Fusion protein targeting PD-L1 and neutralizing Gas6 and application thereof

Abstract

The present disclosure relates to a fusion protein comprising a Gas6 binding moiety and an antigen binding moiety comprising an anti-PD-L1 antibody or an antigen binding fragment thereof. The present disclosure also relates to a method for treating and / or detecting cancer in an individual in need thereof, and a kit for detecting cancer in a sample.

Description

Technical Field

[0001] The present disclosure relates to a novel fusion protein, and more particularly to a fusion protein that targets PD-L1 (programmed death-ligand 1) and neutralizes Gas6 (growth arrest-specific 6). Background Art

[0002] Cancer manipulates the tumor microenvironment for its own benefit. Cancer can trigger an immunosuppressive microenvironment, which is beneficial for its own development, progression, metastasis, and treatment resistance. For decades, immune checkpoint blockers have become a promising cancer immunotherapy. By using immune checkpoint blockers to control the tumor microenvironment of patients, 15% to 25% of patients with different cancers have achieved positive therapeutic effects. For those patients who respond significantly well, the overall survival rate of patients using immune checkpoint blockers even exceeds that of patients receiving standard treatment.

[0003] However, the limitations of PD-L1 / PD-1 blocker monotherapy make combination treatment strategies necessary to expand their application scope, improve their efficacy, and reduce their toxicity. Most frustratingly, combination therapies based on immunotherapy and approved standard therapies are only partially effective and are limited by severe side effects. Therefore, there is a need to develop a novel method for treating cancer. Summary of the Invention

[0004] Embodiments of the present disclosure relate to treating or diagnosing tumors via a fusion protein that includes (i) a Gas6-binding portion and (ii) an antibody or an antigen-binding fragment thereof that binds to an immune checkpoint protein, such as programmed death ligand 1 (PD-L1). Embodiments of the present disclosure relate to the use of such molecules (e.g., for treating cancer) and methods for preparing such molecules.

[0005] Accordingly, the present disclosure provides a fusion protein comprising

[0006] a Gas6-binding portion; and an antigen-binding portion comprising an antibody or an antigen-binding fragment thereof that is specific for an epitope in PD-L1.

[0007] In some embodiments of the present disclosure, the Gas6-binding portion comprises the extracellular domain of an RTK (receptor tyrosine kinase) of the TAM family (Tyro-3, Axl, MerTK).

[0008] In some embodiments of the present disclosure, an antibody or an antigen-binding fragment thereof comprises complementarity-determining regions (CDRs) of a heavy-chain variable region (VH) and complementarity-determining regions of a light-chain variable region, wherein the complementarity-determining regions of the heavy-chain variable region comprise VH-CDR1, VH-CDR2, and VH-CDR3, and the complementarity-determining regions of the light-chain variable region comprise VL-CDR1, VL-CDR2, and VL-CDR3.

[0009] Examples of the antibody or an antigen-binding fragment thereof include (but are not limited to) antibody (a) to (k) or an antigen-binding fragment thereof, 3G10 of US 7,943,743, 12A4 of US 7,943,743, 10A5 of US 7,943,743, 5F8 of US 7,943,743, 10H10 of US 7,943,743, 1B12 of US 7,943,743, 7H1 of US 7,943,743, 11E6 of US 7,943,743, 12B7 of US 7,943,743, 13G4 of US 7,943,743, MDX-1105, MEDI-4736, atezolizumab, durvalumab, avelumab, MDX-1105, envafolimab, cosibelimab, CK-301, CS-1001, SHR-1316, CBT-502, or BGB-A333.

[0010] In some embodiments of the present disclosure, an antibody or an antigen-binding fragment thereof comprises a heavy-chain constant region and a light-chain constant region. In one embodiment of the present disclosure, the constant region contains a mutation at the amino acid position corresponding to N297 of IgG1.

[0011] In some embodiments of the present disclosure, the antigen-binding portion comprises a Fab fragment, an F(ab')2 fragment, a ScFv fragment, a chimeric antibody, or a nanobody.

[0012] In some embodiments of the present disclosure, the antigen-binding portion is multispecific.

[0013] In some embodiments of the present disclosure, the Gas6-binding portion is fused to the antigen-binding portion via a peptide linker.

[0014] In some embodiments of the present disclosure, the Gas6-binding portion is fused to the heavy chain of the antigen-binding portion.

[0015] In some embodiments of the present disclosure, the Gas6-binding portion is fused to the C-terminus of the heavy chain of the antigen-binding portion.

[0016] The present disclosure further provides a pharmaceutical composition, which comprises:

[0017] an effective amount of the fusion protein disclosed herein or the genetically engineered cell disclosed herein; and

[0018] a pharmaceutically acceptable carrier.

[0019] The present disclosure further provides a method for treating, prophylactically treating and / or preventing cancer in an individual in need thereof, which comprises administering to the individual an effective amount of the fusion protein disclosed herein.

[0020] The present disclosure further provides a method for detecting cancer in an individual in need thereof, which comprises contacting a sample derived from the individual with the fusion protein disclosed herein.

[0021] The present disclosure further provides a kit for detecting cancer in a sample, which comprises the fusion protein disclosed herein.

[0022] Examples of cancer include (but are not limited to) bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

[0023] The present disclosure further provides a method for detecting PD-L1 in a sample, which comprises contacting the sample with the fusion protein disclosed herein.

[0024] The present disclosure further provides a method for neutralizing Gas6 in a sample, which comprises contacting the sample with the fusion protein disclosed herein.

[0025] Brief Description of Drawings

[0026] Figure 1 Exhibiting the advantageous spatial configuration of the present disclosure.

[0027] Figure 2 Exhibiting that Example 1 and Example 2 can efficiently capture human Gas6, thus achieving cell growth inhibition in the Ba / F3 cell line overexpressing Axl.

[0028] Figure 3 Exhibiting that Example 3, Example 4, Example 5 and Example 6 can efficiently capture human Gas6, thus achieving cell growth inhibition in the Ba / F3 cell line overexpressing Axl.

[0029] Figure 4 Exhibiting that Example 7, Example 8, Example 9 can efficiently capture human Gas6, thus achieving cell growth inhibition in the Ba / F3 cell line overexpressing Axl.

[0030] Figure 5 The in vivo tumor growth inhibitory function of Example 2 is superior to that of atezolizumab, self-made Axl-Fc, and the combination therapy.

[0031] Figure 6 It is shown that mice intraperitoneally injected with a molecule containing a Gas6-binding moiety exhibit reduced serum Gas6 concentration.

[0032] Figure 7 It is shown that mice achieving complete remission in the Example 2 group exhibit better immune memory when rechallenged with tumors compared to the atezolizumab group. One out of six mice in the atezolizumab group and two out of six mice in the Example 2 group achieved complete remission.

[0033] Figure 8 The impressive in vivo anti-tumor efficacy of Example 3 is shown.

[0034] Embodiment

[0035] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques described herein in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides are well known and commonly used in the art.

[0036] The practice of the present disclosure may employ techniques well within the ordinary skill, including cell biology, cell culture, antibody techniques, and genetic engineering. Such techniques are explained fully in the literature.

[0037] As used in accordance with the present disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings: The term “and / or” as used herein shall be regarded as a specific disclosure of each of the two designated features or components with or without the other. For example, “A and / or B” is regarded as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually recited herein.

[0038] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include plural referents.

[0039] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that includes at least one CDR that specifically binds to or interacts with a particular antigen (e.g., PD-L1). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (VH). In some embodiments, the heavy chain further contains a heavy chain constant region. The heavy chain constant region contains three domains, C H1 、C H2 and C H3 . Each light chain contains a light chain variable region (VL). In some embodiments, the light chain further contains a light chain constant region. The light chain constant region contains one domain (C L1 ). VH and VL can be further divided into hypervariable regions, called CDRs, which are interspersed with more conserved regions, called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FRs of the anti-PD-L1 antibody (or its antigen-binding fragment) may be identical to the human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on the alignment analysis of two or more CDRs.

[0040] As used herein, the term "specific for" or "specifically binds to" means that the antibody does not cross-react to a significant extent with other antigenic determinants.

[0041] As used herein, the term "antigenic determinant" refers to the site on an antigen to which an antibody binds.

[0042] As used herein, the term "complementary determining region (CDR)" refers to contiguous antigen-combining sites found within the variable regions of heavy and light chain polypeptides. Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996) have described CDRs, the definitions of which include overlaps or subsets of amino acid residues when compared to each other.

[0043] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from non-human immunoglobulins and human immunoglobulin constant regions, typically selected from human immunoglobulin templates.

[0044] As used herein, the term "nanobody" refers to an antibody containing a smaller single variable domain (VHH of antibodies obtained from camels and dromedaries). Antibody proteins obtained from members of the camel and dromedary (Camelus baclrianus and Camelus dromaderius) families, including New World members such as the llama species (Lama pacos, Lama glama, and Lama vicugna), have been characterized in terms of size, structural complexity, and antigenicity to human individuals. Some IgG antibodies from this mammalian family as found in nature lack a light chain and are thus structurally different from the typical four-chain quaternary structure of other animal antibodies, which have two heavy chains and two light chains.

[0045] As used herein, the term "antigen-binding fragment" of an antibody and like expressions include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex.

[0046] As applied to polypeptides, the term "substantially similar" or "substantially similarity" means that when a protein sequence is optimally aligned with another (reference) protein sequence using default gap weights by a program such as GAP or BESTFIT, there is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid residue sequence identity with the entire sequence of the reference protein sequence. More preferably, the positions of the residues that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or similarity can be up-regulated to correct for the nature of the conservative substitutions. The manner of making this adjustment is well known to those skilled in the art. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains are cysteine and methionine. More preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine. Alternatively, conservative substitutions are any changes that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0047] Sequence analysis software is commonly used to measure the sequence similarity of polypeptides, which is also referred to as sequence identity. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, the GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine closely related polypeptides, such as sequence homology or sequence identity between homologous polypeptides from organisms of different species or between a wild-type protein and its mutants. The FASTA program of GCG version 6.1 can also be used with preset or recommended parameters to compare polypeptide sequences. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the best overlapping regions between a query sequence and a retrieved sequence and a percentage of sequence identity (Pearson (2000) supra). When comparing the sequences of the present disclosure with a database containing a large number of sequences from different organisms, another more preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, which are incorporated herein by reference in their entireties.

[0048] As used herein, the term “pharmaceutical composition” means a mixture containing a therapeutic agent that is administered to a mammal (e.g., a human) to prevent, treat, or eliminate a specific disease or pathological condition afflicting the mammal.

[0049] As used herein, the term “therapeutically effective amount” or “effective amount” refers to the amount of an antibody that is sufficient to effect such treatment of a disease when administered to a mammal or other subject for treating the disease.

[0050] As used herein, the term “treatment / treating” and its like expressions cover any treatment of a disease in a mammal (specifically a human), and include: (a) preventing the occurrence of the disease in an individual who may be predisposed to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the disease to regress.

[0051] The term “preventing / prevention” is well recognized in the art and, when used in connection with a condition, includes administering an agent prior to the onset of the condition to reduce the incidence or severity of symptoms of a medical condition in an individual or delay its onset, relative to an individual who has not received the agent.

[0052] As used interchangeably herein, the terms "individual", "subject", "person", and "patient" refer to a mammal, including (but not limited to) murine (rat, mouse), non-human primate, human, canine, feline, ungulate (e.g., equine, bovine, ovine, porcine, caprine), etc.

[0053] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of a human, a physician, nurse, nurse practitioner, or individual; in the case of an animal (including non-human mammals), a veterinarian) that an individual requires or would benefit from treatment. This judgment is made based on a variety of factors within the caregiver's area of expertise and includes knowledge that the individual has or will develop a medical condition treatable with the compounds of the present disclosure.

[0054] "Cancer", "tumor", and like terms include pre-cancerous, neoplastic, transformed, and cancer cells, and may refer to solid tumors or non-solid cancers (see, e.g., Edge et al., AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman, Cytology: Diagnostic Principles and Clinical Correlates (3rd ed. 2009)). Cancer includes both benign neoplasms and malignant neoplasms (abnormal growths). "Transformation" refers to a spontaneous or induced phenotypic change, e.g., immortalization of cells, morphological changes, abnormal cell growth, reduced contact inhibition, and anchorage and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994)). Although transformation can be caused by infection with a transforming virus and the incorporation of new genomic DNA or the uptake of foreign DNA, it can also occur spontaneously or after exposure to a carcinogen.

[0055] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and can be used for diagnostic or monitoring assays. The definition encompasses blood and other liquid samples of biological origin; solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny.

[0056] Fusion protein:

[0057] The present disclosure provides a fusion protein that targets both PD-L1 and Gas6. An anti-PD-L1 antibody or PD-L1 binding fragment disclosed herein is fused to a portion of a Gas6 binding moiety (such as the Axl decoy receptor), which specifically binds to Gas6 and acts as a capturer of Gas6. Experimental results show that the bifunctional structure of the fusion protein comprising the anti-PD-L1 antibody or PD-L1 binding fragment fused to the Gas6 binding moiety (also referred to as anti-PD-L1×Gas6 binding moiety) does not interfere with the binding affinity and blocking activity of each part (anti-PD-L1 or Gas6 binding moiety) to its corresponding target. Compared with individual agents, the anti-PD-L1×Gas6 binding moiety motif can be better concentrated to the target tumor. Based on the difference in the binding affinity of the anti-PD-L1 antibody or PD-L1 binding fragment and the Gas6 binding moiety to their corresponding targets, the anti-PD-L1 antibody or PD-L1 binding fragment dominates the tumor targeting of the anti-PD-L1×Gas6 binding moiety. Additionally, compared with the effects produced by the administration of the two agents separately, the anti-PD-L1×Gas6 binding moiety unexpectedly exhibits a synergistic effect in cancer treatment.

[0058] Specifically, the present disclosure provides a fusion protein comprising

[0059] a Gas6 binding moiety; and

[0060] an antigen-binding moiety comprising an antibody or an antigen-binding fragment thereof that is specific for an epitope in PD-L1.

[0061] The fusion protein can bring therapeutic benefits to an individual. The fusion protein disclosed herein can be used as a therapeutic agent for treating and / or diagnosing cancer, which is more fully described herein.

[0062] An inhibitory checkpoint receptor, programmed cell death protein-1 (PD-1) and its ligand PD-L1 are responsible for the exhaustion of T cells in various tumors. Monoclonal antibodies that resist the PD-L1 / PD-1 interaction can thus stimulate immune cells to perform anti-cancer responses. It has been demonstrated that PD-L1 / PD-1 blockers are beneficial as monotherapeutic agents for some patients with cancers characterized by, such as high mutation burden and upregulated PD-L1 expression. However, the high response rate of PD-L1 / PD-1 blockers is limited to patients with certain cancer types. Negative results are detected in some patients who are expected to respond to PD-L1 / PD-1 blockers based on existing biomarkers, which further increases the difficulty of prediction. In addition, although PD-L1 / PD-1 blockers generally may cause relatively mild side effects, there are still about 10% of patients receiving PD-L1 / PD-1 blocker treatment who experience grade 3 to 4 toxicity reactions. The limitations of PD-L1 / PD-1 blocker monotherapy make combination treatment strategies necessary to expand its scope of revelation, improve its efficacy and reduce its toxicity. Most frustratingly, combination therapies based on immunotherapy and approved standard therapies, although partially effective, are limited by the occurrence of severe side effects. Therefore, there is still a need to develop combination therapies with higher survival benefits and fewer side effects.

[0063] Growth arrest-specific 6 is a member of the vitamin K-dependent protein family and is expressed by cancer cells as well as tumor-infiltrating lymphocytes. Gas6 regulates a series of different cellular functions, including cell proliferation, migration, survival, angiogenesis and metabolism, by binding to the TAM family of its receptor RTK. RTK is a cell surface receptor that has the ability to catalyze the phosphorylation of tyrosine residues on target proteins. The TAM family of RTK consists of Axl, Tyro and MerTK. All TAM family receptors have an extracellular ligand-binding domain, a single-pass transmembrane domain and an intracellular kinase domain, as well as a C-terminal tail containing tyrosine. Many studies have shown that the upregulation of Gas6 / TAM can promote the development of several cancers and is involved in cancer therapy resistance. Clinically, the expression of Gas6 and TAM receptors has always predicted a poor prognosis. In addition, Gas6 helps tumors evade immunity by binding to TAM receptors on residual immune cells.

[0064] Gas6 binds to TAM receptors with different affinities, with the highest affinity for Axl. Axl is overexpressed by host cells remaining in the tumor microenvironment, including several types of immune cells, fibroblasts, osteoclasts, and endothelial cells, which helps evade immunity. Elevated expression of the Axl receptor tyrosine kinase in various tumors is associated with a poorer overall survival rate in patients. Endogenous Axl activation induced by tumors and exogenous Axl activation induced by inhibitory immune cells recruited in the tumor microenvironment together contribute to the development of cancer. Through endogenous and exogenous actions, the autocrine and paracrine Gas6 / Axl signaling axis promotes tumor growth, metastasis, immune evasion, and treatment resistance in cancer patients. In addition, it has also been reported that the Gas6 / Axl pathway drives the expression of PD-L1 in cancer cells to prevent T cell activation.

[0065] As pointed out above, preventing Gas6 from binding to TAM has become an important approach for cancer therapy. Simultaneously inhibiting both the PD-L1 / PD-1 and Gas6 / TAM pathways represents a viable solution to the limitations of PD-L1 blockade monotherapy.

[0066] The fusion protein in the present disclosure captures Gas6 by a decoy receptor Gas6-binding portion, which is fused with an antibody portion against PD-L1 and targets the extracellular domain of immune checkpoint proteins on cancer cells and immune cells, thereby locally reducing the activation of endogenous and exogenous TAM kinases in tumors. This fusion protein (sometimes referred to as anti-PD-L1×Gas6-binding portion in the present disclosure) surpasses currently existing anti-cancer strategies, including (but not limited to) administering antibodies and receptors as separate molecules or in combination for various reasons. First, the antibody portion precisely guides the fusion protein to the tumor microenvironment where Gas6, which is concentrated therein, mainly acts through autocrine and paracrine pathways to promote tumor growth, metastasis, and immune evasion. The resulting advantages (e.g., as compared to administering antibodies and receptors as separate molecules) are partly due to the fact that cytokines mainly act through autocrine and paracrine functions in the local environment. The antibody portion guides the cytokine capturer to the tumor microenvironment where it can exert its maximum efficacy by neutralizing local immunosuppressive autocrine or paracrine actions. Second, the fusion protein can address tumor growth, metastasis, and immune evasion with the decoy receptor and at the same time restore local immune surveillance with the antibody portion. In addition, the positive feedback of PD-L1 production caused by Gas6 / Axl axis activation also indicates the synergistic effect of this fusion protein. Therefore, due to the presence of two mechanisms in one entity design, this fusion protein can be applied to a wider range of indications. Finally, as a single entity, this fusion protein can achieve multiple functions at a lower dose. Configuring multiple functions in one entity is also expected to be tolerant to antagonism. In addition, the inventors have screened different spatial arrangements of the fusion protein. The selected configuration shown in the present disclosure is that the Gas6-binding portion is on the C-terminus of the antibody portion, where the antibody portion without a stop codon is followed by the Gas6-binding portion containing a stop codon.

[0067] Overall, the present disclosure has revolutionized anti-PD-L1-based immunotherapy in three aspects: First, the present disclosure precisely drives to the tumor, triggering a synergistic anti-tumor effect by simultaneously blocking the PD-L1 / PD-1 and Gas6 / TAM pathways in the tumor and its microenvironment. Therefore, the fusion protein enhances the efficacy of anti-PD-L1-based immunotherapy by introducing the fused Gas6-binding portion into the entity and allows anti-PD-L1-based immunotherapy to be applied to a wider range of indications; Second, confining the fusion protein in the tumor and its microenvironment to perform multiple functions in one entity achieves the maximum effect, more specifically at the minimum dose. Having multiple functions in one entity form can also enhance its tolerance to antagonism; Finally, the favorable spatial combination of this fusion protein is selected from a variety of possible spatial combination candidates. Therefore, the fusion protein disclosed in the present disclosure is also the actual result predicted by computer simulation, helping to accelerate future innovations of multifunctional proteins that incorporate decoy receptors fused to antibody portions.

[0068] Gas6 binding moiety:

[0069] Gas6 structurally belongs to the plasma vitamin K-dependent protein family. Gas6 has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family. Human Gas6 is a protein consisting of 678 amino acids and is composed of: a vitamin K-dependent carboxyglutamic acid (Gla)-rich domain that mediates binding to the phospholipid membrane, four epidermal growth factor-like domains, and two laminin G-like (LG) domains that mediate binding to the TAM receptors. The sequences of the transcriptional variants of human Gas6 can be obtained at NM_001143946.1, NM_001143945.1, and NM_000820.2 in Genbank, respectively.

[0070] The Gas6 binding moiety provides an isolated fragment that specifically binds to the Gas6 protein. The isolated fragment of the Gas6 binding moiety can bind to the antigenic determinants contained in or presented by one or more amino acid regions that interact with Axl (such as LRMFSGTPVIRLRFKRLQPT (SEQ ID NO:90), EIVGRVTSSGP (SEQ ID NO:91), RNLVIKVN (SEQ ID NO:92), DAVMKIAVA (SEQ ID NO:93), ERGLYHLNLTVGIPFH (SEQ ID NO:94), and WLNGEDTTIQETVVNRM (SEQ ID NO:95), or belong to Gas6 (L295-T317, E356-P372, R389-N396, D398-A406, E413-H429, and W450-M468). Additionally, the isolated fragment of the Gas6 binding moiety is capable of inhibiting or competing with the binding between TAM and Gas6. Among the TAM receptors, wild-type Axl or the soluble Axl (sAxl) variant has the highest in vitro affinity for Gas6, with a K D of 1.0 nM / L, followed by Tyro-3 with approximately equal affinity, and the K D value of MerTK is at least 10-fold lower.

[0071] The Gas6 binding moiety can be any configuration that inhibits the Gas6 / TAM pathway by neutralizing Gas6. Neutralizing Gas6 with a Gas6 binding moiety may be a better strategy than blocking the TAM receptor with an antagonistic molecule, as upregulation of Gas6 production induced by TAM antagonist molecules in a negative feedback loop can be hypothesized. The Gas6 binding moiety can be any smaller fragment of the extracellular domain of the TAM receptor; a partially or fully humanized antibody, or a chimeric antibody; a monoclonal or polyclonal antibody; a fragment of an isolated antibody of the Gas6 binding moiety may contain an antibody region (in the case of an antibody framework or a non-antibody framework) that is sufficient or necessary for the recognizable specific binding of a polypeptide to Gas6; one or more CDRs of the heavy or light chain or a combination thereof; a polypeptide containing a single-chain antibody; a combination of only the variable region or a part of the variable region and the Fc region (such as the CH1 region); or a minibody (such as VL-VH-CH3) or a bispecific antibody.

[0072] The Axl receptor represents a structure common to members of the TAM family, which includes an intracellular tyrosine kinase domain and an extracellular region that juxtaposes immunoglobulin (Ig) repeats and fibronectin type III (FnIII) repeats. The extracellular Ig and Fn motifs are thought to be important in cell adhesion and migration, indicating that the Axl oncogene can contribute to tumor invasion and metastasis in this way. Axl transduces signals from the extracellular matrix to the cytoplasm by binding growth factors such as the vitamin K-dependent protein Gas6. This interaction activates Axl by causing dimerization and autophosphorylation.

[0073] The Gas6 binding moiety in the fusion protein can include any smaller fragment of the extracellular domain of Axl that retains the ability to bind Gas6, which would otherwise bind to the TAM receptor. For example, an Axl fragment spanning two N-terminal Ig domains (designated Ig1 and Ig2) and without carbohydrate modification retains intact Gas6 binding activity. A suitable Gas6 binding moiety for the fusion protein does not include at least the cytoplasmic domain of Axl, and more preferably all or most of the transmembrane domain of Axl, and includes a part of the extracellular domain of Axl, up to the entire extracellular domain. More preferably, the part of the extracellular domain includes at least the major Gas6 binding surface of Axl, and in other embodiments, contains at least the Ig1 and Ig2 domains of Axl, or residues that form a conformational structure sufficient to bind Gas6.

[0074] The native sequence of Axl shown in SEQ ID NO:1 includes Ig1, Ig2, FnIII and intracellular domains, wherein the Ig1 domain sequence is residues 27 - 128 (SEQ ID NO:61), the Ig2 domain sequence is residues 139 - 222 (SEQ ID NO:62), the FnIII domain sequences are residues 225 - 332 (SEQ ID NO:63) and residues 333 - 427 (SEQ ID NO:64), the intracellular domain sequence is residues 473 - 894 (SEQ ID NO:65), tyrosine residues at 779, 821 and 866 autophosphorylate after receptor dimerization and serve as docking sites for intracellular signaling molecules. The native cleavage site for release of sAxl is between residues 437 - 451 (SEQ ID NO:66).

[0075] The Gas6 - binding portion of the fusion protein disclosed in the present disclosure has been modified or rearranged based on the native sequence of Axl. For example, the Gas6 - binding portion can include Ig1 and Ig2 and not include FnIII, wherein the sequence is SEQ ID NO:2.

[0076] The Gas6 - binding portion of the fusion protein can include one or more amino acid modifications in wild - type sAxl, such as one or more amino acid modifications that increase its affinity for Gas6. Amino acid modifications include any naturally occurring or man - made amino acid modifications known in the art or discovered subsequently. Amino acid modifications include any naturally occurring mutations, such as substitutions, deletions, additions, insertions, etc.; replacement of an existing amino acid with another amino acid (e.g., its conservative equivalent); and replacement of one or more existing amino acids with non - natural amino acids or insertion of one or more non - natural amino acids. In some embodiments, the amino acid modifications can include at least 1, 2, 3, 4, 5 or 6 or 10 amino acid mutations or changes. In some exemplary embodiments, one or more amino acid modifications can be used to alter properties of Axl, such as affecting stability, binding activity, specificity and / or thermal stability, etc. In some other embodiments, sAxl does not have a transmembrane domain and optionally does not have an intracellular domain.

[0077] For sufficient binding activity to Gas6, the modification is generally located between the extracellular domain and the transmembrane domain, usually between residues 19 - 437 in SEQ ID NO:1, but it can include or consist essentially of truncated forms from residues 19, 25, 30, 35, 40, 45 or 50 to residues 132, 321, 350, 375, 400, 410, 420, 430, 440 or 450. In some embodiments, the Gas6 binding portion includes one or more amino acid modifications within one or more regions of residues 18 - 130, 10 - 135, 15 - 45, 60 - 65, 70 - 80, 85 - 90, 91 - 99, 104 - 110, 111 - 120, 125 - 130, 21 - 132, 21 - 121, 26 - 132 or 26 - 121 of wild - type Axl (SEQ ID NO:1). In other embodiments, the Gas6 binding portion includes one or more amino acid modifications within one or more regions of residues 20 - 130, 37 - 124 or 141 - 212 of wild - type Axl (SEQ ID NO:1). In some other embodiments, the Gas6 binding portion includes one or more amino acid modifications at one or more of the modifications at residues 19, 23, 26, 27, 32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105, 109, 112, 113, 116, 118, 127 or 129 of wild - type Axl (SEQ ID NO:1), for example, A19T, T23M, E26G, E27G / K, G32S, N33S, T38I, T44A, H6IY, D65N, A72V, S74N, Q78E, V79M, Q86R, D87G, D88N, I90M / V, V92A / G / D, I97R, T98A / P, T105M, Q109R, V112A, F113L, H116R, T118A, G127R / E, E129K and combinations thereof. In some other other embodiments, the Gas6 binding portion includes one or more amino acid modifications or combinations thereof at residues 32, 87, 92 or 127 of wild - type Axl (SEQ ID NO:1), such as G32S, D87G, V92A and / or G127R. In yet some other other embodiments, the Gas6 binding portion includes one or more amino acid modifications or combinations thereof at residues 26, 79, 92, 127 of wild - type Axl (SEQ ID NO:1), such as E26G, V79M, V92A and / or G127E.

[0078] The Gas6 binding portion can be further modified, for example, linked to a wide variety of other oligopeptides or proteins for various purposes. Various post-translational or post-transcriptional modifications can be made to the Gas6 binding portion of the fusion protein. Such modifications can include chemical derivatization of the polypeptide, such as acetylation, amidation, carboxylation, etc. Such modifications can include glycosylation modifications, for example, by exposing the polypeptide to mammalian glycosylation or deglycosylation enzymes. Such modifications can also include phosphorylation of certain amino acid residues, such as phosphotyrosine, phosphoserine, or phosphothreonine. In addition, by using appropriate coding sequences, farnesylation or geranylgeranylation can be provided. In some embodiments, the Gas6 binding portion can be PEGylated, where polyethyleneoxy provides an increased half-life in the bloodstream. The Gas6 binding portion can also be combined with other proteins, such as the Fc of an IgG isotype that can bind complement; with toxins, such as ricin, abrin, diphtheria toxin, or similar toxins; or with specific binding agents that allow targeting of specific portions on target cells. In some other embodiments, the Gas6 binding portion can be modified to improve its resistance to proteolytic degradation or to optimize solubility characteristics or to make it more suitable as a therapeutic agent. For example, the Gas6 binding portion can further include analogs of sAxl variants that contain residues other than the naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids can replace some or all of the amino acid residues. In some other embodiments, the Gas6 binding portion can include two, three, four, five, or six covalently or non-covalently linked identical or different sAxl variants, such that it will have an appropriate size while avoiding undesirable aggregation.

[0079] In some embodiments, the Gas6 binding portion is a fusion protein, for example, in-frame fused with a second polypeptide. The second polypeptide can be a part or all of the Fc region, any suitable polypeptide substantially similar to Fc, or a part or all of albumin, in order to increase the size of the fusion protein and thereby extend the half-life of the fusion protein. In other embodiments, the second polypeptide is suitable for processing the Gas6 binding portion, for example, for purifying the Gas6 binding portion, or stabilizing the Gas6 binding portion in vitro or in vivo. For example, the second polypeptide can be a tag sequence, such as a hexahistidine peptide, to facilitate purification of the fusion polypeptide. Additionally, regions of additional amino acids, especially charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence during purification from host cells or subsequent processing and storage. In addition, a fusion protein having a disulfide-linked dimeric structure can also be more efficient in binding and neutralizing other molecules compared to a monomeric secreted protein or protein fragment alone.

[0080] Antigen binding portion:

[0081] In some embodiments of the present disclosure, the antibody or its antigen-binding fragment comprises the complementarity-determining regions (CDRs) of the heavy-chain variable region (VH) and the complementarity-determining regions of the light-chain variable region (VL), wherein the complementarity-determining regions of the heavy-chain variable region comprise VH-CDR1, VH-CDR2, and VH-CDR3, and the complementarity-determining regions of the light-chain variable region comprise VL-CDR1, VL-CDR2, and VL-CDR3.

[0082] Examples of the antibody or its antigen-binding fragment include (but are not limited to) antibody (a) to (k) or their antigen-binding fragments, 3G10 of US 7,943,743, 12A4 of US 7,943,743, 10A5 of US 7,943,743, 5F8 of US 7,943,743, 10H10 of US 7,943,743, 1B12 of US 7,943,743, 7H1 of US 7,943,743, 11E6 of US 7,943,743, 12B7 of US 7,943,743, 13G4 of US 7,943,743, MDX-1105, MEDI-4736, atezolizumab, durvalumab, avelumab, MDX-1105, envafolimab, cosibelimab, CK-301, CS-1001, SHR-1316, CBT-502, or BGB-A333.

[0083] In some embodiments of the present disclosure, the antibody (a) or its antigen-binding fragment comprises VH-CDR1 of GYSITSDYWN (SEQ ID NO:3) or its substantially similar sequence; VH-CDR2 of YISYTGSTYYNPSLKS (SEQ ID NO:4) or its substantially similar sequence; VH-CDR3 of RGEWLSPFAY (SEQ ID NO:5) or its substantially similar sequence; VL-CDR1 of KSSQSLLYSSNQKNSLA (SEQ ID NO:10) or its substantially similar sequence; VL-CDR2 of WASTRES (SEQ ID NO:11) or its substantially similar sequence; and VL-CDR3 of QQYYTYPFT (SEQ ID NO:12) or its substantially similar sequence.

[0084] In some embodiments of the present disclosure, the antibody (a) or its antigen-binding fragment comprises a VH-CDR1 of GYSITSDYWD (SEQ ID NO: 96) or a substantially similar sequence thereof; a VH-CDR2 of YISYTGSTYYNPSLRS (SEQ ID NO: 97) or a substantially similar sequence thereof; a VH-CDR3 of RGGWLSPFVY (SEQ ID NO: 98) or a substantially similar sequence thereof; a VL-CDR1 of KSRQSLLFSSNQKNSLA (SEQ ID NO: 99) or a substantially similar sequence thereof; a VL-CDR2 of WASTRES (SEQ ID NO: 11) or a substantially similar sequence thereof; and a VL-CDR3 of QQYYTYPFT (SEQ ID NO: 12) or a substantially similar sequence thereof.

[0085] In some embodiments of the present disclosure, the VH of the antibody (a) or its antigen-binding fragment comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4). In some embodiments of the present disclosure, HC-FR1 is EVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 6) or a substantially similar sequence thereof; HC-FR2 is WIRKPPGKGLEYMG (SEQ ID NO: 7) or a substantially similar sequence thereof; HC-FR3 is RITISRDTSKNQYSLKLSSVTAADTAVYYCAR (SEQ ID NO: 8) or a substantially similar sequence thereof; and HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 9) or a substantially similar sequence thereof.

[0086] In some embodiments of the present disclosure, the VL of the antibody (a) or its antigen-binding fragment comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). In some embodiments of the present disclosure, LC-FR1 is DIQMTQSPSSLS ASVGDRVTITC (SEQ ID NO: 13) or a substantially similar sequence thereof; LC-FR2 is WYQQKPGKAPKLLIY (SEQ IDNO: 14) or a substantially similar sequence thereof; LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQ PEDFATYYC (SEQ ID NO: 15) or a substantially similar sequence thereof; and LC-FR4 is FGQGTKLEIK (SEQ ID NO: 16) or a substantially similar sequence thereof.

[0087] In some embodiments of the present disclosure, the VL of the antibody (a) or its antigen-binding fragment comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). In some embodiments of the present disclosure, LC-FR1 is DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 100) or a substantially similar sequence thereof; LC-FR2 is WYQQKPGQPPKLLIY (SEQ ID NO: 101) or a substantially similar sequence thereof; LC-FR3 is GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 102) or a substantially similar sequence thereof; and LC-FR4 is FGQGTKLEIK (SEQ ID NO: 16) or a substantially similar sequence thereof.

[0088] In some embodiments of the present disclosure, the antibody (a) or antigen-binding fragment disclosed above is a selected humanized line of its corresponding mouse line, where the mouse line includes the complementarity-determining regions of the heavy-chain variable region, the complementarity-determining regions of the light-chain variable region, and the frameworks are represented by the following formulas: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4) and (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). The mouse line includes VH-CDR1 of GYSITSDYWN (SEQ ID NO:3) or a substantially similar sequence thereof; VH-CDR2 of YISYTGSTYYNPSLKS (SEQ ID NO:4) or a substantially similar sequence thereof; VH-CDR3 of RGEWLSPFAY (SEQ ID NO:5) or a substantially similar sequence thereof; VL-CDR1 of KSSQSLLYSSNQKNSLA (SEQ ID NO:10) or a substantially similar sequence thereof; VL-CDR2 of WASTRES (SEQ ID NO:11) or a substantially similar sequence thereof; and VL-CDR3 of QQYYTYPFT (SEQ ID NO:12) or a substantially similar sequence thereof; HC-FR1 is QVQLQESGPGLAKPSQTLSLTCSVT (SEQ ID NO:103) or a substantially similar sequence thereof; HC-FR2 is WIRKFPGNKLEFMG (SEQ ID NO:104) or a substantially similar sequence thereof; HC-FR3 is RISITRDTSKNQYYLQLNSVTTEDTATYCAR (SEQ ID NO:105) or a substantially similar sequence thereof; HC-FR4 is WGQGTLVTVSA (SEQ ID NO:106) or a substantially similar sequence thereof; LC-FR1 is DIVMSQSPSSLGVSVGEKITMSC (SEQ ID NO:107) or a substantially similar sequence thereof; LC-FR2 is WYQQKPGQSPKLLIY (SEQ ID NO:108) or a substantially similar sequence thereof; LC-FR3 is GVPDRFTGSGTDFTLTISSVKSEDLAVYYC (SEQ ID NO:109) or a substantially similar sequence thereof; LC-FR4 is FGAGTNLELK (SEQ ID NO:110) or a substantially similar sequence thereof.

[0089] In some embodiments of the present disclosure, the antibody (a) or antigen-binding fragment disclosed above is the selected humanized line of its corresponding mouse line, where the mouse line includes the complementarity-determining regions of the heavy-chain variable region, the complementarity-determining regions of the light-chain variable region, and the frameworks are represented by the following formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4) and (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). The mouse line includes VH-CDR1 of GYSITSDYWD (SEQ ID NO: 96) or a substantially similar sequence thereof; VH-CDR2 of YISYTGSTYYNPSLRS (SEQ ID NO: 97) or a substantially similar sequence thereof; VH-CDR3 of RGGWLSPFVY (SEQ ID NO: 98) or a substantially similar sequence thereof; VL-CDR1 of KSRQSLLFSSNQKNSLA (SEQ ID NO: 99) or a sequence substantially similar thereto; VL-CDR2 of WASTRES (SEQ ID NO: 11) or a sequence substantially similar thereto; and VL-CDR3 of QQYYTYPFT (SEQ ID NO: 12) or a sequence substantially similar thereto; HC-FR1 is EVQLQESGPGLTKPSQTLSLTCSVT (SEQ ID NO: 111) or a substantially similar sequence thereof; HC-FR2 is WIRKFPGNKLEYMG (SEQ ID NO: 112) or a substantially similar sequence thereof; HC-FR3 is RISITRDTSKNQYYLQLSSVTSEDSATYCAR (SEQ ID NO: 113) or a substantially similar sequence thereof; HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 114) or a substantially similar sequence thereof; LC-FR1 is DTVMSQSPSSLGVSVGERVTLTC (SEQ ID NO: 115) or a substantially similar sequence thereof; LC-FR2 is WYQQKPGQSPKLLIY (SEQ ID NO: 116) or a substantially similar sequence thereof; LC-FR3 is GVPDRFTGSGSGTDFTLTISSVKSEDLAVYYC (SEQ ID NO: 117) or a substantially similar sequence thereof; LC-FR4 is FGAGTSLELK (SEQ ID NO: 118) or a substantially similar sequence thereof.

[0090] In some embodiments of the present disclosure, the antibody (b) or its antigen-binding fragment comprises VH-CDR1 of SYIMM (SEQ ID NO:17) or a substantially similar sequence thereof; VH-CDR2 of SIYPSGGITFYADTVKG (SEQ ID NO:18) or a substantially similar sequence thereof; VH-CDR3 of IKLGTVTTVDY (SEQ ID NO:19) or a substantially similar sequence thereof; VL-CDR1 of TGTSSDVGGYNYVS (SEQ ID NO:20) or a substantially similar sequence thereof; VL-CDR2 of DVSNRPS (SEQ ID NO:21) or a substantially similar sequence thereof; and VL-CDR3 of SSYTSSSTRV (SEQ ID NO:22) or a substantially similar sequence thereof.

[0091] In some embodiments of the present disclosure, the antibody (c) or its antigen-binding fragment comprises VH-CDR1 of MYMMM (SEQ ID NO:23) or a substantially similar sequence thereof; VH-CDR2 of SIYPSGGITFYADSVKG (SEQ ID NO:24) or a substantially similar sequence thereof; VH-CDR3 of IKLGTVTTVDY (SEQ ID NO.25) or a substantially similar sequence thereof; VL-CDR1 of TGTSSDVGAYNYVS (SEQ ID NO:26) or a substantially similar sequence thereof; VL-CDR2 of DVSNRPS (SEQ ID NO:27) or a substantially similar sequence thereof; and VL-CDR3 of SSYTSSSTRV (SEQ ID NO:28) or a substantially similar sequence thereof.

[0092] In some embodiments of the present disclosure, the antibody (d) or its antigen-binding fragment comprises VH-CDR1 of SYIMM (SEQ ID NO:29) or a substantially similar sequence thereof; VH-CDR2 of SIYPSGGITFYAPTVKG (SEQ ID NO:30) or a substantially similar sequence thereof; VH-CDR3 of IKLGTVTTVDY (SEQ ID NO:31) or a substantially similar sequence thereof; VL-CDR1 of TGTSSDVGGYNYVS (SEQ ID NO:32) or a substantially similar sequence thereof; VL-CDR2 of DVSNRPS (SEQ ID NO:33) or a substantially similar sequence thereof; and VL-CDR3 of SSYTSSSTRV (SEQ ID NO:34) or a substantially similar sequence thereof.

[0093] In some embodiments of the present disclosure, the VH of antibody (d) or its antigen-binding fragment comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4). In some embodiments of the present disclosure, HC-FR1 is EVQLLESGGLVQPGGSLRLSCAASGFTGS (SEQ ID NO:35) or a substantially similar sequence thereof; HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO:36) or a substantially similar sequence thereof; HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYCAR (SEQ ID NO:37) or a substantially similar sequence thereof; HC-FR4 is WGQGTLVTVSS (SEQ ID NO:38) or a substantially similar sequence thereof. In some embodiments of the present disclosure, the VL of antibody (d) or its antigen-binding fragment comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). In some embodiments of the present disclosure, LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO:39) or a substantially similar sequence thereof; LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO:40) or a substantially similar sequence thereof; LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO:41) or a substantially similar sequence thereof; LC-FR4 is FGTGTKVTVL (SEQ ID NO:42) or a substantially similar sequence thereof.

[0094] In some embodiments of the present disclosure, antibody (e) or its antigen-binding fragment comprises VH-CDR1 of SYIMM (SEQ ID NO:43) or a substantially similar sequence thereof; VH-CDR2 of SIYPSGGITGYADTVKG (SEQ ID NO:44) or a substantially similar sequence thereof; VH-CDR3 of IKLGTVTTVDY (SEQ ID NO:45) or a substantially similar sequence thereof; VL-CDR1 of TGTSSDVGGYNYVS (SEQ ID NO:46) or a substantially similar sequence thereof; VL-CDR2 of DVSNRPS (SEQ ID NO:47) or a substantially similar sequence thereof; and VL-CDR3 of SSYTSSSTRV (SEQ ID NO:48) or a substantially similar sequence thereof.

[0095] In some embodiments of the present disclosure, the antibody (f) or its antigen-binding fragment comprises a heavy chain of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMVWRQAPGKGLEWVSSIYPSGGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVYVSS (SEQ ID NO:49) or a substantially similar sequence thereof, and a light chain of QSALTQPASVSGSPGQSITISCTGTSSDVGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLOAEDEADYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO:50) or a substantially similar sequence thereof.

[0096] In some embodiments of the present disclosure, the antibody (g) or its antigen-binding fragment comprises a heavy chain of EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEVWSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSS (SEQ ID NO:51) or a substantially similar sequence thereof, and a light chain of QSALTQPASVSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO:52) or a substantially similar sequence thereof.

[0097] In some embodiments of the present disclosure, the antibody (h) or its antigen-binding fragment comprises a heavy chain of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:53) or a substantially similar sequence thereof, and a light chain of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:54) or a substantially similar sequence thereof.

[0098] In some embodiments of the present disclosure, the antibody (i) or its antigen-binding fragment comprises a heavy chain of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISP YGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGF DYWGQGTLVTVSS (SEQ ID NO:55) or a substantially similar sequence thereof, and a light chain of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:56) or a substantially similar sequence thereof.

[0099] In some embodiments of the present disclosure, the antibody (j) or its antigen-binding fragment comprises a heavy chain of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISP YGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGF DYWGQGTLVTVSA (SEQ ID NO:57) or a substantially similar sequence thereof, and a light chain of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:58) or a substantially similar sequence thereof.

[0100] In some embodiments of the present disclosure, the antibody (k) or its antigen-binding fragment comprises a heavy chain encoded by SEQ ID NO:59; and a light chain encoded by SEQ ID NO:60.

[0101] In another embodiment, the antibody binds to human, mouse or cynomolgus macaque PD-L1. In a particular aspect, the antibody is capable of blocking the interaction between human, mouse or cynomolgus macaque PD-L1 and the respective human, mouse or cynomolgus macaque PD-1 receptor.

[0102] In another embodiment, the antibody has a KD of 5×10 -9 M or lower, more preferably a KD of 2×10 -9 M or lower and even more preferably a KD of 1×10 -9 M or lower and binds to human PD-L1.

[0103] In addition, other embodiments relate to an anti-PD-L1 antibody or antigen-binding fragment thereof that binds to a functional epitope comprising residues Y56 and D61 of human PD-L1.

[0104] In certain embodiments, the antibody binds to a conformational epitope comprising residues 54-66 and 112-122 of human PD-L1.

[0105] In additional other embodiments, the framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

[0106] In additional other embodiments, the light chain framework sequence is a lambda light chain sequence.

[0107] Constant regions:

[0108] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

[0109] In additional other embodiments, the heavy chain variable region polypeptide, antibody or antibody fragment further comprises at least one CH1 domain. In more specific embodiments, the heavy chain variable region polypeptide, antibody or antibody fragment further comprises CH1, CH2 and CH3 domains.

[0110] In additional other embodiments, the variable region light chain, antibody or antibody fragment further comprises a CL domain.

[0111] In additional other embodiments, the antibody further comprises CH1, CH2, CH3 and CL domains.

[0112] In some embodiments of the present disclosure, the proteins and peptides of the antigen-binding portion comprise a constant region of an immunoglobulin or a fragment, analog, variant, mutant or derivative of a constant region. In more preferred embodiments herein, the constant region is derived from a human immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4 or other classes. In some embodiments, the constant region comprises a CH2 domain. In other embodiments, the constant region comprises CH2 and CH3 domains, or comprises hinge-CH2-CH3. Alternatively, the constant region may comprise all or a portion of the hinge region, CH2 domain and / or CH3 domain.

[0113] In some embodiments, the constant region contains mutations that reduce the affinity for Fc receptors or reduce Fc effector functions. For example, the constant region may contain mutations that eliminate glycosylation sites within the IgG heavy chain constant region. In some embodiments herein, the constant region contains mutations, deletions, or insertions at amino acid positions corresponding to L234, L235, G236, G237, N297, or P331 of IgG1. In certain embodiments, the constant region contains a mutation at the amino acid position corresponding to N297 of IgG1. In alternative embodiments herein, the constant region contains mutations, deletions, or insertions at amino acid positions corresponding to L281, L282, G283, G284, N344, or P378 of IgG1.

[0114] In some embodiments herein, the constant region contains the CH2 domain from a human IgG2 or IgG4 heavy chain. More preferably, the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain. In some embodiments, the mutation changes the N within the QFNS amino acid sequence within the CH2 domain of the IgG2 or IgG4 heavy chain. More preferably, the mutation changes N to G. Alternatively, the mutation changes both F and N within the QFNS amino acid sequence. In some embodiments, the QFNS amino acid sequence is replaced with the QAQs amino acid sequence. The N within the QFNS amino acid sequence corresponds to N297 of IgG1.

[0115] In other embodiments, the constant region includes the CH2 domain and at least a portion of the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, or other classes. More preferably, the hinge region is derived from a human IgG1, IgG2, IgG3, IgG4, or other suitable class. Even more preferably, the hinge region is derived from a human IgG1 heavy chain. In some embodiments, the C within the PKSCDK amino acid sequence of the IgG1 hinge region is altered. In a more preferred embodiment, the PKSCDK amino acid sequence is replaced with the PKSSDK amino acid sequence. In some embodiments, the constant region includes the CH2 domain from a first antibody isotype and the hinge region from a second antibody isotype. In certain embodiments, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain, and the hinge region is derived from an altered human IgG1 heavy chain.

[0116] Alterations of amino acids near the junction of the Fc portion and the non-Fc portion can significantly increase the serum half-life of Fc fusion proteins (PCT Publication WO 01 / 58957). Thus, the linker region of the proteins or polypeptides of the present disclosure may contain alterations, which are preferably within about 10 amino acids of the junction point relative to the naturally occurring sequences of immunoglobulin heavy chains and erythropoietin. These amino acid changes can increase hydrophobicity. In some embodiments, the constant region is derived from an IgG sequence in which the C-terminal K residue has been replaced. More preferably, the C-terminal K of the IgG sequence is replaced with a non-K amino acid such as A or L to further increase the serum half-life. In other embodiments, the constant region is derived from an IgG sequence in which the LSLS amino acid sequence near the C-terminus of the constant region is altered to eliminate potential linked T cell epitopes. For example, in some embodiments, the LSLS amino acid sequence is replaced with an ATAT amino acid sequence. In other embodiments herein, the amino acids within the LSLS segment are replaced with other amino acids such as G or P. Detailed methods for generating amino acid substitutions in the LSLS segment near the C-terminus of IgG1, IgG2, IgG3, IgG4, or other classes have been described in U.S. Patent Publication 2003 / 0166877.

[0117] Suitable hinge regions of the present disclosure can be derived from IgG1, IgG2, IgG3, IgG4, and other classes. The IgG1 hinge region has three C's, two of which are involved in disulfide bonds between the two heavy chains of the antibody. These same C's allow for efficient and consistent disulfide bond formation between the Fc portions. In some embodiments herein, the first C within the human IgG1 hinge region is mutated to another amino acid, more preferably S. The IgG2 isotype hinge region has four disulfide bonds, which tend to promote oligomerization and potentially incorrect disulfide bond formation during secretion in recombinant systems. Suitable hinge regions can be derived from the IgG2 hinge; the first two C's are each more preferably mutated to another amino acid. The hinge region of IgG4 is known to inefficiently form interchain disulfide bonds. However, a suitable hinge region for the fusion protein can be derived from the IgG4 hinge region, which preferably contains mutations that enhance the correct formation of disulfide bonds between the heavy chain-derived portions.

[0118] According to the present disclosure, the constant region may contain CH2 and / or CH3 domains and hinge regions derived from different antibody isotypes, such as hybrid constant regions. For example, in some embodiments, the constant region contains CH2 and / or CH3 domains derived from IgG2 or IgG4 and a mutant hinge region derived from IgG1. Alternatively, a mutant hinge region from another IgG subclass is used for the hybrid constant region. For example, a mutant form of the IgG4 hinge that allows efficient disulfide bond formation between the two heavy chains can be used. The mutant hinge can also be derived from the IgG2 hinge, where the first two C's are each mutated to another amino acid. The assembly of such hybrid constant regions has been described in U.S. Patent Publication No. 2003 / 0044423.

[0119] According to the present disclosure, the constant region may contain one or more mutations described herein. Combinations of mutations in the Fc portion may have additive or synergistic effects in prolonging the serum half-life of the fusion protein and increasing its in vivo potency. Thus, in the exemplary disclosure, the constant region may contain (i) a region derived from an IgG sequence in which the LSLS amino acid sequence is replaced by the ATAT amino acid sequence; (ii) the C-terminal K residue is replaced by A; (iii) CH2 domains and hinge regions derived from different antibody isotypes, such as the CH2 domain may be derived from IgG2 and the hinge region from IgG1; and (iv) mutations that eliminate glycosylation sites within the IgG2-derived CH2 domain, such as the amino acid sequence QAQ S instead of QFNS within the IgG2-derived CH2 domain.

[0120] Antigen-binding fragment:

[0121] In some embodiments of the present disclosure, the antigen-binding fragment comprises a Fab fragment, an F(ab')2 fragment, a ScFv fragment, a chimeric antibody, or a nanobody.

[0122] Proteins and polypeptides may also include antigen-binding fragments. Exemplary antibody fragments include scFv, Fv, Fab, F(ab')2, and single-domain VHH fragments, such as those of camel origin.

[0123] Single-chain antibody fragments (also referred to as single-chain antibodies (scFv)) are recombinant polypeptides that typically bind an antigen or receptor; these fragments contain at least one fragment of VH tethered to at least one fragment of VL, with or without one or more interconnecting linkers. Such linkers can be short flexible peptides that are selected to ensure proper three-dimensional folding of the VH and VL domains after ligation, thereby maintaining the target molecule binding specificity of the intact antibody from which the scFv is derived. Generally, the C-terminus of the VH or VL sequence is covalently linked to the amino terminus of the complementary VH and VL sequences by such peptide linkers.

[0124] Single-chain antibody fragments contain an amino acid sequence having at least one CDR of a full antibody as described in the present disclosure but lacking some or all of the constant domains of those antibodies. These constant domains are not required for antigen binding but form a major part of the structure of the full antibody. Thus, single-chain antibody fragments can overcome some of the problems associated with using antibodies containing some or all of the constant domains. For example, single-chain antibody fragments often do not have unwanted interactions between biomolecules and the heavy-chain constant region or do not have other unwanted biological activities. Additionally, single-chain antibody fragments are significantly smaller than full antibodies and thus may have greater capillary permeability than full antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Additionally, antibody fragments can be produced relatively large-scale in prokaryotic cells, facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes it less likely than full antibodies to stimulate unwanted immune responses in recipients.

[0125] Antibody fragments with binding characteristics identical or comparable to those of full antibodies may also exist. Such fragments may contain one or two Fab fragments or F(ab')2 fragments. Antibody fragments can contain all six CDRs of a full antibody, although fragments containing less than all of such regions, such as fragments with three, four, or five CDRs, are also functional.

[0126] Restoring immune surveillance with anti-PD-L1 antibodies

[0127] T cell inhibitory checkpoint receptors (such as CTLA-4, PD-1, BTLA, LAG-3, TIM-3, and LAIR1) are highly expressed in inducible regulatory T cells and exhausted T cells. Thus, counterparts of T cell inhibitory checkpoint receptors (such as PD-L1 (B7-H1), B7-DC, HVEM, TIM-4, B7-H3, and B7-H4) have been found to be responsible for immune evasion in cancer.

[0128] The antibody portion of the fusion protein helps direct the fusion protein to the tumor and its microenvironment, where the antibody portion relieves the inhibition caused by T cell inhibitory checkpoints. For this purpose, the inventors have examined the anti-tumor efficacy of combinations of the Gas6-binding portion with commercially available and self-made antibodies targeting PD-L1. The inventors found that combining the Gas6-binding portion with an anti-PD-L1 antibody in an entity would not sacrifice the binding affinity and blocking function of the two portions of the anti-PD-L1×Gas6-binding portion. Additionally, the anti-PD-L1×Gas6-binding portion exhibited significant anti-tumor activity, exceeding the activity observed in conventional combination strategies in which the Gas6-binding portion and the anti-PD-L1 antibody were administered separately.

[0129] Spatial configuration:

[0130] The light or heavy chain of an anti-PD-L1 antibody or its PD-L1 binding fragment containing an Fc fragment can be fused to a Gas6 binding moiety. The Gas6 binding moiety can be fused to the N-terminus or C-terminus of the chain of the anti-PD-L1 unit. The anti-PD-L1 unit can have a light chain and a separate heavy chain, or have a light chain and a heavy chain on a single protein chain (such as scFv). Compared with the C-terminal fusion construct, the N-terminal fusion construct exhibits relatively low yields and significantly reduced biological activity. Based on trial and error, a spatial configuration with favorable yields and biological functions is presented in the present disclosure Figure 1 in which the Gas6 binding moiety is fused to the C-terminus of the heavy chain of the anti-PD-L1 unit, which unit comprises a light chain and a separate heavy chain (Type 1) or comprises a light chain and a heavy chain on a single protein chain (Type 2); in both cases the N-terminus of the Gas6 binding moiety is fused to the C-terminus of the heavy chain of the anti-PD-L1 unit through a peptide linker.

[0131] Peptide linker:

[0132] The present disclosure includes the extracellular domain of Axl RTK fused to the C-terminus of the heavy chain of an anti-PD-L1 antibody, scFv or a fragment thereof through a peptide linker. With reference to the extracellular domain, the peptide linker can be entirely an artificial linker or include a part of the N-terminal extracellular fragment of the extracellular domain IPPHVQKSVNNDMIVTDNNGAVKFP (SEQ ID NO:67), and the peptide linker should have a minimum length. If the distance is too short, the stability or activity of the fusion protein is reduced. In some embodiments, the minimum length is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues. In some embodiments, the linker is no longer than 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 170 or 200 amino acid residues.

[0133] A flexible linker, such as one or more GGGGS (SEQ ID NO:68) units, can be added and, in some embodiments, can be used to improve the stability and / or activity of the BfAb. In some embodiments, the flexible linker comprises at least 40%, 50%, 60%, 70% or 80% glycine. In some embodiments, the flexible linker comprises one or more serines. In some embodiments, the flexible linker comprises 1, 2, 3, 4, 5 or 6 SEQ ID NO:68 repeats.

[0134] It has been demonstrated that in some embodiments, the native N-terminal fragment (SEQ ID NO:67) can be replaced with a substituted peptide to increase stability without sacrificing activity or even improving activity. In some embodiments, the substituted peptide is different from SEQ ID NO:67 but has at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity with SEQ ID NO:67.

[0135] An exemplary substituted peptide is IPPHVQXXVNNDMIVTDNXGAVKFP (SEQ ID NO:69), where X is any amino acid other than K, S or N. In some embodiments, substitutions can be made to remove the rigid dipeptide PP, remove potential cleavage sites QK, N and / or K, multiple glycine residues can be added to increase flexibility and / or hydrophobic residues can be reduced. One such example is TAGHTQTSTGGGAITTGTSGAGHGP (SEQ ID NO:70) or a variant having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO:70. In some embodiments, the variant includes at least 4 Gs, no PP dipeptide, no more than 3 hydrophobic amino acid residues selected from the group consisting of I, L, M, F, V, W, Y and P. In some embodiments, the variant includes at least 5 Gs and no more than 1 hydrophobic amino acid residue selected from the group consisting of I, L, M, F, V, W, Y and P.

[0136] In some embodiments, the peptide linker includes a substituted peptide of SEQ ID NO:67. In some embodiments, the peptide linker includes both a flexible linker and a substituted peptide. In some embodiments, the flexible linker is the N-terminus of the substituted peptide. In some embodiments, the flexible linker is the C-terminus of the substituted peptide.

[0137] In some embodiments, the fusion protein at least does not include the entire EEYNTSNPD sequence (SEQ ID NO:71). The fusion protein can remove the entire SEQ ID NO:71 from the extracellular domain of the Gas6 binding portion. In some embodiments, the fusion protein does not include more than 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues of SEQ ID NO:71.

[0138] Method:

[0139] The fusion protein can be produced by any suitable means known in the art or discovered subsequently, such as production from eukaryotic or prokaryotic cells, in vitro synthesis, etc. In the case of protein production from prokaryotic cells, it can be further processed by unfolding (such as heat denaturation, DTT reduction, etc.) and refolded using methods known in the art.

[0140] Methods of making antibodies are well known in the art and are described herein. Antibodies suitable for use in fusion proteins can be obtained from natural sources or produced by hybridoma, recombinant, or chemical synthesis methods, including modifying the constant region function by genetic engineering techniques. Antibodies of fusion proteins can be of any isotype. In certain embodiments, both the variable and constant regions of the antigen-binding portion of the fusion protein are fully human antibodies prepared using the techniques described herein. For example, a fully human antibody against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge but has had its endogenous loci inactivated. Exemplary techniques useful for preparing such antibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140.

[0141] Polypeptides can be prepared by in vitro synthesis using conventional methods, including molecular cloning, antibody phage display libraries, or similar techniques. A variety of commercial synthesis equipment is available. By using a synthesizer, naturally occurring amino acids can be replaced with unnatural amino acids. The particular sequence and manner of preparation will be determined by convenience, economic factors, the desired purity, and the like.

[0142] Polypeptides can also be isolated and purified according to conventional methods for recombinant proteins. Lysates can be prepared from expression hosts and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques.

[0143] Expression vectors containing the coding sequences and appropriate transcriptional / translational control signals can be constructed using methods well known to those skilled in the art. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombinant / genetic recombination. Alternatively, RNA capable of encoding the relevant polypeptide can be chemically synthesized. Direct chemical synthesis methods include, for example, the phosphotriester method, the diethylphosphoramidite method, and the solid support method. Chemical synthesis produces single-stranded oligonucleotides. This can be converted to double-stranded DNA by hybridizing with a complementary sequence or by polymerization using DNA polymerase with the single strand as a template. Although chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences can be obtained by ligating shorter sequences. Alternatively, sub-sequences can be cloned and appropriate sub-sequences cleaved using appropriate restriction enzymes.

[0144] Nucleic acids can be isolated and obtained with a relatively high purity. The nucleic acids of the present disclosure can be provided in a linear molecular form or within a circular molecule, and can be provided within a self-replicating molecule or within a molecule without a replication sequence. The expression of the nucleic acids can be regulated by its own regulatory sequences or by other regulatory sequences known in the art. A variety of techniques available in the art can be used to introduce the nucleic acids of the fusion protein into a suitable host cell, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transport of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and similar techniques.

[0145] Therapeutic indications:

[0146] The present disclosure further provides a method for treating, prophylactically treating, and / or preventing cancer in an individual in need thereof, which comprises administering to the individual an effective amount of the fusion protein disclosed herein.

[0147] The present disclosure further provides a method for neutralizing Gas6 in a sample, which comprises contacting the sample with the fusion protein disclosed herein.

[0148] As described herein, antibodies, variants, or derivatives of the anti-PD-L1×Gas6 binding portion can be used in certain therapeutic and diagnostic methods.

[0149] The fusion protein is further directed to multifunctional molecule- or antibody-based therapies, which involve administering the multifunctional molecules or antibodies of the present disclosure to patients such as animals, mammals, and humans to treat one or more of the diseases or conditions described herein. The therapeutic compounds of the present disclosure include (but are not limited to) the antibodies of the present disclosure (including their variants and derivatives described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives described herein).

[0150] The fusion protein is a method for treating cancer in a patient in need thereof. In one embodiment, the method requires administering to the patient an effective amount of the fusion protein of the present disclosure. In some embodiments, at least one of the cancer cells or surrounding cells in the tumor microenvironment in the patient expresses, overexpresses, or is induced to express PD-L1 and / or Gas6 or Axl. For example, the expression of PD-L1, Gas6, or Axl can be induced by administering a tumor vaccine or radiotherapy.

[0151] Tumors expressing the PD-L1 protein include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urothelial cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, and small cell lung cancer. Tumors that benefit from the Gas6 / Axl axis include lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, glioma, melanoma, prostate cancer, breast cancer, gastric cancer, colon cancer, osteosarcoma, renal cell carcinoma, and thyroid cancer. Additionally, because the Gas6-binding portion of the present disclosure acts as a ligand scavenger for Gas6, the compositions and methods of the present disclosure are applicable to treating any cancer expressing Axl and may be applicable to treating any cancer expressing MerTK and / or Tyro-3.

[0152] Accordingly, the anti-PD-L1 × Gas6-binding portion disclosed herein can be used to treat any one or more of such cancers. Additional diseases or conditions related to increased cell survival that can be treated, prevented, diagnosed, and / or prognosed with the fusion protein or its variants or derivatives include (but are not limited to) the progression and / or metastasis of the following: malignancies and related disorders such as leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease; and solid tumors including (but not limited to) sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, pancreatic cancer, thyroid cancer, endometrial cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, bladder cancer, epithelial carcinoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

[0153] Pharmaceutical composition:

[0154] The present disclosure further provides a pharmaceutical composition comprising: an effective amount of the fusion protein disclosed herein or the genetically engineered cell disclosed herein; and a pharmaceutically acceptable carrier.

[0155] The pharmaceutical compositions of the disclosure are formulated with suitable diluents, carriers, excipients and other agents that provide improved transfer, delivery, tolerability and similar properties. These compositions can be formulated for specific uses such as for veterinary or human medical use. The form of the compositions and excipients, diluents and / or carriers used will depend on the intended use of the antibody and the mode of administration for therapeutic use. Numerous suitable formulations can be found in the prescription collection known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. Also see Powell et al., "Compendium of excipients for parenteral formulations", PDA (1998) J Pharm Sci Technol 52:238-311.

[0156] The dose of the fusion protein administered to a patient can vary depending on the patient's age and size, the target disease, condition, route of administration and similar factors. More preferably, the dose is usually calculated based on body weight or body surface area. When the antibodies of the disclosure are used to treat PD-L1-related conditions or diseases in adult patients, intravenous administration of the antibodies of the disclosure can be advantageous. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and duration for antibody administration can be determined empirically; for example, patient progress can be monitored by periodic assessment and the dose adjusted accordingly. In addition, interspecies scaling of the dose can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0157] A variety of delivery systems are known to those of skill in the art and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include (but are not limited to) intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal mucosa, and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local.

[0158] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, with respect to subcutaneous delivery, pen-type delivery devices are amenable to delivering the pharmaceutical compositions of the present disclosure. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. After all of the pharmaceutical composition within the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be used again. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the reservoir of the pharmaceutical composition is emptied, the entire device is discarded.

[0159] In certain instances, the pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton 1987 CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Disclosures of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Fla. In yet another embodiment, the controlled release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Disclosures of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0160] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying the antibodies or their salts described above in a conventional sterile aqueous medium or oily medium for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers (such as benzyl benzoate, benzyl alcohol, etc.). It is more preferable to fill the injection solution thus prepared into appropriate ampoules.

[0161] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms suitable for fitting the dose of the active ingredient in unit doses. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0162] Detection:

[0163] The present disclosure further provides a method for detecting cancer in an individual in need, which comprises contacting a sample derived from the individual with the fusion protein disclosed herein.

[0164] The present disclosure further provides a kit for detecting cancer in a sample, which comprises the fusion protein disclosed herein.

[0165] The present disclosure further provides a method for detecting PD-L1 in a sample, which comprises contacting the sample with the fusion protein disclosed herein.

[0166] The fusion protein of the present disclosure can also be used, for example, for diagnostic purposes to detect and / or measure PD-L1 or cells expressing PD-L1 in a sample. For example, an anti-PD-L1 antibody or its antigen-binding fragment can be used to diagnose conditions or diseases characterized by abnormal expression of PD-L1 (such as overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for PD-L1 may include, for example, contacting a sample obtained from a patient with the anti-PD-L1 antibody of the present disclosure, wherein the anti-PD-L1 antibody is labeled with a detectable label or a reporter molecule. Alternatively, an unlabeled anti-PD-L1 antibody can be used in combination with a secondary antibody labeled in a detectable manner by itself for diagnostic disclosure. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure PD-L1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0167] The following examples are provided to assist those skilled in the art in practicing the disclosure.

[0168] Examples

[0169] The following examples illustrate the development and use of an anti-PD-L1 × Gas6 binding moiety to inhibit tumor growth by inducing a "hot tumor" microenvironment.

[0170] Example 1

[0171] [Generation of Recombinant DNA]

[0172] Prior to generating recombinant DNA, in silico simulations of the sequences were performed using SnapGene software. Recombinant DNA combining an anti-PD-L1 or anti-PD-L1 (scFv+Fc) (antigen-binding moiety) with a Gas6 capturer (Gas6 binding moiety) was constructed in different spatial arrangements. The constructs were generated by polymerase chain reaction (PCR)-based cloning and plasmid cloning. The process is outlined below: In PCR-based cloning, the insert DNA was amplified by PCR. For digestion of the DNA, the PCR product and the recipient plasmid were incubated with specific restriction enzymes for at least 4 hours and up to overnight, respectively. Phosphatase was applied to prevent recipient plasmid recirculation, and then the digested DNA was run on an agarose gel and gel purified to isolate the DNA of the expected size. T4 DNA ligase was used to fuse the insert DNA with the recipient plasmid. The PCR product in the process described above was replaced with the donor plasmid in plasmid cloning.

[0173] The spatial arrangements and primer sequences used in PCR-based cloning for each form are described below: Form 1 has a Gas6 capturer at the C-terminus of anti-PD-L1. Anti-PD-L1 without a stop codon is followed by a Gas6 capturer containing a stop codon. Primer sequence 1 and primer sequence 2 were used to generate this form.

[0174] The Gas6 capture agent is constructed together with anti-PD-L1 Fc (anti-PD-L1-Fc) to generate the Gas6 capture agent-Fc-based plasmid. Then, the anti-PD-L1 scFv (anti-PD-L1-scFv) is introduced into the Gas6 capture agent-Fc-based plasmid to generate four configurations. Type 2 has the Gas6 capture agent at the C-terminus of the anti-PD-L1-scFv. First, the anti-PD-L1-scFv is introduced into the region encoding the N-terminus of anti-PD-L1-Fc of anti-PD-L1-Fc. Primer sequence 3 is used to generate the insert fragment anti-PD-L1-scFc. Then, the Gas6 capture agent is tagged behind anti-PD-L1-Fc without a stop codon using primer sequence 4 and primer sequence 5.

[0175] Configuration optimization is further applied to Type 1 and Type 2. In Type 1, the recombinant DNA having anti-PD-L1 followed by the Gas6 capture agent at the C-terminus of the heavy chain constant region is modified, while the pTCAE8.3 plasmid is used as the acceptor plasmid. The sequence encoding the heavy chain variable region having anti-PD-L1 and the Gas6 capture agent in the recombinant DNA is replaced. Primer sequence 6 and primer sequence 7 are used to generate the insert fragment DNA for this process. In Type 2, the recombinant DNA having anti-PD-L1-scFv followed by anti-PD-L1-Fc without a stop codon and the Gas6 capture agent is modified, while the pTCAE8.3 plasmid is used as the acceptor plasmid. The sequence encoding the Gas6 capture agent in this recombinant DNA is replaced. Primer sequence 8 and primer sequence 9 are used to generate the insert fragment DNA for this process.

[0176] Table 1: Primer Sequences of the Present Application

[0177] Forward (SEQ ID NO) Reverse (SEQ ID NO) Primer Sequence 1 gagccacaggtgtacactct(72) acggatccgccaggagacag(73) Primer Sequence 2 tgaggatccggcggcggaggatct(74) acggatcctcaccaccagggccacgaga(75) Primer Sequence 3 tccaagcttgaggtccagctggtg(76) cgaattccctcttgatctccact(77) Primer Sequence 4 tgaattcggtggaggcggttca(78) agcggccgccgatccgccaccgcca(79) Primer Sequence 5 tagcggccgctgcccccaggggca(80) tagcggccgcctaccaccagggccacgaga(81) Primer Sequence 6 acccgtcgacgccaccatgg(82) acggatccctactgggggagcactgtg(83) Primer Sequence 7 acccgtcgacgccaccatgg(84) acggatccctatgtgatggtggctgtgcggga(85) Primer Sequence 8 acccgtcgacgccaccatgg(86) gtgcggccgcctactgggggagcactgt(87) Primer Sequence 9 acccgtcgacgccaccatgg(88) gtgcggccgcctatgtgatggtggctgtg(89)

[0178] [Amplification and Verification of Recombinant DNA]

[0179] The recombinant DNA is mixed with DH5α on ice for 20 minutes, and then transformed into DH5α by incubation at 42°C for 45 seconds. Subsequently, the transformed DH5α is incubated in LB medium at 37°C for 1 hour, and then evenly inoculated onto an LB agar plate containing 50 μg / mL ampicillin. The LB agar plate is incubated at 37°C for 16 hours, and then candidate pure lines are selected. Then, the candidate pure lines are amplified and their recombinant DNA is extracted, and tested by performing diagnostic restriction digestion and DNA sequencing.

[0180] [Expression of Related Ab]

[0181] A mammalian expression system is used to produce the relevant Ab. Briefly, recombinant DNA is transfected into mammalian cells by means of chemical substances, lipids, or physical conjugation. Using the drug resistance markers integrated in the recombinant DNA, mammalian cells that have received the recombinant DNA are distinguished from untransfected cells. To select highly proliferating mammalian cells in which the recombinant DNA has been integrated, a scaled-up methotrexate or methionine sulfoximine selection system is used.

[0182] [Purification of the relevant Ab]

[0183] The relevant Ab fused with GFP is purified by affinity column chromatography. Briefly, the anti-GFP mAb is dialyzed into a coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3), and then admixed with the resin in a stoppered container. Then the mixture is tumbled and rotated at room temperature for 2 hours. After washing away the excess anti-GFP mAb, the remaining active groups are treated with a blocking buffer (0.1 M Tris-HCl buffer, pH 8.0). Finally, the resin containing the anti-GFP mAb is packed into a column. To purify the relevant Ab, its lysate supernatant is loaded onto an affinity chromatography column combined with the anti-GFP mAb. Then, the column is washed with a binding buffer (10 column volumes, PBS, 0.13 M NaCl, 0.01 M Na2HPO4, 0.01 M NaH2PO4, pH 7.4), and eluted with an elution buffer (0.1 M glycine-HCl, pH 4.5). The eluate is immediately neutralized by adding a small amount of a neutralization buffer (1 M Tris-HCl, pH 9.0), and then dialyzed (0.15 M PBS, pH 7.4).

[0184] [Analysis of the relevant Ab]

[0185] The purified relevant Ab is analyzed by discontinuous 12% SDS-PAGE and Coomassie Brilliant Blue R250 staining. In addition, size exclusion chromatography combined with dynamic light scattering technology is used to detect the aggregation and denaturation of the relevant Ab with high resolution. The protein content is measured by using the biological software Bandscan 5.0 to estimate the purification and yield.

[0186] We have examined various spatial configurations. The Gas6 capture agent can be fused to the light or heavy chain of an anti-PD-L1 antibody or its PD-L1 binding fragment. Its Gas6 capture agent can be fused to the N-terminus or C-terminus of the chain of the anti-PD-L1 unit. The anti-PD-L1 unit can have a light chain and a separate heavy chain, or have a light chain and a heavy chain on a single protein chain (such as scFv). We unexpectedly found that compared with the C-terminal fusion construct, the N-terminal fusion construct exhibited relatively low yields and significantly reduced bioactivity. Based on trial and error, the spatial configurations with favorable yields and biological functions in this disclosure are presented in Figure 1 wherein the Gas6 capture agent is fused to the C-terminus of the heavy chain of the anti-PD-L1 unit, and the unit comprises a light chain and a separate heavy chain (Type 1) or comprises a light chain and a heavy chain on a single protein chain (Type 2). The exemplary bifunctional molecules presented in this disclosure include bifunctional molecules belonging to Type 1 and Type 2. Exemplary bifunctional molecules belonging to Type 1 are Example 1, Example 3, Example 4, Example 5, and Example 6; and exemplary bifunctional molecules belonging to Type 2 are Example 2, Example 7, Example 8, and Example 9.

[0187] In Example 1 and Example 2, the antibody (j) or its antigen-binding fragment comprises a heavy chain of SEQ ID NO:57 or a substantially similar sequence thereof and a light chain of SEQ ID NO:58 or a substantially similar sequence thereof. In Example 3, Example 4, and Example 7, the antibody (a) or its antigen-binding fragment comprises: VH-CDR1 of SEQ ID NO:3 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO:4 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO:5 or a substantially similar sequence thereof; VL-CDR1 of SEQ ID NO:10 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO:11 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO:12 or a substantially similar sequence thereof. In Example 5, Example 6, Example 8, and Example 9, the antibody (a) or its antigen-binding fragment comprises VH-CDR1 of SEQ ID NO:96 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO:97 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO:98 or a substantially similar sequence thereof; VL-CDR1 of SEQ ID NO:99 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO:11 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO:12 or a substantially similar sequence thereof. In Example 3, Example 5, Example 7, and Example 8, the VH of the antibody (a) or its antigen-binding fragment comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4). In the antibody (a) or its antigen-binding fragment, HC-FR1 is SEQ ID NO:6 or a substantially similar sequence thereof; HC-FR2 is SEQ ID NO:7 or a substantially similar sequence thereof; HC-FR3 is SEQ ID NO:8 or a substantially similar sequence thereof; and HC-FR4 is SEQ ID NO:9 or a substantially similar sequence thereof. The VL of the antibody (a) or its antigen-binding fragment comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). In the antibody (a) or its antigen-binding fragment, LC-FR1 is SEQ ID NO:13 or a substantially similar sequence thereof; LC-FR2 is SEQ ID NO:14 or a substantially similar sequence thereof; LC-FR3 is SEQ ID NO:15 or a substantially similar sequence thereof; and LC-FR4 is SEQ ID NO:16 or a substantially similar sequence thereof.In Example 4, Example 6, and Example 9, the VH of antibody (a) or its antigen-binding fragment contains a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4). In antibody (a) or its antigen-binding fragment, HC-FR1 is SEQ ID NO:6 or a substantially similar sequence thereof; HC-FR2 is SEQ ID NO:7 or a substantially similar sequence thereof; HC-FR3 is SEQ ID NO:8 or a substantially similar sequence thereof; and HC-FR4 is SEQ ID NO:9 or a substantially similar sequence thereof. The VL of antibody (a) or its antigen-binding fragment contains a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). In antibody (a) or its antigen-binding fragment, LC-FR1 is SEQ ID NO:100 or a substantially similar sequence thereof; LC-FR2 is SEQ ID NO:101 or a substantially similar sequence thereof; LC-FR3 is SEQ ID NO:102 or a substantially similar sequence thereof; and LC-FR4 is SEQ ID NO:16 or a substantially similar sequence thereof.

[0188] Example 2:

[0189] [PD-L1 Binding Affinity]

[0190] The binding ability of the bifunctional molecule and its parental mAb was examined by FACS and ELISA. Recombinant human PD-L1 protein at a concentration of 100 ng / well mixed with a coating solution (Seracare, 5150-0014) was evenly seeded onto a 96-well plate. The 96-well culture plate was incubated overnight at 4 °C. The next day, the culture plate was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 between the steps of applying different reagents. After discarding the coating Ab, the culture plate was blocked with PBS containing 3% skim milk at 37 °C for 1 hour. Then, after discarding the blocking buffer, serial dilutions of the relevant Ab / PBS were applied at 37 °C for 1 hour. After discarding the relevant Ab, a secondary antibody, namely horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody, was applied at 37 °C for 1 hour. Finally, color development was completed by the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). Then, the oxidation reaction of TMB driven by HRP was stopped by 1N hydrochloric acid. The optical density (O.D.) at 450 / 650 nm was read to quantify the PD-L1 binding affinity of the bifunctional molecule and the parental Ab. The hPD-L1 binding affinities of the BfAbs are shown in Table 2, where the bifunctional molecule configurations of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 are shown not to interfere with their hPD-L1 binding affinities.

[0191] Table 2: The affinities of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 for human PD-L1 (equivalent to the affinities of their parental antigen-binding fragments) are listed below.

[0192]

[0193] [Gas6 binding affinity]

[0194] The relevant Ab mixed with the coating solution at a concentration of 100 ng / well was evenly inoculated onto a 96-well plate. The 96-well culture plate was incubated overnight at 4°C. The next day, the culture plate was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 between the steps of applying different reagents. After discarding the coated Ab, the culture plate was blocked with PBS containing 3% skim milk at 37°C for 1 hour. Then, after discarding the blocking buffer, serially diluted human Gas6 recombinant protein (hGas6) / PBS at a concentration of 100 μg / well was applied at 37°C for 1 hour. After discarding Gas6, the secondary antibody, namely HRP-conjugated goat anti-human IgG antibody, was applied at 37°C for 1 hour. Finally, color development was completed by the oxidation reaction of TMB. Then, the oxidation reaction of TMB driven by HRP was stopped by 1N hydrochloric acid. The O.D. at 450 / 650 nm was read to quantify the Gas6 binding affinity of the bifunctional molecule and the reference Ab.

[0195] The hGas6 binding affinities of the parental Axl-Fc of the bifunctional molecule (as the Gas6-binding part, where the C-terminus of the Gas6 capturer is fused to the N-terminus of human Fc) and MYD1-72 (reference decoy receptor) are shown in Table 3. The hGas6 binding affinities of the bifunctional molecules are shown in Table 4, where the bifunctional molecule configurations of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 are shown not to hinder their hGas6 binding affinities.

[0196] Table 3: The affinities of the self-made Axl-Fc and the reference MYD1-72 (adapted from US8168415B2) for human Gas6 are listed as follows.

[0197]

[0198] Table 4: The affinities of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 for human Gas6 (comparable to the affinities of their parental Gas6-binding parts) are listed as follows.

[0199]

[0200] Example 3:

[0201] [PD-L1 / PD-1 blockade]

[0202] The PD-1 / PD-L1 blocking functions of the bifunctional molecule and the reference Ab were quantified using a bioanalysis kit (Promega, J1252). Briefly, PD-L1 aAPC / CHO-K1 cells were seeded at 4×10 4 / The cells were seeded at a concentration of 4 / well onto a 96-well plate. Subsequently, the culture plate was incubated overnight at 37 °C. The next day, the relevant Abs at serial dilutions and PD-1 effector cells at a concentration of 5×10

[0203] The PD-L1 / PD-1 blocking activities of the bifunctional molecules are shown in Table 5, where the bifunctional molecule configurations of Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, and Example 9 are shown not to hinder their PD-L1 / PD-1 blocking activities.

[0204] Table 5: Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, and Example 9 can efficiently block human PD-L1 / PD-1 signal transduction.

[0205]

[0206] [Gas6 / Axl Blocking]

[0207] The Gas6 / Axl blocking functions of the BfAbs and reference Abs were quantified using Ba / F3 cell lines expressing human Axl (Ba / F3-hAxl). A mixture consisting of BA / F3-hAxl at a concentration of 2000 cells / well, 10% fetal bovine serum, and hGas6 at 100 pg / well was evenly seeded onto a 96-well culture plate. Subsequently, the relevant Abs at a concentration of 5 ng / well were added to the culture plate. Then, the culture plate was incubated at 37 °C for 72 hours. The cell viability of BA / F3-hAxl was examined using a CCK-8 reagent (Dojindo, CK04), and finally the O.D. at 450 nm was read.

[0208] The Gas6 / Axl blocking activities of the bifunctional molecules and their parental Axl-Fc are shown in Figure 2 、 Figure 3 and Figure 4 where the bifunctional configurations of Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, and Example 9 are shown not to hinder their Gas6 / Axl blocking activities.

[0209] Example 4:

[0210] [Cancer Immunotherapy]

[0211] The EMT-6 (a murine triple-negative breast cancer cell line that spontaneously secretes Gas6) was subcutaneously inoculated into female BALB / cByJNarl mice. Drug administration began when the average tumor size was 50 - 100 mm 3 Five dosing groups were intraperitoneally injected, including hIgG isotype control, atezolizumab, Axl-Fc, the combination of atezolizumab and Axl-Fc, and Example 2, at a dose level of 10 mg / kg, twice a week for three weeks (the actual dose of the treatment was adjusted according to their respective molecular weights). Body weight and tumor volume were measured three to four times a week. Blood samples were collected within 3 hours after drug administration. The in vivo experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Biotechnology Laboratory Development Center and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

[0212] The tumor growth inhibitory activities of Example 2, its parental Ab, and its Axl-Fc (where the parental Ab and Axl-Fc were administered as single agents or in combination) are shown in Figure 5 . As shown in Figure 5, Example 2 was unexpectedly superior in tumor growth inhibitory activity to its parental Ab and Axl-Fc, where the parental Ab and Axl-Fc were administered as single agents or in combination. In the parental Ab group and the combination group of parental Ab and Axl-Fc, 3 out of 6 mice had a tumor volume greater than 500 mm 3 , while in the Example 2 group, only 1 out of 6 mice had a tumor volume greater than 500 mm 3 . A higher complete remission rate was observed in the Example 2 group compared to the parental Ab group. In the parental Ab group, 1 out of 6 mice achieved complete remission, while in the Example 2 group, 2 out of 6 mice achieved complete remission.

[0213] The concentration of murine serum Gas6 was measured ( Figure 6 ). Once a molecule containing the Gas6-binding moiety was administered, the concentration of murine serum Gas6 decreased, indicating that the Gas6-binding moiety disclosed in the present disclosure has an impressive in vivo function.

[0214] The tumor rechallenge study was further performed on the mice that achieved complete remission to examine the immune memory effects of Example 2 and its parental Ab. Briefly, the same tumor cell line (EMT-6) was subcutaneously inoculated into the mice that achieved complete remission in the first round. When rechallenging the tumor, the tumor inoculation was performed on the other side of the mice. Then, body weight and tumor volume were measured three to four times a week. Figure 7 The results shown finally demonstrated that immune memory effects were observed in both Example 2 and its parental Ab. Nevertheless, Example 2 showed a slightly more preferable immune memory response than its parental Ab.

[0215] In the mPD-L1-hPD-L1+EMT-6 syngeneic mouse model, the tumor growth inhibitory function of the present disclosure was confirmed with another Example 3. In Figure 8 it, compared with the vehicle group, the Example 3 group demonstrated the promising anti-cancer effect of the present disclosure.

[0216] The present disclosure provides a bifunctional fusion protein having a novel composition, wherein the novel composition comprises two binding moieties: an anti-PD-L1 antibody or a PD-L1 binding fragment fused to a Gas6 binding moiety at the C-terminus. Different spatial configurations of the bifunctional fusion protein were examined. Supported by the Examples of the present disclosure, the selected anti-PD-L1×Gas6 capturer bifunctional configurations Form 1 and Form 2 herein can be applied to anti-PD-L1 antibodies or PD-L1 binding fragments having different sequences. Moreover, both the Form 1 or Form 2 anti-PD-L1×Gas6 capturer bifunctional fusion proteins demonstrated binding affinities and functions comparable to their parental binding moieties. More importantly, the anti-PD-L1×Gas6 capturer bifunctional fusion proteins disclosed herein unexpectedly demonstrated a synergistic anti-tumor effect: the selected anti-PD-L1×Gas6 capturer bifunctional configurations were superior to the parental binding moieties in terms of tumor growth inhibitory activity, wherein the parental binding moieties were administered as a single agent or in combination. In part, the tumor targeting of the anti-PD-L1×Gas6 capturer bifunctional fusion protein was superior to the combination of the two combined moieties as a single agent. The binding affinities of the anti-PD-L1 antibody or PD-L1 binding fragment and the Gas6 capturer binding moiety to their corresponding targets were different, indicating that the anti-PD-L1 antibody or PD-L1 binding fragment dominates the tumor targeting of the anti-PD-L1×Gas6 capturer fusion protein in the present disclosure.

[0217] Although the present disclosure has been described in connection with the specific embodiments set forth above, many alternatives, modifications, and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications, and variations are considered to be within the scope of the present disclosure.

Claims

1. A fusion protein, which comprises a Gas6 (Growth Arrest-Specific 6) binding portion; and an antigen-binding portion, which comprises an antibody or an antigen-binding fragment thereof that is specific for an epitope in PD-L1 (Programmed Death-Ligand 1).

2. The fusion protein according to claim 1, wherein the Gas6 binding portion comprises the extracellular domain of a receptor tyrosine kinase (RTK) of the TAM (Tyro-3, Axl, MerTK) family.

3. The fusion protein according to claim 1, wherein the Gas6 binding portion comprises the amino acid sequence of SEQ ID NO: 1 or 2 or a variant thereof.

4. The fusion protein according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises complementarity-determining regions (CDRs) of the heavy chain variable region (VH) and complementarity-determining regions of the light chain variable region (VL), wherein the complementarity-determining regions of the heavy chain variable region comprise VH-CDR1, VH-CDR2 and VH-CDR3, and the complementarity-determining regions of the light chain variable region comprise VL-CDR1, VL-CDR2 and VL-CDR3, and the antibody or the antigen-binding fragment thereof is: antibody (a) or an antigen-binding fragment thereof, which comprises VH-CDR1 of SEQ ID NO: 3 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO: 4 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO: 5 or a substantially similar sequence thereof; VL-CDR1 of SEQ ID NO: 10 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO: 11 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO: 12 or a substantially similar sequence thereof, or VH-CDR1 of SEQ ID NO: 96 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO: 97 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO: 98 or a substantially similar sequence thereof; VL-CDR1 of SEQ ID NO: 99 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO: 11 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO: 12 or a substantially similar sequence thereof, antibody (b) or an antigen-binding fragment thereof, which comprises VH-CDR1 of SEQ ID NO: 17 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO: 18 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO: 19 or a substantially similar sequence thereof, VL-CDR1 of SEQ ID NO: 20 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO: 21 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO: 22 or a substantially similar sequence thereof, Antibody (c) or an antigen-binding fragment thereof, comprising VH-CDR1 of SEQ ID NO:23 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO:24 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO:25 or a substantially similar sequence thereof, VL-CDR1 of SEQ ID NO:26 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO:27 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO:28 or a substantially similar sequence thereof, Antibody (d) or an antigen-binding fragment thereof, comprising VH-CDR1 of SEQ ID NO:29 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO:30 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO:31 or a substantially similar sequence thereof, VL-CDR1 of SEQ ID NO:32 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO:33 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO:34 or a substantially similar sequence thereof, Antibody (e) or an antigen-binding fragment thereof, comprising VH-CDR1 of SEQ ID NO:43 or a substantially similar sequence thereof; VH-CDR2 of SEQ ID NO:44 or a substantially similar sequence thereof; VH-CDR3 of SEQ ID NO:45 or a substantially similar sequence thereof, VL-CDR1 of SEQ ID NO:46 or a substantially similar sequence thereof; VL-CDR2 of SEQ ID NO:47 or a substantially similar sequence thereof; and VL-CDR3 of SEQ ID NO:48 or a substantially similar sequence thereof, Antibody (f) or an antigen-binding fragment thereof, comprising a heavy chain of SEQ ID NO:49 or a substantially similar sequence thereof and a light chain of SEQ ID NO:50 or a substantially similar sequence thereof, Antibody (g) or an antigen-binding fragment thereof, comprising a heavy chain of SEQ ID NO:51 or a substantially similar sequence thereof and a light chain of SEQ ID NO:52 or a substantially similar sequence thereof, Antibody (h) or an antigen-binding fragment thereof, comprising a heavy chain of SEQ ID NO:53 or a substantially similar sequence thereof and a light chain of SEQ ID NO:54 or a substantially similar sequence thereof, Antibody (i) or an antigen-binding fragment thereof, comprising a heavy chain of SEQ ID NO:55 or a substantially similar sequence thereof and a light chain of SEQ ID NO:56 or a substantially similar sequence thereof, Antibody (j) or an antigen-binding fragment thereof, comprising a heavy chain of SEQ ID NO:57 or a substantially similar sequence thereof and a light chain of SEQ ID NO:58 or a substantially similar sequence thereof, or Antibody (k) or an antigen-binding fragment thereof, comprising a heavy chain encoded by SEQ ID NO:59 or a substantially similar sequence thereof; and a light chain encoded by SEQ ID NO:60 or a substantially similar sequence thereof.

5. The fusion protein according to claim 4, wherein the VH of the antibody (a) or its antigen-binding fragment comprises a framework represented by the following formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4); the HC-FR1 is SEQ ID NO: 6 or a substantially similar sequence thereof; the HC-FR2 is SEQ ID NO: 7 or a substantially similar sequence thereof; the HC-FR3 is SEQ ID NO: 8 or a substantially similar sequence thereof; and the HC-FR4 is SEQ ID NO: 9 or a substantially similar sequence thereof; and the VL of the antibody (a) or its antigen-binding fragment comprises a framework represented by the following formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4); the LC-FR1 is SEQ ID NO: 13 or a substantially similar sequence thereof; the LC-FR2 is SEQ ID NO: 14 or a substantially similar sequence thereof; the LC-FR3 is SEQ ID NO: 15 or a substantially similar sequence thereof; and the LC-FR4 is SEQ ID NO: 16 or a substantially similar sequence thereof.

6. The fusion protein according to claim 4, wherein the VH of the antibody (a) or its antigen-binding fragment comprises a framework represented by the following formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4); the HC-FR1 is SEQ ID NO: 6 or a substantially similar sequence thereof; the HC-FR2 is SEQ ID NO: 7 or a substantially similar sequence thereof; the HC-FR3 is SEQ ID NO: 8 or a substantially similar sequence thereof; and the HC-FR4 is SEQ ID NO: 9 or a substantially similar sequence thereof; and the VL of the antibody (a) or its antigen-binding fragment comprises a framework represented by the following formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4); the LC-FR1 is SEQ ID NO: 100 or a substantially similar sequence thereof; the LC-FR2 is SEQ ID NO: 101 or a substantially similar sequence thereof; the LC-FR3 is SEQ ID NO: 102 or a substantially similar sequence thereof; and the LC-FR4 is SEQ ID NO: 16 or a substantially similar sequence thereof.

7. The fusion protein according to claim 4, wherein the VH of the antibody (d) or its antigen-binding fragment comprises a framework represented by the following formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4); the HC-FR1 is SEQ ID NO:35 or a substantially similar sequence thereof; the HC-FR2 is SEQ ID NO:36 or a substantially similar sequence thereof; the HC-FR3 is SEQ ID NO:37 or a substantially similar sequence thereof; and the HC-FR4 is SEQ ID NO:38 or a substantially similar sequence thereof; and the VL of the antibody (d) or its antigen-binding fragment comprises a framework represented by the following formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4); the LC-FR1 is SEQ ID NO:39 or a substantially similar sequence thereof; the LC-FR2 is SEQ ID NO:40 or a substantially similar sequence thereof; the LC-FR3 is SEQ ID NO:41 or a substantially similar sequence thereof; and the LC-FR4 is SEQ ID NO:42 or a substantially similar sequence thereof.

8. The fusion protein according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region.

9. The fusion protein according to claim 8, wherein the heavy chain constant region contains a mutation at the amino acid position corresponding to N297 of IgG1.

10. The fusion protein according to claim 1, wherein the antigen-binding portion comprises a Fab fragment, an F(ab')2 fragment, a ScFv fragment, a chimeric antibody, or a nanobody.

11. The fusion protein according to claim 1, wherein the antigen-binding portion is multispecific.

12. The fusion protein according to claim 1, wherein the Gas6-binding portion is fused to the antigen-binding portion via a peptide linker.

13. The fusion protein according to claim 12, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:

71.

14. The fusion protein according to claim 1, wherein the Gas6-binding portion is fused to the heavy chain of the antigen-binding portion.

15. The fusion protein according to claim 1, wherein the Gas6-binding portion is fused to the C-terminus of the heavy chain of the antigen-binding portion.

16. A pharmaceutical composition comprising: an effective amount of the fusion protein according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier.

17. A method for treating, prophylactically treating, and / or preventing cancer in an individual in need thereof, comprising administering to the individual an effective amount of the fusion protein according to any one of claims 1 to 15.

18. The method according to claim 17, wherein the cancer is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

19. A method for detecting cancer in an individual in need thereof, comprising contacting a sample derived from the individual with the fusion protein according to any one of claims 1 to 15.

20. The method according to claim 19, wherein the cancer is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

21. A kit for detecting cancer in a sample, comprising the fusion protein according to any one of claims 1 to 15.

22. The kit according to claim 21, wherein the cancer is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

23. A method for detecting PD-L1 in a sample, comprising contacting the sample with the fusion protein according to any one of claims 1 to 15.

24. A method for neutralizing Gas6 in a sample, comprising contacting the sample with the fusion protein according to any one of claims 1 to 15.

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