, the cancer of blood tissue is lymphoma. In some embodiments, the cancer is mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia (AML), B-lymphoid leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or hairy cell leukemia.

[0118] In all embodiments of the methods and uses provided herein, the protein may comprise a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:25; and the light chain comprises the amino acid sequence of SEQ ID NO:26.

[0119] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent (e.g., a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein) sufficient to reduce or ameliorate the severity and / or duration of cancer, or one or more symptoms thereof, prevent progression of the disease, cause regression of the disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, or enhance or improve the prophylactic or therapeutic effect(s) of another related treatment (e.g., a prophylactic or therapeutic agent) for cancer.

[0120] The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the 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 administration schedule, and other factors known to medical practitioners. Treatment prescription, such as determining the dosage, is within the responsibility of the general practitioner and other physicians and may depend on the severity of the symptoms and / or progression of the disease being treated. Appropriate dosages of antibody-based protein molecules are well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be found herein or in the Physician's Desk Reference (2003). may be used as appropriate for the type of medication being administered. Therapeutically effective amounts or appropriate doses of an antibody-based protein molecule can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dose depends on many factors, including whether the antibody-based protein is for prevention or treatment, the size and location of the area to be treated, the exact nature of the antibody-based protein, and the nature of any detectable label or other molecule attached to the antibody-based protein.

[0121] Typical protein doses range from 100 μg to 1 g for systemic application, and 1 μg to 1 mg for intradermal injection. An initial higher loading dose may be administered, followed by one or more lower doses. In some embodiments, the protein is an IgG1 or IgG4 isotype. Doses for single treatment of adult subjects may be adjusted proportionately for children and infants. Treatment may be repeated at daily, twice weekly, weekly or monthly intervals, at the physician's discretion. Treatment schedules for subjects may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

[0122] Treatment may be periodic, with the period between administrations being about 2 weeks or longer, e.g., about 3 weeks or longer, about 4 weeks or longer, about once a month or longer, about 5 weeks or longer, or about 6 weeks or longer. For example, treatment may be every 2-4 weeks or every 4-8 weeks. Treatment may be given before and / or after surgery and / or may be administered or applied directly to the anatomical site of the surgical or invasive procedure. Suitable formulations and routes of administration are described above.

[0123] In some embodiments, the proteins, multimeric proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may be administered as subcutaneous injections, e.g., using an autoinjector for long-term prevention / treatment.

[0124] In some embodiments, the therapeutic effect of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein may last for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein may last in a subject 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.

[0125] In some embodiments, a subject may be treated with a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein and an additional therapeutic agent or treatment used to treat cancer or a symptom or complication of cancer. The protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein and the additional therapeutic agent or treatment may be administered simultaneously or sequentially.

[0126] In some embodiments, the subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a guinea pig, a mouse or a rat. In some embodiments, the subject is an adult. In some embodiments, the subject is a child.

[0127] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use as a medicament.

[0128] Further provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in the treatment of a disease or disorder.

[0129] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in enhancing an anti-cancer immune response in a subject.

[0130] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in treating cancer in a subject.

[0131] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in ameliorating a symptom of cancer in a subject.

[0132] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in ameliorating a symptom of cancer in a subject, wherein the cancer is gastrointestinal stromal cancer (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, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0133] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in reducing the size of a tumor in a subject.

[0134] Provided herein is a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in inhibiting the growth of a tumor in a subject. definition

[0135] Unless otherwise noted, the terms used herein have definitions as commonly used in the art. Some terms are defined below, and further definitions can be found within the remainder of the detailed description.

[0136] The terms "a" or "an" may refer to one or more of its entities, i.e., to a plurality of referents. Thus, the terms "a", "an", "one and more", and "at least one" are used interchangeably herein. Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one of said elements is present, unless the context clearly requires that only one such element is present.

[0137] Unless otherwise stated or clear from the context, the term "about" means within 10% above or below the reported numerical value (except when such number is greater than 100% or less than 0% of possible values). When used in conjunction with a range of values ​​or a series of values, the term "about" applies to the endpoints of the range or each of the values ​​recited in that series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0138] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout the range of alignment of residues, e.g., nucleotides or amino acids. The "identity fraction" for an aligned segment of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The "percent identity" is the identity fraction x 100. Percent identity can be calculated using the alignment program Clustal Omega (available at ebi.ac.uk / Tools / msa / clustalo) using default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (2011 October 11) Molecular Systems Biology 7:539. For purposes of calculating identity to a sequence, extensions (eg, tags) are not included.

[0139] As used herein, the term "HCDR" refers to a heavy chain complementarity determining region. As used herein, the term "LCDR" refers to a light chain complementarity determining region.

[0140] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another that does not significantly and adversely change the functional activity. A preferred example of a "conservative substitution" is the replacement of one amino acid with another having a value of > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919): [Table 4]

[0141] The term "immunoconjugate" refers to a protein of the present disclosure conjugated to a cytotoxic, cytostatic and / or therapeutic agent.

[0142] The term "isolated molecule" (where the molecule is, for example, a protein, nucleic acid, polynucleotide, or antibody) refers to a molecule that, depending on its source of origin or derivative, is (1) not associated with naturally associated components that accompany it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells 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 the cell from which it naturally originates is "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. Molecular purity or homogeneity can be assayed by many means well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be provided using HPLC or other means well known in the art for purification.

[0143] The terms "inhibit," "block," or "neutralize," as used herein in reference to the biological activity of a protein disclosed herein, refer to the ability of the protein to substantially antagonize, prohibit, suppress, slow, destroy, eliminate, halt, reduce, or reverse, for example, the progression, intensity, or severity of what is being inhibited, including, but not limited to, the binding of PD-L1 to PD-1, or the binding of CD47 to SIRPα.

[0144] As used herein, the terms "treat", "treating" or "treatment of" (and grammatical variations thereof) mean that the severity of the subject's condition is reduced, at least partially ameliorated, or stabilized, and / or some alleviation, reduction, decrease or stabilization of at least one clinical symptom is achieved, and / or there is a delay in the progression of the disease or disorder.

[0145] PD-L1 is also known as programmed cell death ligand 1, CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1, and hPD-L1.

[0146] CD47 is also known as Integrin Associated Protein, IAP, MER6, and Antigenic Surface Determinant Protein OA3.

[0147] As used herein, and unless otherwise stated, the terms "hinge linker," "linker," "hinge," "first linker," and "second linker," and derivatives thereof, refer to a sequence derived from an immunoglobulin hinge region that can link, e.g., two polypeptides, e.g., polypeptides of different Fab regions, and that is separate from any hinge sequence in an immunoglobulin hinge region that may be part of a protein of the invention.

[0148] As used herein, the terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of a disease, disorder and / or clinical symptom(s) in a subject and / or reducing the severity of the onset of a disease, disorder and / or clinical symptom(s) relative to that which would occur in the absence of the compositions and / or methods described herein. Prevention can be complete (e.g., the disease, disorder and / or clinical symptom(s) is / are completely absent). The prevention can also be partial, such that the onset of a disease, disorder and / or clinical symptom(s) in the subject and / or the onset is less severe than that which would occur in the absence of the compositions and / or methods described herein.

[0149] As used herein, a "therapeutically effective amount" is an amount of a protein or pharmaceutical composition provided herein that is effective to treat a disease or disorder or ameliorate a sign or symptom thereof in a subject. A "therapeutically effective amount" may vary depending, for example, on the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the patient to be treated, and the judgment of the prescribing physician.

[0150] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0151] The present disclosure will be further clarified by the following examples, which are merely illustrative of the present disclosure and are in no way intended to be limiting. EXAMPLES

[0152] Working Example Generation of optimized conditionally active therapeutic antibodies Introduction In this example, we successfully generated and demonstrated the utility of an optimized conditionally active antibody (LB101; sequence provided in Table 1), which was well expressed, biophysically stable, highly soluble, and showed improved biological efficacy compared to anti-PD-L1 IgG format antibodies.

[0153] material and method Protein cloning, transient expression, purification, and characterization The DNA sequences encoding the antibodies were cloned via restriction-ligation cloning into expression cassettes encoding separate human IgG1 heavy and light chain constant regions in separate plasmid vectors to generate activatable constructs for expression. The generated antibodies were captured from the clarified supernatant using a HiTrap MabSelect Sure Protein A 5mL column on an AKTA Pure 150 L FPLC system. The eluted protein peak was immediately buffer exchanged into 1x PBS (pH 7.4) by directly loading the eluted Protein A peak fraction onto a HiPrep 26 / 10 desalting column. Protein concentrations were determined by measuring absorbance at 280 nm and 1 μg of each purified protein was analyzed by SDS-PAGE using 4-20% TGX polyacrylamide gradient gels (BioRad, Cat. nr. 456-1093) and 1× Tris / glycine / SDS buffer, separated at 120 V electric field for 1 h under reducing and / or non-reducing conditions. Analytical size-exclusion chromatography was performed to test for the presence of non-covalently bound aggregates and to complement the SDS PAGE analysis. Aliquots of selected clones were analyzed by analytical size-exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 SEC column and 1× PBS (pH 7.4) as running buffer in isocratic mode. Selected proteins were further purified using preparative SEC. Antibody samples up to 1 ml were loaded onto a Superdex 200 Increase 10 / 300 SEC column or a HiLoad 26 / 600 Superdex 200 pg column equilibrated with 1x PBS (pH 7.4). 1 ml fractions of the peak of interest were collected and the main peak fractions were pooled. After size exclusion chromatography, the samples were again analyzed by SDS-PAGE as described above.

[0154] Metalloprotease digestion Protein constructs were incubated with human matrix metalloprotease (MMP) enzyme MMP12 at a ratio of 1% total MMP to protein construct (wt / wt) for 0-24 hours at 37° C. in Tris-buffered saline (pH 7.4) containing 5 mM CaCl2. The reaction was stopped by the addition of 20 mM EDTA and samples were then tested for binding or functional activity as described.

[0155] IgG titration binding ELISA To coat the ELISA plates, the target protein was diluted to 1 μg / ml in PBS (pH 7.4) and added 100 μl / well overnight at 4° C. The coated plates were washed 3 times with PBS (pH 7.4), blocked with 4% skim milk protein in PBS for 1 hour at room temperature (RT) (380 μl / well), and then washed 5 times with PBS-Tween 20 (PBST). Antibody (100 μl / well; diluted in PBST) was then added and incubated for 1 hour at RT. The plates were then washed 3 times with PBS and goat anti-human IgG-HRP was added (100 μl / well) for 1 hour at RT. The plates were then washed 3 times with PBST and twice with PBS, after which 100 μl TMB / well was added. The reaction was stopped by adding 100 μl 2M H2SO4 / well, and the OD was read on a plate reader at 450 nm.

[0156] Phagocytosis potency assay Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation. CD14 positive PBMCs were subsequently isolated via magnetic cell isolation using CD14 microbeads. In parallel, cancer cells were labeled using green CFSE (carboxy-fluorescein diacetate, succinimidyl ester) cell tracking dye. A total of 6.25 × 10 5Labeled Jurkat cells were preincubated in the presence of UH2 antibody in 96-well plates for 1 h at 37 °C in a humidified atmosphere containing 5% CO2. After incubation, 2.5 × 10 5 CD14 positive cells were added to each well and incubated for an additional hour under the same culture conditions. Cells were harvested by vigorous pipetting, stained with viability dye, and fixed using ice-cold 4% paraformaldehyde for 10 minutes. After fixation, cells were blocked with Fc receptor binding inhibitor monoclonal antibody for 10 minutes, then incubated with Alexa Fluor 647 (AF647)-conjugated anti-human CD11b antibody for 30 minutes at room temperature, and further fixed in 4% paraformaldehyde for 5 minutes. Cells were analyzed on a BD Fortessa flow cytometer recording side and forward scatter properties along with CFSE and AF647 fluorescence intensity data. Briefly, cell debris was gated out by scatter properties (SSC-area x FSC-area). Single cells were also gated by SSC-area x SSC-height, then FSC-area x FSC-height. From the remaining single cell population, CFSE and CD11b double positive cells were gated using quadrant gates placed based on the population of CD11b positive cells in the vehicle-treated study. The percentage of CFSE positive cells from the CD11b positive population was calculated and plotted.

[0157] In vivo efficacy analysis in C57B6 / hPD-L1 / hPD1 mice bearing hPD-L1+ MC38 syngeneic tumors In an in vivo multiple-dose efficacy study, atezolizumab, an IgG1 isotype, and LB101 were each administered six times (on days 0, 3, 6, 9, 12, and 15) in C57B6 mice transgenic for human PD-L1 and PD1 bearing tumors generated by subcutaneous inoculation of the mouse cancer cell line MC38 (stably transfected with human PD-L1).2 Dosing was initiated after tumor size was established. Tumor volumes were measured by caliper measurement. Repeats of this study were then performed as above with a full dose titration of LB101 (0.3 mg / kg, 1.5 mg / kg, 4.5 mg / kg, and 8.5 mg / kg).

[0158] PD1 / PD-L1 cell-based antagonism assay The potency of the antibodies in blocking PD1 / PD-L1 interaction was measured using a PD1 / PD-L1 blocking cell-based bioassay (Promega). The day before the assay, PD-L1 aAPC / CHO-K1 cells were thawed and transferred to cell collection medium (90% Ham's F12 / 10% FBS). The cell suspension was dispensed at 100μl / well into each of the inner 60 wells of two 96-well white flat-bottom assay plates. Cell collection medium was added to each of the outer wells and the assay plate and incubated overnight at 37℃ / 5% CO2. On the day of the assay, sample IgG was diluted 4-fold from 100nM to 0.01nM in assay buffer (99% RPMI 1640 / 1% FBS) and 40μl per dilution was added to the assay plate containing PD-L1 aAPC / CHO-K1 cells. Thus, the positive inhibition control was atezolizumab anti-PD-L1 antibody. As a negative inhibition control, the IgG1 isotype was included. PD1 effector cells were then thawed in assay buffer (99% RPMI 1640 / 1% FBS) and the cell suspension was added to the wells of the assay plate containing the PD-L1 aAPC / CHO-K1 cells and the IgG titration samples. The assay plate was incubated in a 37°C / 5% CO2 incubator for 6 hours, equilibrated to ambient temperature for 5-10 minutes, and then 80μl of Bio-Glo TM Reagent (Promega) was added, and the assay plate was incubated at ambient temperature for an additional 5-30 min, and the luminescence signal was subsequently measured at 10, 20 and 30 min.

[0159] Evaluation of LB101 binding to human Fcγ receptors Binding of test antibodies to high and low affinity human Fcγ receptors was assessed by single cycle analysis using a Biacore® T200 running at a flow rate of 30 μl / min. Human Fc receptors, FcγRI, FcγRIIA (both 167R and 167H polymorphs), FcγRIIB, FcγRIIIA (both 176F and 176V polymorphs) and FcγRIIIB, were captured on a CM5 sensor chip that was previously coupled using a His capture kit using standard amine chemistry. A 5-point 3-fold dilution range of test antibodies (analytes) with no regeneration between concentrations was used for each receptor tested. In all cases, test antibodies were passed over the chip at increasing concentrations at 30 μl / min, followed by one dissociation step. After dissociation, the chip was regenerated with an injection of glycine (pH 1.5). The signal from the reference channel Fc1 (blank) was subtracted from the signal of the receptor-loaded Fc to correct for differences in non-specific binding to the reference surface. Sensorgrams were analyzed with 1:1 kinetics for the high affinity Fcγ receptor hFcγRI and by steady-state binding for the low affinity Fcγ receptor.

[0160] Affinity analysis of LB101 binding to human and mouse CD47 To evaluate the binding of LB101, LB101 after 16 hours of MMP12 digestion, and a positive control anti-CD47 Fab (a recombinant Fab using the variable domain sequence used in LB101) to human and mouse CD47, a multi-cycle kinetic analysis was performed on a Biacore® T200 at 25°C. HBS-P+ supplemented with 0.1% BSA was used as the running buffer and for ligand and analyte dilution. LB101 antibody and control anti-CD47 Fab samples were diluted to 1.0 μg / mL in running buffer and loaded at the start of each cycle onto Fc2, Fc3, and Fc4 of a series S CM5 chip that had previously been coupled with anti-human Fab capture antibody using standard amine chemistry. Ligand was captured at a flow rate of 10 μl / min, resulting in an immobilization level (RL) of approximately 60 RU. The surface was then stabilized. Multi-cycle kinetic data were obtained using either human or mouse CD47 when analytes were injected at a flow rate of 30 μl / min. An eight-point two-fold dilution range of 50.0 nM to 0.39 nM for human CD47 and 500.0 nM to 3.9 nM for mouse CD47 was prepared in running buffer. For each concentration, the association phase was monitored for 180 s and the dissociation phase was measured for 300 s. Regeneration of the sensor chip surface was performed between cycles using 10 mM glycine, pH 2.1. The signal from reference channel Fc1 (no ligand captured) was subtracted from that of Fc2-4 to correct for bulk effects and differences in non-specific binding to the reference surface. The signal from each CD47 blank run (ligand captured but not antigen) was subtracted to correct for differences in surface stability. Human CD47 binding was analyzed using a 1:1 binding analysis due to the high affinity interaction between the antibody and antigen. Mouse CD47 binding was analyzed using a steady-state assay.

[0161] Results and Discussion Protein construct design principles A key issue limiting efficacy for CD47 targeted drugs is that this protein is expressed on many different cell classes in the body (e.g., red blood cells, platelets, and endothelial cells (among others)). This off-tumor target expression often results in dose-limiting side effects risk and an antigen "sink" effect. The CD47 "sink" minimizes the amount of drug that penetrates the tumor (even when large doses of IgG are given). Binding of blood cells by anti-CD47 is also a significant toxicity risk, potentially causing multiple forms of cytopenias and even hemagglutination in patients. These factors minimize the potential safety and efficacy of anti-CD47 antibodies.

[0162] The LB101 anti-PD-L1 / CD47 protein construct (Figure 1) aims to overcome the peripheral sink and toxicity issues by minimizing binding of CD47 outside of diseased tissues. This effect is achieved by adding PD-L1 and a linker on top (n-terminal) of the CD47 binding domain. Use of the appropriate upper domain / linker combination results in a configuration that completely blocks binding activity at the lower CD47 domain. The PD-L1 domain then drives enrichment in the PD-L1-enriched tumor microenvironment, and 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 and expose the CD47 binding domain, thereby conditionally activating CD47 binding activity in the tumor but not in the periphery. The combined biological functions of LB101 thereby provide the molecule with the potential to enhance both innate and adaptive immune responses against cancer cells, as outlined in Figure 2.

[0163] The LB101 design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, with or without effector function capabilities. In this construct, four polypeptide chains code for four Fab domains (2x Fab A, 2x Fab B), four linker sequences, with or without an immunoglobulin hinge region and an Fc domain. The linkers in the LB101 design are proteolytically cleavable and can be cleaved sequentially. An initial "fast" cleavage results in an intact structure, creating an intermediate active state, which allows Fab A and Fab B derived from a single protein construct to bind their cognate targets. The cleaved linker derived from an immunoglobulin hinge sequence may also recruit increased immune effector functions (ADCC, CDC and ADCP) at the cell membrane via endogenous anti-hinge antibodies. This is a known phenomenon in human patients with (and even without) underlying autoreactive disease.

[0164] Protein construct cloning, expression, and characterization To generate LB101, DNA cassettes of each construct type (Table 1) were synthesized and cloned into expression vectors encoding human IgG1 heavy and light chains. The anti-PD-L1 variable domain sequences used in the protein constructs disclosed herein are the variable domain sequences provided in U.S. Patent No. 7,943,743 B2. The anti-CD47 variable domain sequences used in the protein constructs disclosed herein are provided in U.S. Patent No. 10,683,350 B2. The proteins were produced by transient transfection of CHO cells and then purified by Protein A affinity and size-exclusion chromatography. The purified proteins demonstrated high purity and homogeneity by SDS-PAGE (Figure 3A) and analytical size-exclusion chromatography (SEC) (Figure 3B), demonstrating that the LB101 constructs can be expressed and purified as intact, stable products in a single process.

[0165] LB101 in vitro functional characterization Purified LB101 protein was incubated in the presence or absence of human MMP12 enzyme at 37°C for 0, 2, 4, 8, or 24 hours, and then tested for its binding signals of human PD-L1 and human CD47 by ELISA (Figure 4). All samples showed high binding signals to human PD-L1, clearly indicating that the LB101 domain that binds PD-L1 is fully active. Samples without added MMP12 did not show any binding signals for CD47. Incubation with MMP12 for 2, 4, 8, and 24 hours clearly showed an increase in CD47 binding, suggesting that this binding signal is induced by cleavage of the linker between PD-L1 and the CD47 domain.

[0166] To examine the effect of CD47 binding and blocking by MMP12-activated LB101, CFSE-labeled A549 cells (which express PD-L1 and CD47) were mixed with freshly isolated human CD14+ cells in the presence of titrated IgG1 isotype, CD47 IgG4, LB101 without MMP12 treatment, LB101 with MMP12 treatment for 4 hours, and vehicle. Myeloid cells undergoing phagocytosis were observed by CD11b / CFSE staining in flow cytometry (Figure 5A). This analysis showed that all samples induced weak or no phagocytosis except for the CD47 IgG4 positive control and LB101 with MMP12 treatment, clearly indicating that the phagocytosis-promoting ability of LB101 relies on enzyme activation. This phagocytosis assay was then repeated using the PD-L1+ / CD47+ myeloid cell line HEL as target cells. Again, all samples induced weak or no phagocytosis except for the CD47 IgG4 positive control and LB101 with MMP12 treatment, further demonstrating that the phagocytosis-promoting ability of LB101 relies on enzyme activation and that the PD-L1 binding activity of LB101 alone is insufficient to drive strong phagocytic activity.

[0167] In vitro efficacy analysis in the MC38 syngeneic tumor environment In an in vitro multiple-dose efficacy study, equimolar doses of isotype control IgG1 (5 mg / kg), LB101 (8.5 mg / kg) and atezolizumab (5 mg / kg) were each administered six times (every 3 days) in C57B6 mice transgenic for human PD-L1 and PD1 bearing tumors generated by subcutaneous inoculation of the murine cancer cell line MC38 (stably transfected with human PD-L1). 2 After establishing the basal concentration, dosing was started and the animals were 2The tumor volumes were measured by caliper measurements (Figure 6A). This study clearly showed that the IgG1 isotype control allowed rapid and uniform outgrowth of MC38 tumors, with 0 / 16 animals in this group showing tumor regression. As for the atezolizumab group, tumor growth was slowed in the group, but only 1 / 16 in the group showed tumor regression. In the LB101 group, in contrast, tumor growth was strongly inhibited in the group, with 14 / 16 showing tumor regression. This efficacy difference was repeated at higher doses of atezolizumab (10 mg / kg) and LB101 (17 mg / kg), with survival rates similar to those of the 5 mg / kg and 8.5 mg / kg groups outlined above (Figure 6B, Figure 6C). This indicated that even high doses of atezolizumab could not achieve the efficacy observed for LB101. Thus, the high efficacy of LB101 at the moderate dose of 8.5 mg / kg is most likely driven by protease activation in the MC38 tumor microenvironment, resulting in PD-L1 and CD47 blockade, and stimulating the involvement of both adaptive and innate immunity, as outlined in Figure 2. This interpretation was further supported by the analysis of tumor growth curves of individual mice in the 5 mg / kg and 8.5 mg / kg groups (Figure 6D). Here, for example, it was observed that some tumors continued to regress for 10 days after day 15 (when treatment ended).

[0168] A repeat of this study was then performed as above, but using a full dose titration of LB101. In a dose titration analysis of LB101 versus isotype control IgG, both the 0.3 mg / kg and 1.5 mg / kg doses were found to be less effective, while strong tumor growth inhibition was observed at 4.5 mg / kg, with the strongest activity observed at 8.5 mg / kg (Figure 6E).

[0169] PD-L1 bioactivity of LB101 Isotype control, atezolizumab and LB101 IgG were tested for their ability to antagonize PD-L1 / PD1 signaling in the Promega PD1 cell signaling bioassay (Figure 7). In this analysis, the IgG1 isotype did not block PD-L1 / PD1 binding, while atezolizumab and LB101 (with or without MMP12 treatment for 1 or 2 hours) showed similarly potent signaling inhibition.

[0170] "Rechallenge" analysis in the MC38 syngeneic tumor environment To perform the "re-challenge" experiments, naive transgenic hPD-L1 / hPD1 C57B6 mice (control group, n=10), and non-naive mice that had previously shown complete hPD-L1+ MC38 tumor regression when treated with LB101 (8.5 mg / kg, n=13) or LB101 (17 mg / kg, n=9) (Figure 6A, Figure 6B), were re-implanted with hPD-L1+ MC38 cells and tumor growth was measured over 18 days (Figure 8). This analysis clearly demonstrated that LB101-treated animals did not grow tumors. In contrast, all control animals (10 / 10) had tumors ranging from 330 to 1100 mm by day 18. 3 By day 28, all control animals (10 / 10) had tumors measuring approximately 3000 mm 3 Tumors with an average size of 100 μg / kg were established, whereas all LB101 animals remained tumor free (FIG. 9).

[0171] Analysis of the immunomodulatory effects of LB101 in the MC38 syngeneic tumor environment To examine the immunomodulatory effects of LB101 in tumors compared to controls, equimolar doses of isotype control IgG1 (5 mg / kg), LB101 (8.5 mg / kg) and atezolizumab (5 mg / kg) were each administered four times (every 3 days) in C57B6 mice (n=8 mice / group) transgenic for human PD-L1 and PD1 bearing tumors generated by subcutaneous inoculation of the murine cancer cell line MC38 (stably transfected with human PD-L1). 2 Treatment was initiated after the establishment of a PD-L1 / ...

[0172] Analysis of LB101 pharmacokinetics and safety in cynomolgus monkeys A dose-ranging (DRF) study of LB101 in cynomolgus monkeys was performed to establish pharmacokinetic and safety parameters. Male and female monkeys were dosed at 5 mg / kg, 20 mg / kg, or 50 mg / kg LB101, once every 7 days, for four doses. Blood samples were collected to measure LB101 pharmacokinetics (Figure 11A). This clearly showed that LB101 exhibited linear dose-dependent concentrations over the 7 days following dosing. Body weight measurements (Figure 11B) did not show any weight loss in any of the animals. Most importantly, factors known to be at risk of change due to CD47 blockade, such as red blood cell levels (RBC, Figure 11C), hemoglobin levels (HBG, Figure 11D), platelet levels (Figure 11E), and neutrophil levels (Figure 11F), all remained unchanged and within normal levels in all dosing schemes. These findings clearly demonstrated that the CD47-binding ability of LB101 was indeed completely restricted in the periphery and that the activation level of LB101 was extremely low, since CD47-blocking IgG was associated with nonlinear PK and severe anemia at doses above 1 mg / kg.

[0173] Evaluation of LB101 binding to human Fcγ receptors The binding of the control antibody, LB101, and MMP12-digested LB101 to high and low affinity human Fcγ receptors was examined using Biacore® biosensor technology (Table 2). Significant binding of the positive control human IgG1, trastuzumab, was observed to both high and low affinity human Fcγ receptors. LB101 was found to bind with similar affinity to trastuzumab for all receptors. Overall, LB101 digested with MMP12 for 2 hours retained the ability to bind to all Fcγ receptors with similar affinity to LB101, even though MMP12-digested LB101 showed slightly reduced affinity (KD>3-fold) for hFcγRIIA167 and hFcγRIIB (Table 2). [Table 2]

[0174] Affinity analysis of LB101 binding to human and mouse CD47 Table 3 summarizes the data for LB101 to human and mouse CD47, LB101 after 16 hour MMP12 digestion, and a positive control anti-CD47 Fab. The LB101 sample clearly showed no binding to either human or mouse CD47. Unlocked LB101 (digested for 16 hours) bound to both human and mouse CD47. The affinity for mouse CD47 was found to be lower than the affinity for human CD47, and thus the K D Got the value. [Table 3] NB = no binding observed NA = Not Applicable (Steady State Binding Method)

[0175] Although the present invention has been described with its preferred specific embodiments, it should be understood that the foregoing description and the following examples are intended to be illustrative and not limiting of the scope of the invention. It will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the scope of the invention, and furthermore, other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains. In addition to the embodiments described herein, the present invention contemplates and claims those inventions that result from combinations of the features of the inventions cited herein and the features of the cited prior art references that complement the features of the invention. Similarly, it is recognized that any described material, feature, or article may be used in combination with any other material, feature, or article, and such combinations are considered to be within the scope of the invention. The disclosures of each patent, patent application, and publication cited or described in this document are each incorporated herein by reference in their entirety for all purposes.

[0176] Numbered embodiments Notwithstanding the appended claims, the present disclosure includes the following numbered embodiments:

[0177] Embodiment 1. A protein comprising a heavy chain and 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 CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; 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-CD47 VL domain, and a second immunoglobulin light chain constant region; wherein the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; wherein said anti-CD47 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein said anti-CD47 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; wherein said first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; protein.

[0178] Embodiment 2. The anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO: 21, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 22; the anti-CD47 VH domain comprises the amino acid sequence of SEQ ID NO: 23 and the anti-CD47 VL domain comprises the amino acid sequence of SEQ ID NO: 24; 2. The protein of embodiment 1.

[0179] Embodiment 3. The heavy chain comprises the amino acid sequence of SEQ ID NO:25; The light chain comprises the amino acid sequence of SEQ ID NO: 26. 3. The protein according to embodiment 1 or 2.

[0180] Embodiment 4. The protein of embodiment 1 or 2, wherein the immunoglobulin heavy chain constant region is an IgG, IgE, IgM, IgD, IgA, or IgY constant region.

[0181] Embodiment 5. The protein of embodiment 1 or 2, wherein the immunoglobulin heavy chain constant region is an IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 constant region.

[0182] Embodiment 6 The protein of embodiment 1 or 2, wherein the immunoglobulin heavy chain constant region is immunologically inert.

[0183] Embodiment 7. The protein of embodiment 1 or 2, wherein the immunoglobulin heavy chain constant region is 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, or a human IgG4 constant region comprising the amino acid substitution S228P, where numbering is according to the EU index as in Kabat.

[0184] Embodiment 8. The protein according to any one of embodiments 1 to 7, wherein the first linker comprises the amino acid sequence of SEQ ID NO:13.

[0185] Embodiment 9. The protein of any one of embodiments 1 to 8, wherein the second linker comprises the amino acid sequence of SEQ ID NO:14.

[0186] Embodiment 10. A multimeric protein that is a dimer of two identical proteins, where each protein is a protein according to any one of embodiments 1-9.

[0187] Embodiment 11. An immunoconjugate comprising a protein of any one of embodiments 1 to 9 linked to a therapeutic agent.

[0188] Embodiment 12. The immunoconjugate of embodiment 11, 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.

[0189] Embodiment 13. A pharmaceutical composition comprising a protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, or an immunoconjugate according to embodiment 11 or 12, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0190] Embodiment 14: The protein according to any one of embodiments 1 to 9. (a) the heavy chain amino acid sequence; (b) the light chain amino acid sequence; or (c) the amino acid sequences of both the heavy chain and the light chain; A nucleic acid molecule encoding

[0191] Embodiment 15. The nucleic acid molecule of embodiment 14, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:28, or both the nucleotide sequences of SEQ ID NO:27 and SEQ ID NO:28.

[0192] Embodiment 16. An expression vector comprising the nucleic acid molecule of embodiment 15.

[0193] Embodiment 17. A recombinant host cell comprising the nucleic acid molecule of embodiment 15 or the expression vector of embodiment 16.

[0194] Embodiment 18. A method of producing a protein, the method comprising culturing a recombinant host cell comprising the expression vector of embodiment 16 under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0195] Embodiment 19. A method for enhancing an anti-cancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13.

[0196] Embodiment 20. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13.

[0197] Embodiment 21. A method for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein described in any one of embodiments 1 to 9, a multimeric protein described in embodiment 10, an immunoconjugate described in embodiment 11 or 12, or a pharmaceutical composition described in embodiment 13.

[0198] Embodiment 22. The method of any one of embodiments 19 to 21, wherein the cancer is gastrointestinal stromal cancer (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, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0199] Embodiment 23. A method for reducing tumor size in a subject, comprising administering to the subject a therapeutically effective amount of a protein described in any one of embodiments 1 to 9, a multimeric protein described in embodiment 10, an immunoconjugate described in embodiment 11 or 12, or a pharmaceutical composition described in embodiment 13.

[0200] Embodiment 24. A method for inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of a protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13.

[0201] Embodiment 25. A protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13, for use in enhancing an anti-cancer immune response in a subject.

[0202] Embodiment 26. A protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13, for use in treating cancer in a subject.

[0203] Embodiment 27. A protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13, for use in ameliorating a cancer symptom in a subject.

[0204] Embodiment 28. The protein, multimeric protein, immunoconjugate or pharmaceutical composition according to any one of embodiments 25 to 26 for use, wherein the cancer is gastrointestinal stromal cancer (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, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0205] Embodiment 29. A protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13, for use in reducing tumor size in a subject.

[0206] Embodiment 30. A protein according to any one of embodiments 1 to 9, a multimeric protein according to embodiment 10, an immunoconjugate according to embodiment 11 or 12, or a pharmaceutical composition according to embodiment 13, for use in inhibiting tumor growth in a subject.

Claims

1. A protein comprising a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a CH domain, a first linker, an anti-CD47 VH domain, and an immunoglobulin heavy chain constant region; wherein the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD47 VL domain, and a second immunoglobulin light chain constant region; wherein the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; wherein the anti-CD47 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:9; wherein the anti-CD47 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; wherein said first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; protein.

2. the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO: 21, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 22; the anti-CD47 VH domain comprises the amino acid sequence of SEQ ID NO: 23, and the anti-CD47 VL domain comprises the amino acid sequence of SEQ ID NO: 24; The protein of claim 1.

3. The heavy chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:25; the light chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 26; The protein of claim 1.

4. the heavy chain comprises the amino acid sequence of SEQ ID NO: 25; the light chain comprises the amino acid sequence of SEQ ID NO: 26; The protein of claim 1.

5. The protein described in claim 1, wherein the heavy chain comprises an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:

27.

6. The protein described in claim 1, wherein the light chain comprises an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:

28.

7. 2. The protein of claim 1, wherein the immunoglobulin heavy chain constant region is an IgG, IgE, IgM, IgD, IgA, or IgY constant region.

8. The protein of claim 1 , wherein the immunoglobulin heavy chain constant region is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 constant region.

9. The protein of claim 1 , wherein the immunoglobulin heavy chain constant region is immunologically inert.

10. 2. The protein of claim 1, wherein the immunoglobulin heavy chain constant region is 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, or a human IgG4 constant region comprising the amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat.

11. The protein of claim 1 , wherein the first linker comprises the amino acid sequence of SEQ ID NO:

13.

12. The protein of claim 1 , wherein the second linker comprises the amino acid sequence of SEQ ID NO:

14.

13. A multimeric protein that is a dimer of two identical proteins, wherein each protein is a protein according to claim 1.

14. An immunoconjugate comprising the protein of claim 1 linked to a therapeutic agent.

15. The immunoconjugate of claim 14, 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.

16. A pharmaceutical composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, and a pharmaceutically acceptable carrier, diluent or excipient.

17. The protein of claim 1 (a) the heavy chain amino acid sequence; (b) the light chain amino acid sequence; or (c) the amino acid sequences of both the heavy chain and the light chain; A nucleic acid molecule encoding

18. 18. The nucleic acid molecule of claim 17, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:28, or both the nucleotide sequences of SEQ ID NO:27 and SEQ ID NO:

28.

19. 19. An expression vector comprising the nucleic acid molecule of claim 18.

20. 20. A recombinant host cell comprising the nucleic acid molecule of claim 18 or the expression vector of claim 19.

21. 1. A method for producing a protein, said method comprising: Culturing a recombinant host cell containing the expression vector of claim 19 under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating said protein from said host cell or culture; A method that encompasses

22. A composition comprising the protein of any one of claims 1 to 12, the multimeric protein of claim 13, or the immunoconjugate of claim 14 or 15, or a pharmaceutical composition comprising the protein of any one of claims 1 to 12, the multimeric protein of claim 13, or the immunoconjugate of claim 14 or 15, and a pharmaceutically acceptable carrier, diluent or excipient, for use in enhancing an anti-cancer immune response in a subject.

23. A composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, or a pharmaceutical composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, and a pharmaceutically acceptable carrier, diluent or excipient, for use in treating cancer in a subject.

24. 24. The composition of claim 23, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, cancer of the central nervous system, cancer of the peripheral nervous system, 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 intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

25. A composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, or a pharmaceutical composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, and a pharmaceutically acceptable carrier, diluent or excipient, for use in reducing tumor size in a subject.

26. A composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, or a pharmaceutical composition comprising a protein according to any one of claims 1 to 12, a multimeric protein according to claim 13, or an immunoconjugate according to claim 14 or 15, and a pharmaceutically acceptable carrier, diluent or excipient, for use in inhibiting tumor growth in a subject.

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