Fusion proteins that target PD-L1 and neutralize GAS6 and uses thereof

Abstract

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

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE

[0001] This disclosure relates to novel fusion proteins, and in particular to fusion proteins that target PD-L1 (programmed death-ligand 1) and neutralize Gas6 (growth arrest-specific 6). [Background technology]

[0002]

[0002] Cancer manipulates the tumor microenvironment for its own benefit. Cancer can induce an immunosuppressive microenvironment to benefit its own development, progression, metastasis, and treatment resistance. Immune checkpoint inhibitors have emerged as a promising cancer immunotherapy for decades. By exploiting a patient's tumor microenvironment with immune checkpoint inhibitors, favorable outcomes have been achieved in 15 to 25 percent of patients with various cancers. Overall survival in patients treated with immune checkpoint inhibitors has even exceeded that of standard treatment, especially in patients who respond significantly.

[0003] However, monotherapy using PD-L1 / PD-1 inhibitors has limitations, necessitating combination treatment strategies to broaden its scope, improve efficacy, and reduce toxicity. Most unfortunately, approved standard therapies and immunotherapy-based combination therapies, despite their partial efficacy, are limited by the occurrence of severe side effects. Therefore, there is a need to develop novel approaches to treat cancer. Summary of the Invention

[0004]

[0004] Embodiments of the present disclosure relate to the treatment or diagnosis of tumors via fusion proteins, such as fusion proteins comprising (i) a Gas6-binding moiety and (ii) an antibody or 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 uses of such molecules (e.g., to treat cancer) and methods of making such molecules.

[0005]

[0005] Therefore, the present disclosure provides: and an antigen-binding portion comprising an antibody or antigen-binding fragment thereof specific for an epitope in PD-L1.

[0006]

[0006] In some embodiments of the present disclosure, the Gas6 binding moiety comprises the extracellular domain of a RTK (receptor tyrosine kinase) of the TAM family (Tyro-3, Axl, MerTK).

[0007]

[0007] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) of a heavy chain variable region (VH) and a complementarity determining region of a light chain variable region (VL), wherein the complementarity determining region of the heavy chain variable region comprises VH-CDR1, VH-CDR2 and VH-CDR3, and the complementarity determining region of the light chain variable region comprises VL-CDR1, VL-CDR2 and VL-CDR3.

[0008]

[0008] For example, examples of antibodies or antigen-binding fragments include antibodies (a) to (k) or antigen-binding fragments thereof, 3G10 of U.S. Patent No. 7,943,743, 12A4 of U.S. Patent No. 7,943,743, 10A5 of U.S. Patent No. 7,943,743, 5F8 of U.S. Patent No. 7,943,743, 10H10 of U.S. Patent No. 7,943,743, 1B12 of U.S. Patent No. 7,943,743, and 7H of U.S. Patent No. 7,943,743. No. 7,943,743, 11E6 of U.S. Pat. No. 7,943,743, 12B7 of U.S. Pat. No. 7,943,743, 13G4 of U.S. Pat. No. 7,943,743, MDX-1105, MEDI-4736, atezolizumab, durvalumab, avelumab, MDX-1105, embafolimab, cosibelimab, CK-301, CS-1001, SHR-1316, CBT-502, BGB-A333, and the like.

[0009] 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, hi one embodiment of the present disclosure, the constant region has a mutation at the amino acid position corresponding to N297 of IgG1.

[0010] In some embodiments of the present disclosure, the antigen-binding portion comprises a Fab fragment, a F(ab')2 fragment, an ScFv fragment, a chimeric antibody, or a nanobody.

[0011]

[0011] In some embodiments of the present disclosure, the antigen-binding moiety is multispecific.

[0012]

[0012] In some embodiments of the present disclosure, the Gas6 binding moiety is fused to the antigen binding moiety via a peptide linker.

[0013]

[0013] In some embodiments of the present disclosure, the Gas6 binding moiety is fused to the heavy chain of the antigen binding moiety.

[0014]

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

[0015]

[0015] The present disclosure further provides: an effective amount of a fusion protein disclosed herein or a genetically engineered cell disclosed herein; and Pharmaceutically acceptable carrier The present invention provides a pharmaceutical composition comprising:

[0016]

[0016] The present disclosure further provides a method for treating, prophylactically treating, and / or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein disclosed herein.

[0017]

[0017] The present disclosure further provides a method for detecting cancer in a subject in need thereof, the method comprising the step of contacting a sample derived from the subject with the fusion protein disclosed herein.

[0018]

[0018] The present disclosure further provides a kit for detecting cancer in a sample, comprising a fusion protein according to the present invention.

[0019]

[0019] Examples of cancer include, but are not limited to, bladder cancer, liver cancer, colon cancer, rectal cancer, uterine 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.

[0020]

[0020] The present disclosure further provides a method for detecting PD-L1 in a sample, the method comprising contacting the sample with a fusion protein disclosed herein.

[0021]

[0021] The present disclosure further provides a method for neutralizing Gas6 in a sample, the method comprising contacting the sample with a fusion protein disclosed herein. [Brief explanation of the drawings]

[0022] [Figure 1] 1A and 1B illustrate preferred spatial configurations of the present invention. [Figure 2] FIG. 1 shows that Example 1 and Example 2 can efficiently capture human Gas6 and thus achieve cell growth inhibition in Axl-overexpressing Ba / F3 cell line. [Figure 3] This figure shows that Examples 3, 4, 5, and 6 can efficiently capture human Gas6 and thus achieve cell growth inhibition in the Axl-overexpressing Ba / F3 cell line. [Figure 4] FIG. 1 shows that Examples 7, 8 and 9 can efficiently capture human Gas6 and thus achieve cell growth inhibition in the Axl-overexpressing Ba / F3 cell line. [Figure 5] FIG. 1 shows that the in vivo tumor growth inhibitory function of Example 2 exceeds that of atezolizumab, in-house Axl-Fc, and combination therapy. [Figure 6]FIG. 1 shows that mice injected intraperitoneally with a molecule containing a Gas6-binding moiety exhibited reduced serum Gas6 concentrations. [Figure 7] 1 shows that mice that achieved complete remission in the Example 2 group exhibited better immunological memory during rechallenge than the atezolizumab group, where 1 of 6 mice in the atezolizumab group and 2 of 6 mice in the Example 2 group achieved complete remission. [Figure 8] 1 shows the impressive in vivo antitumor effect of Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0023]

[0030] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art.

[0024]

[0031] The practice of the present disclosure may employ techniques within the skill of the art, including conventional techniques of cell biology, cell culture, antibody technology, and genetic engineering, which are fully explained in the literature.

[0025]

[0032] As utilized in accordance with the present disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated: As used herein, the term "and / or" shall be construed as specifically disclosing each of the two specified features or components, either with or without the other feature or component. For example, "A and / or B" shall be construed as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0026]

[0033] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027]

[0034] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains 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 is comprised of three domains: C, C, and L. H1 , C H2 and C H3 Each light chain comprises a light chain variable region (VL). In some embodiments, the light chain further comprises a light chain constant region. The light chain constant region comprises one domain (C L1 VH and VL can be further subdivided into regions of hypervariability, called CDRs, interspersed with more conserved regions, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an anti-PD-L1 antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0028]

[0035] As used herein, the terms "specific for" or "specifically binds to" mean that the antibody does not cross-react to a significant extent with other epitopes.

[0029]

[0036] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0030]

[0037] As used herein, the term "complementarity-determining region (CDR)" refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US 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), where the definitions include overlapping or subsets of amino acid residues when compared against each other.

[0031]

[0038] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, typically selected from a human immunoglobulin template.

[0032]

[0039] As used herein, the term "nanobody" refers to antibodies comprising small single variable domains (VHHs) of antibodies obtained from camelids and dromedaries. Antibody proteins obtained from species of camelids and dromedaries (Camelus baclrianus and Camelus dromedarius), including New World species such as llamas (alpacas, llamas, and vicunas), have been characterized with respect to size, structural complexity, and antigenicity to human subjects. Certain IgG antibodies from mammals of this family found in nature lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure with two heavy chains and two light chains of antibodies from other animals.

[0033]

[0040] As used herein, the term "antigen-binding fragment" of an antibody and like terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0034]

[0041] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of amino acid residues over the entire sequence of another (reference) protein sequence when the protein sequence is optimally aligned with the reference protein sequence, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill 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: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has 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.

[0035]

[0042] Sequence similarity of polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its variants. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to databases containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0036]

[0043] As used in this disclosure, the term "pharmaceutical composition" means a mixture containing therapeutic agents that is administered to a mammal, such as a human, to prevent, treat, or eliminate a particular disease or condition from which the mammal suffers.

[0037]

[0044] As used herein, the terms "therapeutically effective amount" or "effective amount" refer to the amount of an antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.

[0038]

[0045] As used herein, the terms "treatment," "treating," and the like encompass any treatment of disease in a mammal, particularly a human, and include (a) preventing disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting disease, i.e., halting the development of the disease; and (c) palliating disease, i.e., causing regression of the disease.

[0039]

[0046] The terms "preventing" or "prevention," when used in reference to a condition, are art-recognized and include administering an agent prior to the onset of the condition to reduce the frequency or severity of, or delay the onset of symptoms of, a medical condition in a subject compared to a subject not receiving the agent.

[0040]

[0047] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0041]

[0048] 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, bedside nurse, or individual; in the case of an animal, including a non-human mammal, a veterinarian) that a subject needs or will benefit from treatment. This judgment is within the expertise of the caregiver, but is made based on a variety of factors, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the compounds of the present disclosure.

[0042]

[0049] Terms such as "cancer," "tumor," and the like include precancerous cells, neoplastic cells, transformed cells, and cancer cells, and can 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 and malignant neoplasms (abnormal growths). "Transformation" refers to spontaneous or induced phenotypic changes, such as cellular immortalization, morphological changes, abnormal cell proliferation, loss of contact inhibition and anchorage, and / or malignant transformation (see Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed., 1994)). Transformation can result from infection with a transforming virus and uptake of new genomic DNA, or uptake of exogenous DNA, but can also occur spontaneously or after exposure to carcinogens.

[0043]

[0050] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and can be used in a diagnostic or monitoring assay. This definition includes 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.

[0044]

[0051] Fusion proteins:

[0052] The present disclosure provides fusion proteins that target both PD-L1 and Gas6. The anti-PD-L1 antibodies or PD-L1-binding fragments disclosed herein are fused to a Gas6-binding moiety (e.g., an Axl decoy receptor) that specifically binds to Gas6 and functions as a trap for Gas6. Experimental results demonstrate that the bifunctional structure of the fusion protein (also referred to as an anti-PD-L1×Gas6 binding moiety) comprising an anti-PD-L1 antibody or PD-L1-binding fragment fused to a Gas6-binding moiety does not interfere with the binding affinity and inhibitory activity of either entity (anti-PD-L1 or Gas6 binding moiety) for its corresponding target. The anti-PD-L1×Gas6 binding moiety motif can target tumors better than the individual agents. Based on the difference in binding affinity between the anti-PD-L1 antibody or PD-L1-binding fragment and the Gas6-binding moiety for their corresponding targets, the anti-PD-L1 antibody or PD-L1-binding fragment dominates the tumor targeting effect of the anti-PD-L1×Gas6-binding moiety. Furthermore, the anti-PD-L1×Gas6-binding moiety unexpectedly exhibits synergistic effects in cancer treatment compared with the effects of administering the two agents separately.

[0045]

[0053] In particular, the present disclosure provides: Gas6 binding moiety; and Antigen-binding portion, including an antibody or antigen-binding fragment thereof, specific for an epitope in PD-L1 The present invention provides a fusion protein comprising:

[0046]

[0054] The fusion proteins can provide a therapeutic benefit to a subject. The fusion proteins disclosed herein can be used as therapeutic agents for treating and / or diagnosing cancer, as described more fully herein.

[0047]

[0055] Programmed cell death protein-1 (PD-1), an inhibitory checkpoint receptor, and its ligand, PD-L1, are involved in T cell exhaustion in various tumors. Therefore, monoclonal antibodies that block the PD-L1 / PD-1 interaction can stimulate immune cells to mount anticancer responses. PD-L1 / PD-1 inhibitors as monotherapy have proven beneficial in patients with certain cancers characterized by high mutational burden and upregulated PD-L1 expression. However, the high response rate of PD-L1 / PD-1 inhibitors is limited to patients with specific cancer types. A subset of patients predicted to respond to PD-L1 / PD-1 inhibitors based on available biomarkers further confounds predictions and is associated with negative outcomes. Furthermore, despite relatively mild side effects, PD-L1 / PD-1 inhibitors can generally cause grade 3–4 toxicity in approximately 10% of patients treated with PD-L1 / PD-1 inhibitors. Due to limitations of monotherapy using PD-L1 / PD-1 inhibitors, combination treatment strategies are needed to broaden their scope, improve efficacy, and reduce toxicity. Unfortunately, approved standard therapies and immunotherapy-based combination therapies, despite their partial efficacy, are limited by the occurrence of severe side effects. Therefore, the development of combination therapies with improved survival rates and fewer side effects remains desirable.

[0048]

[0056] Growth arrest-specific 6, a member of the vitamin K-dependent protein family, is expressed by cancer cells and tumor-infiltrating leukocytes. Gas6 regulates diverse cellular functions, including cell proliferation, migration, survival, angiogenesis, and metabolism, by binding to its receptor, the TAM family of RTKs. RTKs are cell surface receptors capable of catalyzing the phosphorylation of tyrosine residues on target proteins. The TAM family of RTKs consists of Axl, Tyro, and MerTK. All TAM family receptors possess an extracellular ligand-binding domain, a single-pass transmembrane domain, an intracellular kinase domain, and a tyrosine-containing C-terminal tail. Numerous studies have shown that 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 consistently predicts poor prognosis. Furthermore, Gas6 aids tumor immune evasion by binding to TAM receptors on residual immune cells.

[0049]

[0057] Gas6 binds to TAM receptors with varying affinities, with Axl being the most potent. Axl is overexpressed by host cells remaining in the tumor microenvironment, including several immune cell types, fibroblasts, osteoclasts, and endothelial cells, and has been shown to promote immune evasion. Increased expression of the Axl receptor tyrosine kinase in various tumors correlates with poor overall patient survival. Endogenous Axl activation by tumors and extrinsic Axl activation by suppressor immune cells recruited to the tumor microenvironment contribute to cancer proliferation. Through both endogenous and extrinsic effects, the autocrine and paracrine Gas6 / Axl signaling axis promotes tumor growth, metastasis, immune evasion, and therapeutic resistance in cancer patients. Furthermore, the Gas6 / Axl pathway has also been reported to drive PD-L1 expression in cancer cells to prevent T cell activation.

[0050]

[0058] As mentioned above, avoiding Gas6 binding to TAMs has emerged as an important approach for cancer therapy. Simultaneous inhibition of the PD-L1 / PD-1 and Gas6 / TAM pathways offers a reasonable solution to the limitations of monotherapy involving PD-L1 inhibition.

[0051]

[0059] The fusion protein disclosed herein enables the local reduction of TAM kinase activation in tumors, both endogenously and exogenously, by capturing Gas6 with a Gas6-binding moiety, a decoy receptor fused to anti-PD-L1, an antibody moiety that targets the extracellular domain of an immune checkpoint protein found on cancer cells and immune cells. This fusion protein, sometimes referred to herein as the anti-PD-L1×Gas6-binding moiety, surpasses currently available anti-cancer strategies, including but not limited to, administering the antibody and receptor as separate molecules or in combination, for several reasons. First, the antibody moiety precisely targets the fusion protein to the tumor microenvironment, where the concentrated Gas6 functions primarily via autocrine and paracrine pathways to increase tumor growth, metastasis, and immune evasion. The resulting advantage (e.g., over administering the antibody and receptor as separate molecules) is due, in part, to the cytokine's preferential function in the local environment via autocrine and paracrine functions. The antibody portion neutralizes local immunosuppressive autocrine or paracrine effects, thereby targeting cytokine traps to the tumor microenvironment where they may be most effective. Second, the fusion protein can address tumor growth, metastasis, and immune evasion using a decoy receptor, while simultaneously restoring local immune surveillance using the antibody portion. Furthermore, the positive feedback of PD-L1 production through activation of the Gas6 / Axl axis also predicts a synergistic effect of this fusion protein. Therefore, due to the two mechanisms in the design of a single entity, this fusion protein can be used for broader applications. Finally, as a single entity, multifunctionality can be achieved using this fusion protein at a lower dosage. Having multifunctionality in a single entity is also expected to increase tolerance to resistance. Furthermore, different spatial configurations of the fusion protein were screened by the inventors. The selected conformation shown in this disclosure has a Gas6 binding moiety at the C-terminus of the antibody portion, with the antibody portion lacking a stop codon followed by a Gas6 binding moiety containing a stop codon.

[0052]

[0060] Overall, this disclosure revolutionizes anti-PD-L1-based immunotherapy in three ways. First, it precisely targets tumors to induce synergistic anti-tumor effects resulting from simultaneous inhibition of the PD-L1 / PD-1 and Gas6 / TAM pathways in tumors and their microenvironments. Thus, the fusion protein enhances the efficacy of anti-PD-L1-based immunotherapy and enables broader application of anti-PD-L1-based immunotherapy by incorporating a fused Gas6-binding moiety into the entity. Second, the fusion protein, limited to tumors and their microenvironments, exerts multifunctionality in a single entity, achieving maximum efficacy with minimal dosage and greater specificity. Multifunctionality in a single entity format can also enhance tolerance to resistance. Finally, the preferred spatial combination of this fusion protein was selected from various possible spatial combinations. Therefore, the fusion protein disclosed in this disclosure is also a practical result of in silico prediction and will help accelerate future innovation of multifunctional proteins containing antibody moieties fused to decoy receptors.

[0053]

[0061] Gas6 connection part:

[0062] Gas6 structurally belongs to the family of plasma vitamin K-dependent proteins. Gas6 possesses growth factor-like properties through its interaction with the TAM family of receptor tyrosine kinases. Human Gas6 is a 678-amino acid protein consisting of a vitamin K-dependent carboxyglutamic acid (Gla)-rich domain that mediates binding to phospholipid membranes, four epidermal growth factor-like domains, and two laminin G-like (LG) domains that mediate binding to TAM receptors. The sequences of human Gas6 transcript variants can be accessed at GenBank under NM_001143946.1, NM_001143945.1, and NM_000820.2, respectively.

[0054]

[0063] The Gas6-binding portion provides an isolated fragment thereof that specifically binds to the Gas6 protein. The isolated fragment of the Gas6-binding portion can bind to an epitope contained in or presented by one or more amino acid residues that interact with Axl (e.g., 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 belongs to Gas6 (L295-T317, E356-P372, R389-N396, D398-A406, E413-H429, and W450-M468). Furthermore, the isolated fragment of the Gas6-binding portion can inhibit or compete with the binding between TAM and Gas6. Among the TAM receptors, wild-type Axl or the soluble Axl (sAxl) variant exhibited a K of 1.0 nM / L for Gas6. D It has the highest in vitro demonstrated affinity for Tyro-3, followed by a nearly equal affinity for MerTK and a K D have an affinity at least 10 times lower than the value.

[0055]

[0064] The Gas6-binding moiety may be any conformation that inhibits the Gas6 / TAM pathway by neutralizing Gas6. Using a Gas6-binding moiety to neutralize Gas6 may be a better strategy than inhibiting the TAM receptor with an antagonist molecule, because upregulation of Gas6 production induced by a TAM antagonist molecule in a negative feedback loop may be expected. The Gas6-binding moiety may 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. The isolated antibody fragment of the Gas6-binding moiety may contain the region of the antibody (in the context of either an antibody scaffold or a non-antibody scaffold) sufficient or necessary for recognizable specific binding of the 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 variable region alone or a variable region combined with a portion of the Fc region (e.g., a CH1 region), or a minibody (e.g., VL-VH-CH3) or diabody.

[0056]

[0065] The Axl receptor displays a structure shared among TAM family members, containing an intracellular tyrosine kinase domain and an extracellular region juxtaposed with immunoglobulin (Ig) and fibronectin type III (FnIII) ​​repeats. The extracellular Ig and Fn motifs are thought to be important in cell adhesion and migration, representing a means by which the Axl oncogene contributes to tumor invasiveness and metastasis. Axl transduces signals from the extracellular matrix to the cytoplasm by binding to growth factors such as the vitamin K-dependent protein Gas6. This interaction activates Axl by causing its dimerization and autophosphorylation.

[0057]

[0066] The Gas6-binding moiety in the fusion protein can comprise 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 the two N-terminal Ig domains (designated Ig1 and Ig2) and lacking carbohydrate modifications retains full Gas6-binding activity. A Gas6-binding moiety suitable for use in a fusion protein excludes at least the cytoplasmic domain of Axl, preferably all or most of the transmembrane domain of Axl, and includes a portion of, or even the entire extracellular domain of Axl. Preferably, the portion of the extracellular domain includes at least the major Gas6-binding surface of Axl; in other embodiments, it contains at least the Ig1 and Ig2 domains of Axl, or residues therein that form a conformation sufficient to bind Gas6.

[0058]

[0067] The native sequence of Axl, shown in SEQ ID NO: 1, contains Igl, Ig2, FnIII, and intracellular domains. The Igl 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 sequence is residues 225-332 (SEQ ID NO: 63) and residues 333-427 (SEQ ID NO: 64), and the intracellular domain sequence is residues 473-894 (SEQ ID NO: 65). The tyrosine residues at positions 779, 821, and 866 are autophosphorylated during 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).

[0059]

[0068] The Gas6-binding portion of the fusion protein disclosed herein is modified or reconstructed based on the native sequence of Axl. For example, the Gas6-binding portion can include Ig1 and Ig2 and exclude FnIII, and the sequence is SEQ ID NO: 2.

[0060]

[0069] The Gas6-binding portion of the fusion protein can include one or more amino acid modifications in wild-type sAxl, e.g., one or more amino acid modifications that increase its affinity for Gas6. Amino acid modifications include any naturally occurring or artificial amino acid modifications known in the art or discovered in the future. Amino acid modifications include any naturally occurring mutation, e.g., substitution, deletion, addition, insertion, etc.; replacing an existing amino acid with another amino acid, e.g., its conservative equivalent; and replacing one or more existing amino acids with or inserting one or more unnatural amino acids. In some embodiments, the amino acid modifications can include at least 1, 2, 3, 4, 5, 6, or 10 amino acid mutations or changes. In some exemplary embodiments, one or more amino acid modifications can be used to alter the properties of sAxl, such as affecting its stability, binding activity, specificity, and / or thermal stability. In some other embodiments, sAxl lacks a transmembrane domain and, optionally, an intracellular domain.

[0061]

[0070] For sufficient binding activity to Gas6, the modifications are usually between the extracellular domain and the transmembrane domain, generally between residues 19-437 of SEQ ID NO: 1, but may also comprise or consist essentially of truncations 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 moiety comprises one or more amino acid modifications within one or more of the following regions of wild-type Axl (SEQ ID NO: 1): 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. In other embodiments, the Gas6 binding moiety comprises 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 moiety comprises one or more amino acid modifications, e.g., A19T, A20T, A21T, A22T, A23T, A24T, A25T, A26T, A27T, A28T, A29T, A30T, A31T, A32T, A33T, A34T, A35T, A36T, A37T, A38T, A39T, A40T, A41T, A42T, A43T, A44T, A45T, A46T, A47T, A48T, A49T, A49T, A50T, A51T, A52T, A53T, A54T, A55T, A56T, A57T, A58T, A59T, A60T, A61T, A62T, A63T, A64T, A65T, A66T, A67T, A68T, A69 ... In some other embodiments, the Gas6 binding moiety comprises one or more amino acid modifications at residue 32, 87, 92, or 127 of wild-type Axl (SEQ ID NO: 1), e.g., G32S, D87G, V92A, and / or G127R. In yet some other embodiments, the Gas6 binding moiety comprises one or more amino acid modifications at residues 26, 79, 92, 127, or combinations thereof, of wild-type Axl (SEQ ID NO: 1), e.g., E26G, V79M, V92A, and / or G127E.

[0062]

[0071] Gas6-binding moieties can be further modified, e.g., conjugated to a wide variety of other oligopeptides or proteins for various purposes. Various post-translational or post-transcriptional modifications can be performed on the Gas6-binding moiety of the fusion protein. Such modifications can include chemical derivatization of the polypeptide, such as acetylation, amidation, or carboxylation. Such modifications can include glycosylation modification, e.g., by exposing the polypeptide to mammalian glycosylation or deglycosylation enzymes. Such modifications can also include phosphorylation of specific amino acid residues (e.g., phosphotyrosine, phosphoserine, or phosphothreonine). Furthermore, farnesylation or prenylation can be achieved by using appropriate coding sequences. In some embodiments, the Gas6-binding moiety can be PEGylated, with the polyethyleneoxy group having the effect of extending half-life in the blood. Gas6-binding moieties can also be combined with other proteins, such as the Fc of the IgG isotype, which may be complement binding, toxins such as ricin, abrin, or diphtheria toxin, or specific binding agents that enable targeting to specific moieties on target cells. In some other embodiments, the Gas6 binding moiety can be modified to improve resistance to proteolysis, optimize solubility properties, or make it more suitable as a therapeutic agent. For example, the Gas6 binding moiety can further include analogs of sAxl variants containing residues other than natural L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids can be substituted for some or all of the amino acid residues. In yet other embodiments, the Gas6 binding moiety can include two, three, four, five, or six covalently or non-covalently linked sAxl variants of the same or different sAxl variants to achieve the appropriate size but avoid undesired aggregation.

[0063]

[0072] In some embodiments, the Gas6-binding moiety is a fusion protein, e.g., fused in-frame with a second polypeptide. The second polypeptide may be part or all of an Fc region, any suitable polypeptide substantially similar to Fc, or part or all of an albumin protein to increase the size of the fusion protein and thereby extend its half-life. In other embodiments, the second polypeptide is useful for handling the Gas6-binding moiety, e.g., for purifying the Gas6-binding moiety or stabilizing the Gas6-binding moiety in vitro or in vivo. For example, the second polypeptide may be a marker sequence, such as a hexahistidine peptide, to facilitate purification of the fusion polypeptide. Furthermore, adding additional amino acids, particularly a region of charged amino acids, to the N-terminus of the polypeptide can improve stability and durability during purification from host cells or subsequent handling and storage. Furthermore, fusion proteins with disulfide-bonded dimeric structures may also be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone.

[0064]

[0073] Antigen binding part:

[0074] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) of a heavy chain variable region (VH) and a complementarity determining region of a light chain variable region (VL), wherein the complementarity determining region of the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the complementarity determining region of the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3.

[0065]

[0075] For example, examples of antibodies or antigen-binding fragments include antibodies (a) to (k) or antigen-binding fragments thereof, 3G10 of U.S. Patent No. 7,943,743, 12A4 of U.S. Patent No. 7,943,743, 10A5 of U.S. Patent No. 7,943,743, 5F8 of U.S. Patent No. 7,943,743, 10H10 of U.S. Patent No. 7,943,743, 1B12 of U.S. Patent No. 7,943,743, 7H1 of U.S. Patent No. 7,943,743, No. 7,943,743, 11E6, U.S. Patent No. 7,943,743, 12B7, U.S. Patent No. 7,943,743, 13G4, MDX-1105, MEDI-4736, atezolizumab, durvalumab, avelumab, MDX-1105, embafolimab, cosibelimab, CK-301, CS-1001, SHR-1316, CBT-502, BGB-A333, and the like.

[0066]

[0076] In some embodiments of the present disclosure, the antibody (a) or antigen-binding fragment thereof comprises a VH-CDR1 of GYSITSDYWN (SEQ ID NO: 3) or a substantially similar sequence thereof; a VH-CDR2 of YISYTGSTYYNPSLKS (SEQ ID NO: 4) or a substantially similar sequence thereof; a VH-CDR3 of RGEWLSPFAY (SEQ ID NO: 5) or a substantially similar sequence thereof; a VL-CDR1 of KSSQSLLYSSNQKNSLA (SEQ ID NO: 10) 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.

[0067]

[0077] In some embodiments of the present disclosure, the antibody (a) or antigen-binding fragment thereof 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.

[0068]

[0078] In some embodiments of the present disclosure, the VH of antibody (a) or an antigen-binding fragment thereof 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.

[0069]

[0079] In some embodiments of the present disclosure, the VL of antibody (a) or antigen-binding fragment thereof 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 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 13) or a substantially similar sequence thereof; LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 14) or a substantially similar sequence thereof; LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (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.

[0070]

[0080] In some embodiments of the present disclosure, the VL of antibody (a) or an antigen-binding fragment thereof 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.

[0071]

[0081] In some embodiments of the present disclosure, the antibody (a) or antigen-binding fragment disclosed above is a selected humanized clone of its corresponding murine clone, which comprises heavy chain variable region complementarity determining regions, light chain variable region complementarity determining regions, and frameworks represented by the 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 clone comprises a VH-CDR1 of GYSITSDYWN (SEQ ID NO: 3) or a substantially similar sequence thereof; a VH-CDR2 of YISYTGSTYYNPSLKS (SEQ ID NO: 4) or a substantially similar sequence thereof; a VH-CDR3 of RGEWLSPFAY (SEQ ID NO: 5) or a substantially similar sequence thereof; a VL-CDR1 of KSSQSLLYSSNQKNSLA (SEQ ID NO: 10) 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; an HC-FR1 of QVQLQESGPGLAKPSQTLSLTCSVT (SEQ ID NO: 103) or a substantially similar sequence thereof; and an HC-FR2 of WIRKFPGNKLEFMG (sequence HC-FR1 is RISITRDTSKNQYYLQLNSVTTEDTATYYCAR (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 GVPDRFTGSGSGTDFTLTISSVKSEDLAVYYC (SEQ ID NO: 109) or a substantially similar sequence thereof; and LC-FR4 is FGAGTNLELK (SEQ ID NO: 110) or a substantially similar sequence thereof.

[0072]

[0082] In some embodiments of the present disclosure, the antibody (a) or antigen-binding fragment disclosed above is a selected humanized clone of its corresponding murine clone, which comprises heavy chain variable region complementarity determining regions, light chain variable region complementarity determining regions, and frameworks represented by the 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 clone 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; an HC-FR1 of EVQLQESGPGLTKPSQTLSLTCSVT (SEQ ID NO: 111) or a substantially similar sequence thereof; and an HC-FR2 of WIRKFPGNKLEYMG ( HC-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; and LC-FR4 is FGAGTSLELK (SEQ ID NO: 118) or a substantially similar sequence thereof.

[0073]

[0083] In some embodiments of the present disclosure, antibody (b) or an antigen-binding fragment thereof comprises a VH-CDR1 of SYIMM (SEQ ID NO: 17) or a substantially similar sequence thereof; a VH-CDR2 of SIYPSGGITFYADTVKG (SEQ ID NO: 18) or a substantially similar sequence thereof; a VH-CDR3 of IKLGTVTTVDY (SEQ ID NO: 19) or a substantially similar sequence thereof, a VL-CDR1 of TGTSSDVGGYNYVS (SEQ ID NO: 20) or a substantially similar sequence thereof; a VL-CDR2 of DVSNRPS (SEQ ID NO: 21) or a substantially similar sequence thereof; and a VL-CDR3 of SSYTSSSTRV (SEQ ID NO: 22) or a substantially similar sequence thereof.

[0074]

[0084] In some embodiments of the present disclosure, antibody (c) or an antigen-binding fragment thereof comprises a VH-CDR1 of MYMMM (SEQ ID NO: 23) or a substantially similar sequence thereof; a VH-CDR2 of SIYPSGGITFYADSVKG (SEQ ID NO: 24) or a substantially similar sequence thereof; a VH-CDR3 of IKLGTVTTVDY (SEQ ID NO: 25) or a substantially similar sequence thereof, a VL-CDR1 of TGTSSDVGAYNYVS (SEQ ID NO: 26) or a substantially similar sequence thereof; a VL-CDR2 of DVSNRPS (SEQ ID NO: 27) or a substantially similar sequence thereof; and a VL-CDR3 of SSYTSSSTRV (SEQ ID NO: 28) or a substantially similar sequence thereof.

[0075]

[0085] In some embodiments of the present disclosure, antibody (d) or an antigen-binding fragment thereof comprises a VH-CDR1 of SYIMM (SEQ ID NO: 29) or a substantially similar sequence thereof; a VH-CDR2 of SIYPSGGITFYAPTVKG (SEQ ID NO: 30) or a substantially similar sequence thereof; a VH-CDR3 of IKLGTVTTVDY (SEQ ID NO: 31) or a substantially similar sequence thereof, a VL-CDR1 of TGTSSDVGGYNYVS (SEQ ID NO: 32) or a substantially similar sequence thereof; a VL-CDR2 of DVSNRPS (SEQ ID NO: 33) or a substantially similar sequence thereof; and a VL-CDR3 of SSYTSSSTRV (SEQ ID NO: 34) or a substantially similar sequence thereof.

[0076]

[0086] In some embodiments of the present disclosure, the VH of antibody (d) or an antigen-binding fragment thereof 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 EVQLLESGGGLVQPGGSLRLSCAASGFTGS (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 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 37) or a substantially similar sequence thereof; and 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 an antigen-binding fragment thereof 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; and LC-FR4 is FGTGTKVTVL (SEQ ID NO: 42) or a substantially similar sequence thereof.

[0077]

[0087] In some embodiments of the present disclosure, antibody (e) or an antigen-binding fragment thereof comprises a VH-CDR1 of SYIMM (SEQ ID NO: 43) or a substantially similar sequence thereof; a VH-CDR2 of SIYPSGGITGYADTVKG (SEQ ID NO: 44) or a substantially similar sequence thereof; a VH-CDR3 of IKLGTVTTVDY (SEQ ID NO: 45) or a substantially similar sequence thereof, a VL-CDR1 of TGTSSDVGGYNYVS (SEQ ID NO: 46) or a substantially similar sequence thereof; a VL-CDR2 of DVSNRPS (SEQ ID NO: 47) or a substantially similar sequence thereof; and a VL-CDR3 of SSYTSSSTRV (SEQ ID NO: 48) or a substantially similar sequence thereof.

[0078]

[0088] In some embodiments of the present disclosure, the antibody (f) or antigen-binding fragment thereof comprises a heavy chain of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMVWRQAPGKGLEWVSSIYPSGGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVYVSS (SEQ ID NO: 49), or a substantially similar sequence thereof, and a light chain of QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLOAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 50), or a substantially similar sequence thereof.

[0079]

[0089] In some embodiments of the present disclosure, the antibody (g) or antigen-binding fragment thereof 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.

[0080]

[0090] In some embodiments of the disclosure, the antibody (h) or antigen-binding fragment thereof 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.

[0081]

[0091] In some embodiments of the present disclosure, antibody (i) or an antigen-binding fragment thereof comprises a heavy chain of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 55), or a substantially similar sequence thereof, and a light chain of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 56), or a substantially similar sequence thereof.

[0082]

[0092] In some embodiments of the present disclosure, antibody (j) or an antigen-binding fragment thereof comprises a heavy chain of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 57), or a substantially similar sequence thereof, and a light chain of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 58), or a substantially similar sequence thereof.

[0083]

[0093] In some embodiments of the present disclosure, the antibody (k) or antigen-binding fragment thereof comprises a heavy chain encoded by SEQ ID NO:59 and a light chain encoded by SEQ ID NO:60.

[0084]

[0094] In another embodiment, the antibody binds to human, mouse, or cynomolgus PD-L1. In a specific disclosure, the antibody can inhibit the interaction between human, mouse, or cynomolgus PD-L1 and the respective human, mouse, or cynomolgus PD-1 receptor.

[0085]

[0095] In another embodiment, the antibody is administered at a concentration of 5×10 -9 KD of 10 or less, preferably 2 x 10 -9 KD of 1×10 or less, and even more preferably 1×10 -9 Binds to human PD-L1 with a KD of ≤M

[0086]

[0096] Yet other embodiments relate to anti-PD-L1 antibodies, or antigen-binding fragments thereof, that bind to a functional epitope comprising residues Y56 and D61 of human PD-L1.

[0087]

[0097] In a specific embodiment, the antibody binds to a conformational epitope comprising residues 54-66 and 112-122 of human PD-L1.

[0088]

[0098] In yet other embodiments, the framework sequences are derived from human consensus framework sequences or human germline framework sequences.

[0089]

[0099] In still further embodiments, the light chain framework sequences are lambda light chain sequences.

[0090]

[0100] Constant region:

[0101] 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.

[0091]

[0102] In still further embodiments, the heavy chain variable region polypeptide, antibody, or antibody fragment further comprises at least a CH1 domain. In more specific embodiments, the heavy chain variable region polypeptide, antibody, or antibody fragment further comprises a CH1, CH2, and CH3 domain.

[0092]

[0103] In still further embodiments, the variable region light chain, antibody, or antibody fragment further comprises a CL domain.

[0093]

[0104] In still further embodiments, the antibody further comprises a CH1, a CH2, a CH3, and a CL domain.

[0094]

[0105] In some embodiments of the present disclosure, the antigen-binding proteins and peptides comprise an immunoglobulin constant region or a fragment, analog, variant, mutant, or derivative of a constant region. In preferred embodiments herein, the constant region is derived from a human immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other class. In some embodiments, the constant region comprises a CH2 domain. In other embodiments, the constant region comprises CH2 and CH3 domains, or comprises a hinge-CH2-CH3 domain. Alternatively, the constant region can comprise all or a portion of the hinge region, CH2 domain, and / or CH3 domain.

[0095]

[0106] In some embodiments, the constant region has a mutation that reduces affinity for an Fc receptor or reduces an Fc effector function. For example, the constant region can have a mutation that eliminates a glycosylation site within the constant region of an IgG heavy chain. In some embodiments herein, the constant region contains a mutation, deletion, or insertion at an amino acid position corresponding to L234, L235, G236, G237, N297, or P331 of IgG1. In a specific embodiment, the constant region contains a mutation at an amino acid position corresponding to N297 of IgG1. In alternative embodiments herein, the constant region contains a mutation, deletion, or insertion at an amino acid position corresponding to L281, L282, G283, G284, N344, or P378 of IgG1.

[0096]

[0107] In some embodiments herein, the constant region contains a CH2 domain derived from a human IgG2 or IgG4 heavy chain. Preferably, the CH2 domain has a mutation that eliminates a glycosylation site within the CH2 domain. In some embodiments, the mutation changes N in the QFNS amino acid sequence within the CH2 domain of the IgG2 or IgG4 heavy chain. Preferably, the mutation changes N to G. Alternatively, the mutation changes both F and N in the QFNS amino acid sequence. In some embodiments, the QFNS amino acid sequence is replaced with a QAQS amino acid sequence. N in the QFNS amino acid sequence corresponds to N297 of IgG1.

[0097]

[0108] In other embodiments, the constant region comprises a CH2 domain and at least a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, for example, IgG1, IgG2, IgG3, IgG4, or other class. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable class. More preferably, the hinge region is derived from a human IgG1 heavy chain. In some embodiments, the C in the PKSCDK amino acid sequence of the IgG1 hinge region is altered. In a preferred embodiment, the PKSCDK amino acid sequence is replaced with a PKSSDK amino acid sequence. In some embodiments, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In certain embodiments, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain, while the hinge region is derived from a modified human IgG1 heavy chain.

[0098]

[0109] Altering amino acids near the junction between the Fc portion and the non-Fc portion can dramatically increase the serum half-life of an Fc fusion protein (PCT Publication WO 01 / 58957). Therefore, the junction region of a protein or polypeptide of the present disclosure can contain alterations, preferably within about 10 amino acids of the junction, compared to the naturally occurring sequences of an immunoglobulin heavy chain and erythropoietin. These amino acid changes can result in increased hydrophobicity. In some embodiments, the constant region is derived from an IgG sequence in which the C-terminal K residue has been substituted. Preferably, the C-terminal K of the IgG sequence is substituted with a non-K amino acid, such as A or L, to further increase 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 has been altered to eliminate potential junctional T-cell epitopes. For example, in some embodiments, the LSLS amino acid sequence is substituted with an ATAT amino acid sequence. In other embodiments herein, amino acids within the LSLS segment are substituted with other amino acids, such as G or P. Detailed methods for making amino acid substitutions in the LSLS segment near the C-terminus of IgG1, IgG2, IgG3, IgG4, or other classes are described in US Patent Publication No. 2003 / 0166877.

[0099]

[0110] Hinge regions suitable for the present disclosure may be derived from IgG1, IgG2, IgG3, IgG4, or other classes. The IgG1 hinge region has three Cs, two of which are involved in disulfide bonds between the two heavy chains of the antibody. These same Cs allow for efficient and consistent disulfide bond formation between the Fc portions. In some embodiments herein, the first C in the human IgG1 hinge region is mutated to another amino acid, preferably S. The IgG2 isotype hinge region has four disulfide bonds, which tend to promote oligomerization and possibly incorrect disulfide bonding during secretion in recombinant systems. A suitable hinge region may be derived from an IgG2 hinge, with the first two Cs each preferably mutated to another amino acid. The hinge region of IgG4 is known to form interchain disulfide bonds inefficiently. However, a suitable hinge region for the fusion protein may preferably be derived from an IgG4 hinge region with mutations that enhance the correct formation of disulfide bonds between the heavy chain-derived moieties.

[0100]

[0111] According to the present disclosure, the constant region can contain CH2 and / or CH3 domains and hinge regions derived from different antibody isotypes, e.g., 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 derived from another IgG subclass is used in the hybrid constant region. For example, a mutant form of the IgG4 hinge that allows efficient disulfide bonding between the two heavy chains can be used. The mutant hinge can also be derived from an IgG2 hinge in which the first two Cs have been mutated to different amino acids. The assembly of such hybrid constant regions is described in U.S. Patent Application Publication No. 2003 / 0044423.

[0101]

[0112] According to the present disclosure, the constant region can have one or more mutations described herein. Combinations of mutations in the Fc portion can have additive or synergistic effects on extending the serum half-life and increasing the in vivo potency of the fusion protein. Thus, in an exemplary disclosure, the constant region can contain (i) a region derived from an IgG sequence in which the LSLS amino acid sequence is replaced with the ATAT amino acid sequence; (ii) a C-terminus in which the K residue is replaced with A; (iii) a CH2 domain and hinge region derived from a different isotype, for example, the CH2 domain can be derived from IgG2, but the hinge region is derived from IgG1; and (iv) a mutation that removes the glycosylation site in the IgG2-derived CH2 domain, for example, a substitution of the amino acid sequence QFNS to QAQS in the IgG2-derived CH2 domain.

[0102]

[0113] Antigen binding fragment:

[0114] In some embodiments of the present disclosure, the antigen-binding fragment comprises a Fab fragment, a F(ab')2 fragment, an ScFv fragment, a chimeric antibody, or a nanobody.

[0103]

[0115] Proteins and polypeptides can also include antigen-binding fragments. Exemplary antibody fragments include ScFv, Fv, Fab, F(ab')2, and single domain VHH fragments such as those of camelid origin.

[0104]

[0116] Single-chain antibody fragments, also known as single-chain antibodies (ScFv), are recombinant polypeptides that typically bind to antigens or receptors; these fragments contain at least one VH fragment connected to at least one VL fragment, with or without one or more interconnecting linkers. Such linkers may be short, flexible peptides selected to ensure proper three-dimensional folding of the VH and VL domains after they are joined, so as to maintain the target molecule binding specificity of the whole antibody from which the ScFv is derived. Generally, the C-terminus of the VH or VL sequence is covalently linked to the amino acid termini of the complementary VH and VL sequences by such a peptide linker.

[0105]

[0117] Single-chain antibody fragments contain an amino acid sequence comprising at least one CDR of a whole antibody described in this disclosure, but lack some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding but constitute a major portion of the overall antibody structure. Thus, single-chain antibody fragments can overcome some of the problems associated with the use of antibodies containing some or all of the constant domains. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and heavy-chain constant regions or other undesired biological activity. Furthermore, single-chain antibody fragments are significantly smaller than whole antibodies and therefore have greater capillary permeability than whole antibodies, which may allow single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Antibody fragments can also be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely than whole antibodies to provoke an undesired immune response in recipients.

[0106]

[0118] Fragments of antibodies may also exist that have binding characteristics that are the same as or equivalent to those of the whole antibody. Such fragments may contain either or both of the Fab or F(ab')2 fragments. Antibody fragments may contain all six CDRs of the whole antibody, although fragments containing less than all of such regions, e.g., 3, 4, or 5 CDRs, are also functional.

[0107]

[0119] Recovery immune surveillance using anti-PD-L1 antibodies

[0120] T cell inhibitory checkpoint receptors, such as CTLA-4, PD-1, BTLA, LAG-3, TIM-3, and LAIR1, are highly expressed in induced regulatory T cells and exhausted T cells. Therefore, their counterparts, such as PD-L1 (B7-H1), B7-DC, HVEM, TIM-4, B7-H3, and B7-H4, have been found to be involved in immune evasion in cancer.

[0108]

[0121] The antibody portion of the fusion protein helps target the fusion protein to the tumor and its microenvironment, where it relieves the inhibition exerted by the T cell inhibitory checkpoint. To this end, the inventors investigated the anti-tumor effects of combining a Gas6-binding moiety with both commercially available and in-house antibodies targeting PD-L1. The inventors discovered that combining the Gas6-binding moiety and anti-PD-L1 antibody in a single entity did not compromise the binding affinity or inhibitory function of either moiety in the anti-PD-L1 x Gas6 binding moiety. Furthermore, the anti-PD-L1 x Gas6 binding moiety demonstrated significant anti-tumor activity that exceeded that observed with conventional combination strategies in which the Gas6-binding moiety and anti-PD-L1 antibody were administered separately.

[0109]

[0122] Spatial conformation:

[0123] Either 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 either the N-terminus or C-terminus of a chain of an anti-PD-L1 unit. The anti-PD-L1 unit can either have a light chain and a separate heavy chain, or have the light chain and heavy chain on a single protein chain (e.g., ScFv). N-terminal fusion constructs have demonstrated relatively low productivity and significantly reduced biological activity compared to C-terminal fusion constructs. In the present disclosure, spatial conformations with preferred yield and biological function based on trial and error are shown in Figure 1, in which the Gas6-binding moiety is fused to the C-terminus of the heavy chain of an anti-PD-L1 unit comprising either a light chain and a separate heavy chain (Format 1) or a light chain and heavy chain on a single protein chain (Format 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 via a peptide linker.

[0110]

[0124] Peptide Linker:

[0125] The present disclosure includes an Axl RTK extracellular domain fused to the C-terminus of the heavy chain of an anti-PD-L1 antibody, ScFv, or fragment thereof via a peptide linker. The peptide linker, which may be an entirely artificial linker or may comprise a portion of the extracellular fragment IPPHVQKSVNNDMIVTDNNGAVKFP (SEQ ID NO: 67) N-terminal to the ectodomain, should have a minimum length. If the distance is too short, the stability or activity of the fusion protein will decrease. 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 more 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.

[0111]

[0126] The inclusion of a flexible linker, e.g., one or more GGGGS (SEQ ID NO: 68) units, can be useful for the stability and / or activity of BfAbs in some embodiments. 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 repeats of SEQ ID NO: 68.

[0112]

[0127] In some embodiments, it is shown that the native N-terminal fragment (SEQ ID NO:67) can be replaced with a replacement peptide to increase stability without sacrificing activity or even improving activity. In some embodiments, the replacement peptide differs from SEQ ID NO:67 but has at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO:67.

[0113]

[0128] An example of a replacement peptide is IPPHVQXXVNNDMIVTDNXGAVKFP (SEQ ID NO: 69), where X is any amino acid except K, S, or N. In some embodiments, substitutions can be made to remove the rigid dipeptide PP, removing potential cleavage sites QK, N, and / or K, including multiple glycine residues to increase flexibility and / or reducing hydrophobic residues. 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 to SEQ ID NO: 70. In some embodiments, the variant contains at least four Gs, zero PP dipeptides, and no more than three hydrophobic amino acid residues selected from the group consisting of I, L, M, F, V, W, Y, and P. In some embodiments, the variant comprises at least five Gs and no more than one hydrophobic amino acid residue selected from the group consisting of I, L, M, F, V, W, Y, and P.

[0114]

[0129] In some embodiments, the peptide linker comprises a replacement peptide of SEQ ID NO: 67. In some embodiments, the peptide linker comprises both a flexible linker and a replacement peptide. In some embodiments, the flexible linker is N-terminal to the replacement peptide. In some embodiments, the flexible linker is C-terminal to the replacement peptide.

[0115]

[0130] In some embodiments, the fusion protein does not include at least the entire sequence of EEYNTSNPD (SEQ ID NO: 71). The fusion protein may have all of SEQ ID NO: 71 removed from the extracellular domain of the Gas6 binding moiety. 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.

[0116]

[0131] method:

[0132] Fusion proteins can be produced by any suitable means known in the art or hereafter discovered, for example, they can be produced from eukaryotic or prokaryotic cells, or they can be synthesized in vitro. If the protein is produced by a prokaryotic cell, it can be further treated by unfolding, e.g., heat denaturation, DTT reduction, etc., and further refolded using methods known in the art.

[0117]

[0133] Methods for producing 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 constant region function through genetic engineering techniques. The antibodies of the 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 produced using techniques such as those described herein. For example, fully human antibodies 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 whose endogenous gene locus has been disabled. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent Nos. 6,150,584; 6,458,592; and 6,420,140.

[0118]

[0134] Polypeptides can be prepared by in vitro synthesis using conventional methods, including molecular cloning, antibody phage display libraries, or similar techniques. A variety of commercially available synthesis equipment is available. The use of synthesizers allows for the substitution of unnatural amino acids for naturally occurring amino acids. The particular sequence and mode of preparation will be determined by convenience, economy, required purity, etc.

[0119]

[0135] The polypeptides can also be isolated and purified according to conventional methods for recombinant proteins: a lysate can be prepared from the expression host and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques.

[0120]

[0136] Methods well known to those skilled in the art can be used to construct expression vectors containing a coding sequence and appropriate transcriptional / translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Alternatively, RNA capable of encoding a polypeptide of interest can be chemically synthesized. Direct chemical synthesis methods include, for example, the phosphotriester method, the diethyl phosphoramidite method, and the solid support method. Chemical synthesis produces single-stranded oligonucleotides. This single-stranded oligonucleotide can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Chemical synthesis of DNA is often limited to sequences of approximately 100 bases, but longer sequences can be obtained by ligating shorter sequences. Alternatively, subsequences can be cloned and cleaved using appropriate restriction enzymes.

[0121]

[0137] Nucleic acids can be isolated and obtained with substantial purity. The nucleic acids of the present disclosure can be provided as linear molecules or contained within circular molecules, and can be provided within molecules capable of self-replicating or without replication sequences. Expression of the nucleic acid can be regulated by the nucleic acid itself or by other regulatory sequences known in the art. Nucleic acids for fusion proteins can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin-polycation-mediated DNA transfer, transfection using naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular delivery of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene guns, calcium phosphate-mediated transfection, etc.

[0122]

[0138] Therapeutic indications:

[0139] The present disclosure further provides a method for treating, prophylactically treating, and / or preventing cancer in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein disclosed herein.

[0123]

[0140] The present disclosure further provides a method for neutralizing Gas6 in a sample, the method comprising contacting the sample with a fusion protein disclosed herein.

[0124]

[0141] As described herein, antibodies, variants, or derivatives of the anti-PD-L1xGas6 binding moiety may be used in certain therapeutic and diagnostic methods.

[0125]

[0142] The fusion proteins are further directed to multifunctional molecule or antibody-based therapies, including 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 disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0126]

[0143] The fusion protein is a method for treating cancer in a patient in need thereof. The method, in one embodiment, involves administering to the patient an effective amount of a fusion protein of the present disclosure. In some embodiments, at least one of the cancer cells or surrounding cells in the patient's tumor microenvironment expresses, overexpresses, or is induced to express PD-L1 and / or Gas6 or Axl. Induction of PD-L1, Gas6, or Axl expression can be achieved, for example, by administration of a tumor vaccine or radiation therapy.

[0127]

[0144] Tumors that express the PD-L1 protein include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral 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 may benefit from the Gas6 / Axl axis include lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, glioma, melanoma, prostate cancer, breast cancer, gastric cancer, colorectal cancer, osteosarcoma, renal cell carcinoma, and thyroid cancer. Furthermore, because the Gas6-binding moieties of the present disclosure act as ligand traps for Gas6, the compositions and methods of the present disclosure are useful for treating any cancer in which Axl is expressed, and potentially any cancer in which MerTK and / or Tyro-3 are expressed.

[0128]

[0145] Accordingly, the anti-PD-L1×Gas6 binding moieties of the present disclosure can be used to treat any one or more such cancers. Additional diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed using the fusion proteins, or variants or derivatives thereof, include malignancies and related disorders such as leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, as well as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, and the like. These include, but are not limited to, progression and / or metastasis of solid tumors, including, but not limited to, sarcomas and carcinomas such as 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, germ cell carcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, epithelial carcinoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

[0129]

[0146] Pharmaceutical Composition:

[0147] The present disclosure further provides a pharmaceutical composition comprising an effective amount of a fusion protein disclosed herein or a genetically engineered cell disclosed herein and a pharmaceutically acceptable carrier.

[0130]

[0148] The pharmaceutical compositions of the present disclosure are formulated with appropriate diluents, carriers, excipients, and other agents that provide improved mobility, delivery, tolerance, etc. The compositions can be formulated for a particular use, such as veterinary use or human pharmaceutical use. The form of the composition and the excipients, diluents, and / or carriers used will depend on the intended use of the antibody and, in the case of therapeutic applications, the mode of administration. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, California), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0131]

[0149] The dose of the fusion protein administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. When the antibodies of the present disclosure are used to treat a PD-L1-associated condition or disease in an adult patient, it may be advantageous to administer the antibodies of the present disclosure intravenously. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering antibodies can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation, and the dose can be adjusted accordingly. Furthermore, scaling of dosages between species can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0132]

[0150] Various delivery systems are known 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, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction 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, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0133]

[0151] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is readily disclosed for delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. After all of the pharmaceutical composition in 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 delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. After the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0134]

[0152] In some situations, pharmaceutical compositions can be delivered in a sustained 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, a polymeric material can be used; see Medical Disclosure of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a sustained release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Disclosure of Controlled Release, supra, Vol. 2, pp. 115-138). Other sustained release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0135]

[0153] Injectable formulations include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like. These can be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (hydrogenated castor oil polyoxyethylene (50 mol) adduct)]. Oily media include, for example, sesame oil and soybean oil, and can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable formulations prepared in this manner are preferably filled into appropriate ampoules.

[0136]

[0154] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a dosage form of a unit dose adapted to fit the dose of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0137]

[0155] detection:

[0156] The present disclosure further provides a method of detecting cancer in a subject in need thereof, comprising contacting a sample from the subject with a fusion protein disclosed herein.

[0138]

[0157] The present disclosure further provides a kit for detecting cancer in a sample, comprising a fusion protein according to the present invention.

[0139]

[0158] The present disclosure further provides a method of detecting PD-L1 in a sample, the method comprising contacting the sample with a fusion protein disclosed herein.

[0140]

[0159] Fusion proteins of the present disclosure can also be used to detect and / or measure PD-L1 or PD-L1-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-PD-L1 antibody or antigen-binding fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression of PD-L1 (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for PD-L1 can include, for example, contacting a sample obtained from a patient with an anti-PD-L1 antibody of the present disclosure, where the anti-PD-L1 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-PD-L1 antibody can be used in diagnostic assays in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The PD-L1 may be a radioisotope such as I; 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).

[0141]

[0160] The following examples are provided to aid those skilled in the art in practicing the present disclosure. [Example]

[0142]

[0162] The following example illustrates the development and use of anti-PD-L1xGas6 binding moieties to inhibit tumor growth by inducing a "hot tumor" microenvironment.

[0143]

[0163] Example 1

[0164] [Generation of recombinant DNA]

[0165] Prior to generating recombinant DNA, in silico simulation of the sequence was performed using SnapGene software. Recombinant DNAs combining either anti-PD-L1 or anti-PD-L1 (ScFv+Fc) (antigen-binding moiety) with Gas6 trap (Gas6-binding moiety) were constructed in different spatial combinations. The constructs were generated by polymerase chain reaction (PCR)-based cloning and plasmid cloning. This process is briefly described as follows: In PCR-based cloning, the insert DNA was amplified by PCR. To digest the DNA, the PCR product and recipient plasmid were incubated with specific restriction enzymes for at least 4 hours and overnight, respectively. After applying phosphatase to prevent the recipient plasmid from recircularizing, the digested DNA was run on an agarose gel and gel-purified to isolate DNA of the expected size. The insert DNA was fused to the recipient plasmid using T4 DNA ligase. The PCR product in the above process was replaced with the donor plasmid in the plasmid cloning step.

[0144]

[0166] The spatial arrangement and primer sequences used for PCR-based cloning in each format are described below. Format 1 has a Gas6 trap at the C-terminus of anti-PD-L1. Anti-PD-L1 lacking a stop codon is followed by a Gas6 trap containing a stop codon. Primer sequence 1 and primer sequence 2 were used to generate this format.

[0145]

[0167] A Gas6 trap was constructed with anti-PD-L1 Fc (anti-PD-L1-Fc) to generate a Gas6 trap-Fc-based plasmid. Anti-PD-L1 ScFv (anti-PD-L1-ScFv) was then introduced into the Gas6 trap-Fc-based plasmid to generate four conformations. Format 2 has a Gas6 trap at the C-terminus of the anti-PD-L1-ScFv. The anti-PD-L1-ScFv was first introduced into the region of anti-PD-L1-Fc encoding the N-terminus of anti-PD-L1-Fc. Primer sequence 3 was used to generate inserted anti-PD-L1-scFc. Primer sequence 4 and primer sequence 5 were then used to tag the Gas6 trap onto the end of anti-PD-L1-Fc lacking a stop codon.

[0146]

[0168] The steric optimization was further applied to Format 1 and Format 2. In Format 1, recombinant DNA was modified with anti-PD-L1 conjugated to a Gas6 trap at the C-terminus of the heavy chain constant region, and pTCAE8.3 plasmid was used as the recipient plasmid. The sequences encoding the anti-PD-L1 heavy chain variable region and Gas6 trap in the recombinant DNA were replaced. Primer Sequence 6 and Primer Sequence 7 were used to generate the insert DNA for this process. In Format 2, recombinant DNA was modified with anti-PD-L1-ScFv followed by anti-PD-L1-Fc lacking a stop codon and a Gas6 trap, while pTCAE8.3 plasmid was used as the recipient plasmid. The sequence encoding the Gas6 trap in this recombinant DNA was replaced. Primer Sequence 8 and Primer Sequence 9 were used to generate the insert DNA for this process.

[0147]

[0169] [Table 1]

[0148]

[0170] [Amplification and verification of recombinant DNA]

[0171] The recombinant DNA was mixed with DH5α on ice for 20 minutes and then transformed into DH5α by incubation at 42°C for 45 seconds. The transformed DH5α was then incubated in LB medium at 37°C for 1 hour and then uniformly plated onto LB agar plates containing 50 μg / mL ampicillin. The LB agar plates were incubated at 37°C for 16 hours, after which candidate clones were selected. The candidate clones were then grown, and their recombinant DNA was extracted and tested by diagnostic restriction digestion and DNA sequencing.

[0149]

[0172] [Expression of target Ab]

[0173] Mammalian expression systems were used to produce the Abs of interest. Briefly, recombinant DNA was transfected into mammalian cells using chemicals, lipids, or physical engagement. A drug resistance marker incorporated into the recombinant DNA was used to distinguish mammalian cells that had taken up the recombinant DNA from non-transfected cells. A scaled-up methotrexate or methionine sulfoximine selection system was used to select for highly proliferative mammalian cells that had integrated the recombinant DNA.

[0150]

[0174] [Purification of target Ab]

[0175] The target Ab fused to GFP was purified by affinity column chromatography. Briefly, the anti-GFP mAb was dialyzed against coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) and then blended with the resin in a stoppered container. The mixture was then rotated upside down at room temperature for 2 hours. After washing away excess anti-GFP mAb, the remaining active groups were treated with blocking buffer (0.1 M Tris-HCl buffer, pH 8.0). The anti-GFP mAb-containing resin was finally packed into a column. To purify the target Ab, the lysate supernatant was loaded onto an anti-GFP mAb-bound affinity chromatography column. The column was then washed with binding buffer (10 column volumes, PBS, 0.13 M NaCl, 0.01 M Na2HPO4, 0.01 M NaH2PO4, pH 7.4) and eluted with elution buffer (0.1 M glycine-HCl, pH 4.5). The eluted fractions were immediately neutralized by adding a small amount of neutralization buffer (1 M Tris-HCl, pH 9.0) and then dialyzed (0.15 M PBS, pH 7.4).

[0151]

[0176] [Analysis of target Abs]

[0177] The purified Abs were analyzed by discontinuous 12% SDS-PAGE and Coomassie Brilliant Blue R250 staining. Furthermore, size-exclusion chromatography coupled with dynamic light scattering technology was used to detect aggregation and denaturation of the Abs with high resolution. Purification and yield were estimated by determining protein content using the biosoftware Bandscan 5.0.

[0152]

[0178] The inventors investigated various spatial conformations. The Gas6 trap can be fused to either the light or heavy chain of an anti-PD-L1 antibody or its PD-L1-binding fragment. The fused Gas6 trap can be fused to either the N-terminus or C-terminus of the chain of the anti-PD-L1 unit. The anti-PD-L1 unit can either have a light chain and a separate heavy chain, or the light chain and heavy chain on a single protein chain (e.g., ScFv). The inventors unexpectedly found that N-terminal fusion constructs exhibit relatively low productivity and significantly reduced biological activity compared with C-terminal fusion constructs. Based on trial and error, spatial conformations with preferred yields and biological functions in the present invention are shown in Figure 1, in which the Gas6 trap is fused to the C-terminus of the heavy chain of an anti-PD-L1 unit containing either a light chain and a separate heavy chain (Format 1) or the light chain and heavy chain on a single protein chain (Format 2). Exemplary bifunctional molecules provided herein include bifunctional molecules belonging to Format 1 and Format 2. Exemplary bifunctional molecules belonging to Format 1 are Examples 1, 3, 4, 5, and 6, and exemplary bifunctional molecules belonging to Format 2 are Examples 2, 7, 8, and 9.

[0153]

[0179] In Examples 1 and 2, antibody (j) or an antigen-binding fragment thereof 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 Examples 3, 4, and 7, antibody (a) or an antigen-binding fragment thereof comprises a VH-CDR1 of SEQ ID NO: 3 or a substantially similar sequence thereof, a VH-CDR2 of SEQ ID NO: 4 or a substantially similar sequence thereof, a VH-CDR3 of SEQ ID NO: 5 or a substantially similar sequence thereof, a VL-CDR1 of SEQ ID NO: 10 or a substantially similar sequence thereof, a VL-CDR2 of SEQ ID NO: 11 or a substantially similar sequence thereof, and a VL-CDR3 of SEQ ID NO: 12 or a substantially similar sequence thereof. In Examples 5, 6, 8, and 9, antibody (a) or an antigen-binding fragment thereof comprises a VH-CDR1 of SEQ ID NO: 96 or a sequence substantially similar thereto, a VH-CDR2 of SEQ ID NO: 97 or a sequence substantially similar thereto, a VH-CDR3 of SEQ ID NO: 98 or a sequence substantially similar thereto, a VL-CDR1 of SEQ ID NO: 99 or a sequence substantially similar thereto, a VL-CDR2 of SEQ ID NO: 11 or a sequence substantially similar thereto, and a VL-CDR3 of SEQ ID NO: 12 or a sequence substantially similar thereto. In Examples 3, 5, 7, and 8, the VH of antibody (a) or an antigen-binding fragment thereof comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4). In antibody (a) or an antigen-binding fragment thereof, HC-FR1 is SEQ ID NO: 6 or a sequence substantially similar thereto, HC-FR2 is SEQ ID NO: 7 or a sequence substantially similar thereto, HC-FR3 is SEQ ID NO: 8 or a sequence substantially similar thereto, and HC-FR4 is SEQ ID NO: 9 or a sequence substantially similar thereto. The VL of antibody (a) or an antigen-binding fragment thereof comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4).In antibody (a) or an antigen-binding fragment thereof, LC-FR1 is SEQ ID NO: 13 or a sequence substantially similar thereto, LC-FR2 is SEQ ID NO: 14 or a sequence substantially similar thereto, LC-FR3 is SEQ ID NO: 15 or a sequence substantially similar thereto, and LC-FR4 is SEQ ID NO: 16 or a sequence substantially similar thereto. In Examples 4, 6, and 9, the VH of antibody (a) or an antigen-binding fragment thereof comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4). In antibody (a) or an antigen-binding fragment thereof, HC-FR1 is SEQ ID NO: 6 or a sequence substantially similar thereto, HC-FR2 is SEQ ID NO: 7 or a sequence substantially similar thereto, HC-FR3 is SEQ ID NO: 8 or a sequence substantially similar thereto, and HC-FR4 is SEQ ID NO: 9 or a sequence substantially similar thereto. The VL of antibody (a) or an antigen-binding fragment thereof comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4). In antibody (a) or an antigen-binding fragment thereof, LC-FR1 is SEQ ID NO: 100 or a sequence substantially similar thereto, LC-FR2 is SEQ ID NO: 101 or a sequence substantially similar thereto, LC-FR3 is SEQ ID NO: 102 or a sequence substantially similar thereto, and LC-FR4 is SEQ ID NO: 16 or a sequence substantially similar thereto.

[0154]

[0180] Example 2

[0181] [PD-L1 binding affinity]

[0182] The binding capacity of the bifunctional molecules and their parent mAbs was examined by FACS and ELISA. Recombinant human PD-L1 protein mixed with coating solution (Seracare, 5150-0014) at a concentration of 100 ng / well was uniformly plated onto a 96-well plate. The 96-well plate was incubated overnight at 4°C. The next day, the plate was washed between different reagent application steps using phosphate-buffered saline (PBS) containing 0.05% Tween 20. After discarding the coating Ab, the plate was blocked with 3% skim milk in PBS at 37°C for 1 hour. Then, after discarding the blocking buffer, serially diluted Abs of interest in PBS were applied at 37°C for 1 hour. A secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody, was applied at 37°C for 1 hour after discarding the Abs of interest. Finally, color was developed by oxidation with 3,3',5,5'-tetramethylbenzidine (TMB). The HRP-driven oxidation of TMB was then quenched with 1N hydrochloride. The optical density (OD) readings at 450 / 650 nm were used to quantify the PD-L1 binding affinities of the bifunctional molecules and parent Abs. The hPD-L1 binding affinities of the BfAbs are shown in Table 2, and the conformations of the bifunctional molecules of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 did not impede their hPD-L1 binding affinities.

[0155]

[0183] [Table 2]

[0156]

[0184] [Gas6 binding affinity]

[0185] The target Abs mixed with the coating solution at a concentration of 100 ng / well were uniformly plated onto a 96-well plate. The 96-well plate was incubated overnight at 4°C. The next day, the plate was washed between different reagent application steps with phosphate-buffered saline (PBS) containing 0.05% Tween 20. After discarding the coating Abs, the plate was blocked with PBS containing 3% skim milk for 1 h at 37°C. After discarding the blocking buffer, serially diluted human Gas6 recombinant protein (hGas6) at a concentration of 100 μg / well in PBS was applied for 1 h at 37°C. After discarding the Gas6, a secondary antibody, HRP-conjugated goat anti-human IgG antibody, was applied for 1 h at 37°C. Finally, color development was achieved by the oxidation of TMB. The HRP-driven oxidation of TMB was then stopped with 1 N hydrochloride. The OD at 450 / 650 nm was read to quantify the Gas6 binding affinity of the bifunctional molecules and the reference Ab.

[0157]

[0186] The hGas6 binding affinities of the parent bifunctional molecule, Axl-Fc (which serves as the Gas6-binding moiety, with the C-terminus of the Gas6 trap 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, and the conformations of the bifunctional molecules of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 did not impair their hGas6 binding affinities.

[0158]

[0187] [Table 3]

[0159]

[0188] [Table 4]

[0160]

[0189] Example 3

[0190] [PD-L1 / PD-1 inhibitors]

[0191] A bioassay kit (Promega, J1252) was used to quantify the PD-1 / PD-L1 inhibitory function of the bifunctional molecules and reference Abs. Briefly, PD-L1 aAPC / CHO-K1 cells were plated onto a 96-well plate at 4 × 10 4 The plates were then incubated overnight at 37°C. The next day, serially diluted Abs of interest and PD-1 effector cells were added at a concentration of 5 × 10 4 The cells were plated at a concentration of 1000 μg / well and incubated at 37°C for 6 hours. Finally, we examined T cell receptor (TCR)- and nuclear factor of activated T cells (NFAT)-mediated activation of luciferase activity in effector cells. TCR / NFAT signaling was inhibited by PD-L1 / PD-1 signaling. Therefore, the higher the luminescence value of effector cells, the more effectively the Ab inhibited PD-L1 / PD-1 signaling.

[0161]

[0192] The PD-L1 / PD-1 inhibitory activities of the bifunctional molecules are shown in Table 5, and the three-dimensional structures of the bifunctional molecules of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 did not impair their PD-L1 / PD-1 inhibitory activities.

[0162]

[0193] [Table 5]

[0163]

[0194] [Gas6 / Axl inhibition]

[0195] The human Axl-expressing Ba / F3 cell line (Ba / F3-hAxl) was used to quantify the Gas6 / Axl inhibitory functions of BfAbs and reference Abs. A mixture consisting of Ba / F3-hAxl at a concentration of 2000 cells / well, 10% fetal bovine serum, and 100 μg / well hGas6 was uniformly plated onto a 96-well plate. The Ab of interest was then added to the plate at a concentration of 5 ng / well. The plate was then incubated at 37°C for 72 hours. The cell viability of Ba / F3-hAxl was examined using CCK-8 reagent (Dojindo, CK04), and the OD at 450 nm was finally read.

[0164]

[0196] The Gas6 / Axl inhibitory activity of the bifunctional molecules and their parent Axl-Fc is shown in Figures 2, 3, and 4, and the bifunctional conformations of Examples 1, 2, 3, 4, 5, 6, 7, 8, and 9 showed no impairment to their Gas6 / Axl inhibitory activity.

[0165]

[0197] Example 4

[0198] [Cancer immunotherapy]

[0199] EMT-6 (a mouse triple-negative breast cancer cell line that spontaneously secretes Gas6) was subcutaneously inoculated into female BALB / cByJNarl mice. Drug administration was performed at 50-100 mmHg. 3The mice were started at an average tumor size of 1000 mg / kg. Five treatment groups, including hIgG isotype control, atezolizumab, Axl-Fc, atezolizumab combined with Axl-Fc, and Example 2, were intraperitoneally injected twice weekly for three weeks at a dose level of 10 mg / kg (the actual treatment dosage was adjusted according to their respective molecular weights). Body weight and tumor volume were measured three to four times weekly. Blood samples were collected three hours after drug administration. The in vivo experiments were reviewed and approved by the Development Center for Biotechnology Laboratories Institutional Animal Care and Use Committee and were conducted in accordance with the Guide for Care and Use of Laboratory Animals.

[0166]

[0200] The tumor growth inhibitory activity of Example 2 and its parent Ab, as well as its Axl-Fc, is shown in Figure 5. The parent Ab and its Axl-Fc were administered as single agents or in combination. As shown in Figure 5, Example 2 was unexpectedly superior in tumor growth inhibitory activity to the parent Ab and its Axl-Fc administered as single agents or in combination. In the parent Ab group and the parent Ab and Axl-Fc combination group, 3 of 6 mice showed tumor growth of 500 mm 3 In group 2, only one of six mice had a tumor volume of 500 mm 3 Compared with the parental Ab, a higher complete remission rate was observed in the Example 2 group, with 1 of 6 mice achieving complete remission in the parental Ab group and 2 of 6 mice achieving complete remission in the Example 2 group.

[0167]

[0201] Serum Gas6 concentrations in mice were measured (FIG. 6). Serum Gas6 concentrations in mice were reduced when molecules containing Gas6-binding moieties were administered, demonstrating the impressive in vivo functionality of the Gas6-binding moieties disclosed in the present disclosure.

[0168]

[0202] Mice that achieved complete remission were further enrolled in a rechallenge study to examine the immune memory effect of Example 2 and its parent Ab. Briefly, the same tumor cell line (EMT-6) was subcutaneously inoculated into mice, and complete remission was achieved the first time. Tumor inoculation was performed on the contralateral side of the mice during rechallenge. Body weight and tumor volume were then measured 3-4 times a week. The results shown in Figure 7 conclusively demonstrate that an immune memory effect was observed in both Example 2 and its parent Ab. Nevertheless, Example 2 exhibits a slightly better immune memory response than its parent Ab.

[0169]

[0203] The tumor growth inhibitory function of the present invention was confirmed in an mPD-L1-hPD-L1+EMT-6 syngeneic mouse model in another embodiment, Example 3. Compared with the vehicle group, the Example 3 group showed promising anti-cancer effects of the present invention in Figure 8.

[0170]

[0204] The present disclosure provides bifunctional fusion proteins with novel compositions, which comprise two binding moieties: an anti-PD-L1 antibody or PD-L1-binding fragment fused at its C-terminus to a Gas6-binding moiety. Different spatial conformations of the bifunctional fusion proteins were explored. As supported by the examples in this disclosure, selected anti-PD-L1 x Gas6 Trap bifunctional conformations, Format 1 and Format 2, disclosed herein can be applied to anti-PD-L1 antibodies or PD-L1-binding fragments with different sequences. Furthermore, anti-PD-L1 x Gas6 Trap bifunctional fusion proteins in Format 1 or Format 2 showed comparable binding affinity and functionality to both parent binding moieties. More importantly, the anti-PD-L1 x Gas6 Trap bifunctional fusion proteins disclosed herein unexpectedly demonstrated synergistic anti-tumor effects, with selected anti-PD-L1 x Gas6 Trap bifunctional conformations demonstrating superior tumor growth inhibitory activity to the parent binding moieties administered as single agents or in combination. The superior tumor targeting effect of the anti-PD-L1×Gas6-Trap bifunctional fusion protein relative to the combination of the two binding moieties as single agents is partly responsible. As suggested by the difference in binding affinity between the anti-PD-L1 antibody or PD-L1-binding fragment and the Gas6-Trap binding moiety for their corresponding targets, the anti-PD-L1 antibody or PD-L1-binding fragment dominated the tumor targeting effect of the anti-PD-L1×Gas6-Trap fusion protein in this disclosure.

[0171]

[0205] While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and variations will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are intended to fall within the scope of the present invention.

Claims

1. a Gas6 (growth arrest specific 6) binding moiety; and and an antigen-binding portion comprising an antibody or antigen-binding fragment thereof specific for an epitope in PD-L1 (programmed death ligand 1).

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

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

6. the VH of the antibody (a) or an antigen-binding fragment thereof comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4), wherein the HC-FR1 is SEQ ID NO: 6 or a sequence substantially similar thereto, the HC-FR2 is SEQ ID NO: 7 or a sequence substantially similar thereto, the HC-FR3 is SEQ ID NO: 8 or a sequence substantially similar thereto, and the HC-FR4 is SEQ ID NO: 9 or a sequence substantially similar thereto; 5. The fusion protein of claim 4, wherein the VL of the antigen-binding fragment comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4), wherein the LC-FR1 is SEQ ID NO: 100 or a sequence substantially similar thereto, the LC-FR2 is SEQ ID NO: 101 or a sequence substantially similar thereto, the LC-FR3 is SEQ ID NO: 102 or a sequence substantially similar thereto, and the LC-FR4 is SEQ ID NO: 16 or a sequence substantially similar thereto.

7. the VH of the antibody (d) or antigen-binding fragment thereof comprises a framework represented by the formula: (HC-FR1)-(VH-CDR1)-(HC-FR2)-(VH-CDR2)-(HC-FR3)-(VH-CDR3)-(HC-FR4), wherein the HC-FR1 is SEQ ID NO: 35 or a sequence substantially similar thereto, the HC-FR2 is SEQ ID NO: 36 or a sequence substantially similar thereto, the HC-FR3 is SEQ ID NO: 37 or a sequence substantially similar thereto, and the HC-FR4 is SEQ ID NO: 38 or a sequence substantially similar thereto; 5. The fusion protein of claim 4, wherein the VL of the antigen-binding fragment thereof comprises a framework represented by the formula: (LC-FR1)-(VL-CDR1)-(LC-FR2)-(VL-CDR2)-(LC-FR3)-(VL-CDR3)-(LC-FR4), wherein the LC-FR1 is SEQ ID NO: 39 or a sequence substantially similar thereto, the LC-FR2 is SEQ ID NO: 40 or a sequence substantially similar thereto, the LC-FR3 is SEQ ID NO: 41 or a sequence substantially similar thereto, and the LC-FR4 is SEQ ID NO: 42 or a sequence substantially similar thereto.

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

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

10. The antigen-binding portion may be a Fab fragment, F(ab') ２ The fusion protein of claim 1 , comprising a fragment, an ScFv fragment, a chimeric antibody, or a nanobody.

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

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

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

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

16. an effective amount of the fusion protein of any one of claims 1 to 15; and a pharmaceutically acceptable carrier.

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

18. 18. The method of 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. 16. A method for detecting cancer in a subject in need thereof, comprising contacting a sample derived from said subject with the fusion protein of any one of claims 1 to 15.

20. 20. The method of 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 of any one of claims 1 to 15.

22. 22. The kit of 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. 16. A method for detecting PD-L1 in a sample, the method comprising contacting the sample with the fusion protein of any one of claims 1 to 15.

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

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