Compositions of LANCL1 antibodies and methods of their use in treatment of liver cancer

Abstract

Compositions comprising an anti-LANCL1 antibody, functional variants thereof, and methods of use thereof are disclosed. An exemplary anti-LANCL1 antibody has a heavy chain variable region containing CDR SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 8, and a light chain variable region containing CDR SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24, respectively. Another exemplary anti-LANCL1 antibody has heavy chain variable regions respectively containing CDR SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and light chain variable regions respectively containing CDR SEQ ID NO: 22, SEQ ID NO: 73 and SEQ ID NO: 25. The antibody may be a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody. The disclosed compositions may be used to treat a subject in need thereof, such as a subject diagnosed with liver cancer or at risk of developing liver cancer, such as HCC.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 696,454, filed September 19, 2024, which application is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present invention is generally in the field of liver cancer, and specifically in the field of therapeutic antibodies for treating liver cancer. BACKGROUND

[0004] Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide, and more specifically, the second and third leading cause of cancer death in China and Hong Kong, respectively. Despite surgical resection and liver transplantation being available, most HCCs are diagnosed at an advanced stage and thus are inoperable. The median survival of inoperable HCC patients is only a few weeks. Even with surgery, the tumor recurrence rate is high. Even after surgical resection of the cancer, the 5-year survival rate of HCC patients is only about 20%. The high mortality rate of HCC is attributed in part to its aggressive behavior and the lack of promising curative therapies. HCC has well-known resistance to traditional chemotherapy, and thus treatment options for advanced HCC are limited.

[0005] Tumor initiation is critical in both liver carcinogenesis and tumor recurrence derived from post-treatment residual tumor cell populations. Targeting cell surface proteins responsible for tumor initiation is a potential strategy for cancer treatment. Existing cell surface proteins that promote liver tumor initiation include CD13, CD24, CD47, CD90, CD133, and EpCAM. However, these proteins are limited in their application for transformation. For example, the expression of some of these proteins, such as CD90, is low, whether in very low percentage of positive cells in tumors or in very few patient samples showing detectable expression, suggesting that the proteins play a minor role in HCC initiation in certain patient cohorts. Moreover, certain surface proteins are not only detectable in HCC tumors, but also at high levels in non-tumor liver tissue. Targeting these proteins can have non-specific cytotoxic effects on surrounding non-tumor liver tissue. Thus, there is an urgent need for more effective therapies to precisely target cell surface proteins for treatment of HCC.

[0006] It is an object of the present invention to provide monoclonal antibodies that specifically bind to a LanC-like glutathione S-transferase 1 (LANCL1) protein or a portion of a human LANCL1 protein.

[0007] It is another object of the present invention to provide compositions for treating HCC in a subject in need thereof.

[0008] It is a further object of the present application to provide methods for treating HCC in a subject.

[0009] It is yet another object of the present application to provide methods for inhibiting HCC tumor growth in a subject in need thereof.

[0010] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed anywhere before the priority date of each claim of this application.

[0011] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. SUMMARY

[0012] Liver tumor initiating cells (LTIC) are a subpopulation of self-renewing cells in hepatocellular carcinoma (HCC) that contribute to tumor initiation and can be viewed as the cause of HCC therapeutic resistance (Yi, et al., Cancer Treat Rev., 39:290-296 (2013); Visvader and Lindeman, Nat Rev Cancer, 8:755-768 (2008); Tirino et al., FASEB J., 27: 13-24 (2013))). LTIC are marked by unique cell surface markers that are functional and capable of activating downstream cellular signaling to exert their effects. The problem is that existing LTIC markers are limited in their ability to translate into applications, thereby hindering the development of therapeutic applications against LTIC for cancer therapy. The disclosed compositions and methods solve this problem by providing human monoclonal antibodies that reduce LTIC surface marker activity and reduce downstream signaling pathways triggered by these surface markers, thereby reducing hepatocellular carcinoma initiation.

[0013] It is recognized that developing human monoclonal antibodies that target and inhibit LanC-like glutathione S-transferase 1 (LANCL1), a LTIC marker that is commonly found in hepatocellular carcinoma patients, is useful for reducing HCC cell proliferation, reducing HCC development, and / or increasing survival rates in liver cancer patients. To date, all available antibodies against LANCL1 are only capable of use in Western blot, immunohistochemistry, or immunofluorescence applications. None of the existing available LANCL1 antibodies possess cancer therapeutic capabilities.

[0014] Disclosed are antibodies or antigen-binding fragments thereof that immunospecifically bind to a surface or transmembrane unit of a human LanC-like glutathione S-transferase 1 (LANCL1) protein and are capable of blocking or reducing the activity of LANCL1 in vitro or in a recipient subject or patient. These antibodies and other molecules generally comprise six complementarity determining regions (CDRs). In preferred forms, the antibodies and other molecules immunospecifically bind to the N-terminal extracellular region of the amino acid sequence SEQ ID NO: 1. In some forms, the N-terminal extracellular region of LANCL1 has the amino acid sequence SEQ ID NO: 2.

[0015] The anti-LANCL1 antibodies contain a heavy chain variable region having three complementarity determining regions (CDRs) and a light chain variable region having three CDRs. In one particular form, the three heavy chain variable region CDRs comprise DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), S GDGYYFAS (SEQ ID NO: 8), respectively, and the three light chain variable region CDRs contain RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), respectively. In another particular form, the three heavy chain variable region CDRs comprise DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9), respectively, and the three light chain variable region CDRs contain RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25), respectively.

[0016] The disclosed subject matter can also relate to chimeric and / or humanized antibodies and fragments, or human antibodies and fragments. Most preferably, such molecules will have sufficient affinity and avidity to be capable of binding to extracellular LANCL1 when present in a subject’s body.

[0017] Also provided are nucleic acids encoding the antibodies or antigen-binding fragments thereof.

[0018] Also disclosed are pharmaceutical compositions containing the anti-LANCL1 antibodies.

[0019] Also disclosed are methods for treating a subject in need thereof (e.g., a subject diagnosed with a liver cancer, such as hepatocellular carcinoma (HCC), or a subject at risk of developing a liver cancer, such as HCC).

[0020] The methods generally comprise administering to a subject a pharmaceutical composition containing an effective amount of an anti-LANCLl antibody. In some forms, the pharmaceutical composition is effective to reduce proliferation of tumor initiating cells, sphere formation of hepatocellular carcinoma cells, and / or block or reduce activity of LANCL1.

[0021] Additional advantages of the disclosed methods and compositions will be set forth in part in the description that follows, and in part will be obvious from the description, or can be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by the elements and combinations specifically pointed out in the appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and together with the description, serve to explain the principles of the disclosed methods and compositions.

[0023] Figures 1A-1F Generation and screening of LANCL1 monoclonal antibodies is shown. Figure 1A Table of ELISA results for hybridoma clone supernatants. Figure 1B Protein blot of denatured GST-tagged human LANCL1 1-42 aa peptide overexpressed in E. coli BL21 lysate probed with individual hybridoma clone supernatants at 1 : 1000 dilution. Ladder scale in kDa. Figure 1C Protein blot of non-denatured GST-tagged human LANCL1 1-42 aa peptide expressed in E. coli BL21 lysate probed with individual hybridoma clone supernatants at 1 : 1000 dilution. Ladder scale in kDa. Figure 1D Table summary of detection capability by individual hybridoma clone supernatants of both denatured and non-denatured GST-tagged human LANCL1 1-42 aa peptide overexpressed in E. coli BL21 lysate. Figure 1E Protein blot of non-denatured His-tagged full-length human LANCL1 protein overexpressed in PLC / PRF / 5 lysate probed with individual hybridoma clone supernatants at 1 : 250 dilution. Ladder scale in kDa. Figure 1F Protein blot of both denatured and non-denatured His-tagged full-length human LANCL1 protein overexpressed in PLC / PRF / 5 lysate probed with individual hybridoma clone supernatants at 1 : 100 dilution. Ladder scale in kDa.

[0024] Figure 2 LANCL1 monoclonal antibodies 4D9 and 19G4 were shown to inhibit spheroid formation of PLC / PRF / 5 HCC cell line. 700 PLC / PRF / 5 cells were seeded in 96 well plates in a volume of 100 μΐ. Three independent experiments were performed (unpaired t-test).

[0025] Figures 3A-3C The effect of LANCL1 monoclonal antibodies 4D9 and 19G4 on the growth of PLC / PRF / 5 HCC xenografts was tested in vivo. 5e5 PLC / PRF / 5 HCC cells in 0.5X growth factor-reduced Matrigel were injected subcutaneously in a single site on the flank of each NOD-SCID mouse. When palpable tumors were observed at day 9 post-PLC / PRF / 5 cell injection, a first dose of monoclonal antibody and corresponding IgG control was injected intraperitoneally at 12 mg / kg. Two doses were administered weekly for two weeks as indicated. Figure 3A Mouse body weight was not affected by the administration of 4D9 and 19G4 monoclonal antibodies compared to IgG control. Figure 3B The 19G4 (but not 4D9) monoclonal antibody significantly inhibited PLC / PRF / 5 xenograft growth compared to the 12 mg / kg dose of IgG control as observed at day 19 post-PLC / PRF / 5 cell injection (unpaired t-test). Figure 3C Tumors collected at endpoint (day 21 post-PLC / PRF / 5 cell injection) showed reduced tumor size for 4D9 and 19G4 monoclonal antibody treatment compared to IgG control.

[0026] Figures 4A-4C The effect of LANCL1 monoclonal antibody 19G4 on a mouse orthotopic HCC model was tested. 1 x 10 6 Luciferase-labeled PLC / PRF / 5 HCC cells in IX Matrigel were injected orthotopically into the left lobe of the liver of each NOD-SCID mouse. When tumor formation was observed by luciferase signal imaging 2 weeks post-orthotopic injection, mice were randomized into groups to receive 19G4 monoclonal antibody, corresponding mouse IgG control or no treatment. Antibodies were injected intraperitoneally at 60 mg / kg. Two doses were administered weekly for two weeks. Figure 4A , 4B Liver tumor size was indicated at endpoint by luciferase signal imaging of mice and extracted livers, respectively. Figure 4C) The 19G4 monoclonal antibody significantly inhibited lung metastasis of PLC / PRF / 5 HCC cells compared to IgG control as determined by luciferase signal imaging of extracted lung tissue (unpaired t-test).

[0027] Figures 5A-5B . By ( Figure 5A ) cytotoxicity assays (which facilitated the determination of IC50) and ( Figure 5B ) annexin V assays by flow cytometry were used to evaluate the cytotoxic activity of the 19G4 monoclonal antibody on liver cancer cells. Figure 5A ) In cytotoxicity assays, 2000 PLC / PRF / 5 cells seeded in 96-well plates were treated with eight different indicated concentrations of the 19G4 monoclonal antibody or IgG control per well, respectively. After 24 hours, cell viability was assessed using DAPI staining followed by cell counting. The IC50 value of the 19G4 monoclonal antibody was determined to be 35.29 pg / ml. Figure 5B ) The cytotoxic activity of the 19G4 monoclonal antibody was further validated by annexin V / PI staining. In these assays, cells were seeded at 10,000 cells / well in 24-well plates with four wells per condition, and the wells of cells were treated with 128 pg / ml of the 19G4 monoclonal antibody or IgG control immediately after cell seeding, respectively. For each condition, a total of 1 x 10 5 PLC / PRF / 5 cells were harvested for flow cytometry, with 10,000 events analyzed per sample. The 48-hour treatment of the 19G4 monoclonal antibody showed a trend of increased apoptosis compared to the IgG control, but it did not yet reach statistical significance. Flow cytometry plots of a representative experiment are shown in the left panel, while the right panel shows the summary of three independent experiments (paired t-test).

[0028] Figures 6A-6B The effect of the 19G4 monoclonal antibody on the migratory and invasive capacity of HCC cells was evaluated by transwell migration and invasion assays, respectively. Figure 6A ) In the transwell migration assay, 1 x 10 5 PLC / PRF / 5 cells were seeded in the upper chamber, and 128 pg / mL of the 19G4 monoclonal antibody was applied to both the upper and lower chambers. The lower chamber contained 10% FBS as a chemoattractant, while the upper chamber had serum-free medium. After 6 hours of incubation at 37°C, the number of cells that had migrated across the membrane to the bottom surface of the membrane in the transwell was stained and counted in random view. The 19G4 antibody significantly reduced HCC cell migration compared to the IgG control. Figure 6B ) In the transwell invasion assay, 5 x 10 5The upper chamber was pre-coated with 3 mg / ml Matrigel prior to the addition of the PLC / PRF / 5 cells. All other conditions were identical to the migration assay except for the prolonged incubation period of 72 hours. The 19G4 antibody significantly reduced HCC cell invasion compared to the IgG control. Three independent experiments were performed for all assays and statistical significance was analyzed by paired t-test.

[0029] Figures 7A-7B Flow cytometry analysis was performed to determine the mean fluorescence intensity (MFI) of the total cell population and the percentage of cells positive for LANCL1 expression in non-permeabilized PLC / PRF / 5 cells collected at different days of spheroid and regular adherent culture using the 19G4 monoclonal antibody. Figure 7A Figure 7B The expression of LANCL1 increased in spheroid culture but not in adherent culture, indicating enrichment of LANCL1 -positive cells in spheroid culture.

[0030] Figures 8A-8C Schematic representation of the humanization of the 19G4 monoclonal antibody. Figure 8A Method to design the humanized monoclonal antibody. Figure 8B Structure of the original non-humanized 19G4 antibody and the humanized chimeric 19G4 monoclonal antibody. Figure 8C Structure of different variants of the humanized 19G4 monoclonal antibody. CDR, complementarity determining region. Figure created in BioRender.

[0031] Figures 9A-9C Affinity measurements of the humanized 19G4 monoclonal antibody variants by Biacore. Figure 9A Schematic representation of the Biacore measurements. Figure 9B Analytes, ligands and capture molecules used in the Biacore measurements. Figure 9C Raw Biacore data of the affinity measurements of each humanized antibody variant. Figures 9D-9E Results of the Biacore affinity measurements of the humanized 19G4 monoclonal antibody variants. The equilibrium dissociation constant (KD) indicating the overall binding affinity was calculated by the following formula: KD = kd / ka; however, the dissociation rate constant kd indicates the speed at which unbinding occurs, while the association rate constant ka indicates the speed at which binding occurs. The closer the KD value of an antibody variant to the KD value of the chimeric humanized antibody (VH+VL), the more similar their affinity for the LANCL1 1-42 aa region.

[0032] Figures 10A-10B Figure 10A ​​Results of post-translational modification (PTM) risk prediction for the heavy chain of the humanized 19G4 antibody variants. Amino acid residues highlighted in blue have a medium risk of PTM, while amino acid residues highlighted in green have a low risk of PTM. Figure 10B Results of post-translational modification (PTM) risk prediction for the light chain of the humanized 19G4 antibody variants. Amino acid residues highlighted in blue have a medium risk of PTM, while amino acid residues highlighted in green have a low risk of PTM.

[0033] Figures 11A-11B Spheroid formation assay of various humanized 19G4 monoclonal antibody variants was tested using PLC / PRF / 5 HCC cell line. The number of spheroids formed at day 8 ( Figure 11A ) and day 11 ( Figure 11B ) were counted. Humanized antibody variants VH2+VL2 and VH3+VL2 significantly inhibited spheroid formation, similar to the chimeric humanized antibody VH+VL. Three independent experiments were performed, where a paired t-test was performed for statistical significance analysis. DETAILED DESCRIPTION

[0034] The disclosed methods and compositions can be more readily understood by reference to the following detailed description, taken in conjunction with the accompanying drawings, and wherein the same reference numerals designate similar elements, and the included drawings.

[0035] I. DEFINITIONS

[0036] The term "antibody" is used in the broadest sense of the term. Thus, "antibody" can be naturally occurring or man-made, such as a monoclonal antibody produced by conventional hybridoma technology. Antibodies include monoclonal and polyclonal antibodies as well as fragments and polymers containing antigen binding domains and / or one or more complementarity determining regions of these antibodies. The term "antibody" as used herein refers to any form of antibody or antigen binding fragment or recombinant protein thereof, and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they specifically bind to a target antigen. Any specific antibody can be used in the methods and compositions provided herein. The term "antibody" encompasses immunoglobulin molecules having a "variable region" antigen recognition site. Thus, the term "antibody" encompasses molecules having at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule, which combine to form a specific binding site for a target antigen. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Patent No. 6,005,079), single-chain Fv (scFv) (see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fv (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to the disclosed antibodies and anti-anti-Id antibodies). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0037] The term "monoclonal antibody" or "MAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that can be present in minor amounts.

[0038] The term "variable region" is intended to distinguish such domains of immunoglobulins from domains common to antibodies generally, such as the antibody Fc domain. The variable region refers to the portion of the light and / or heavy chain of an antibody that specifically binds antigen as defined herein and includes, for example, the amino acid sequences of the CDRs; i.e., CDR1, CDR2, and CDR3, as well as the framework regions (FRs). For example, the variable region can include three or four FRs (e.g., FR1, FR2, FR3, and optional FR4), as well as three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. The variable region includes the "hypervariable region" whose residues are responsible for antigen binding.

[0039] The hypervariable region includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., according to Kabat, generally about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and about residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or from a "hypervariable loop" (e.g., according to Chothia, residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). Conventions that include corrected or alternative numbering systems for the variable domain include not only Kabat and Chothia, but also IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27: 55-77), Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196: 901-917; Chothia, et al. (1989), Nature 342: 877-883), and AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309: 657-670). For convenience, examples of binding proteins of the present disclosure can also be labeled according to Kabat, Chothia, or IMGT. These examples are expressly so indicated.

[0040] “Framework region” or “FR” residues are those variable domain residues other than the hypervariable region residues herein defined.

[0041] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the complementarity determining regions (“CDRs”) of the antibody and optionally framework residues of the “variable region” antigen recognition site of the antibody and exhibit the ability to immunospecifically bind an antigen. Such fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments, as well as mutants, naturally occurring variants, and fusion proteins comprising the “variable region” antigen recognition site of an antibody and a heterologous protein (e.g., a toxin, an antigen recognition site of a different antigen, an enzyme, a receptor or receptor ligand, etc.). For example, the term antigen binding fragment can be used to refer to recombinant single-chain Fv fragments (scFv) as well as di- (di-scFv) and tri- (tri-scFV) valent versions thereof. Such fragments can be produced via various methods known in the art.

[0042] As used herein, the term “single-chain Fv” or “scFv” means a single-chain variable fragment that includes a light chain variable region (VL) and a heavy chain variable region (VH) in a single polypeptide chain connected by a linker that enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of a single polypeptide chain to associate with one another to form an Fv). The VL and VH regions can be derived from a parent antibody or can be chemically synthesized or recombinantly synthesized.

[0043] As used herein, the term “constant region” refers to the portion of an antibody’s heavy or light chain other than the variable region. In a heavy chain, the constant region generally comprises multiple constant domains and a hinge region, e.g., the IgG constant region includes the following linked components, constant heavy C H 1, a linker, C H 2, and C H 3. In a heavy chain, the constant region comprises the Fc. In a light chain, the constant region generally includes one constant domain (CL1).

[0044] The term “fragment crystallizable” or “Fc” or “Fc region” or “Fc portion” (which are used interchangeably herein) refers to the region of an antibody that comprises at least one constant domain and is generally (but not necessarily) glycosylated and capable of binding to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of the five isotypes: a, d, e, g, or m. Exemplary heavy chain constant regions are g1 (IgG1), g2 (IgG2), and g3 (IgG3), or hybrids thereof.

[0045] A "constant domain" is a domain in an antibody whose sequence is highly conserved among antibodies of the same type (e.g., IgG or IgM or IgE). The constant region of an antibody typically comprises multiple constant domains, e.g., the constant region of a gamma, alpha, or delta heavy chain includes two constant domains.

[0046] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy chains and light chains including an Fc region. The constant domains can be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0047] A "chimeric antibody" is a molecule in which different portions of the antibody come from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chain shuffling (U.S. Patent No. 5,565,332).

[0048] As used herein, the term "humanized antibody" refers to an immunoglobulin that contains a human framework region and one or more core-receptor regions (CDRs) derived from a non-human (typically mouse or rat) immunoglobulin. The non-human immunoglobulin that provides the CDR is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor."

[0049] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0050] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains its essential characteristics. A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the difference is limited, making the sequences of the reference polypeptide and the variant very similar overall and identical in many regions. The difference in amino acid sequence between the variant and the reference polypeptide can be due to one or more modifications (e.g., substitution, addition, and / or deletion). The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of a polypeptide can be naturally occurring, such as allelic variants, or they can be variants of unknown origin.

[0051] The structure of the polypeptides disclosed herein can be modified and altered, and molecules with similar characteristics to the polypeptides can still be obtained (e.g., conserved amino acid substitutions). For example, certain amino acids in the sequence can be substituted by other amino acids without significant loss of activity. Because the interaction capabilities and properties of the polypeptide determine its biological functional activity, certain amino acid sequence substitutions can be made in the polypeptide sequence, and polypeptides with similar properties can still be obtained.

[0052] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is known in the art. Certain amino acids can be substituted for other amino acids in certain proteins without appreciable loss of biologic activity according to the principle that the hydropathic amino acid index is conserved. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0053] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, for example, enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that the substitution of amino acids can occur within the molecule without appreciable loss of biologic activity according to the principle that the hydropathic index of an amino acid is conserved. In making such changes, amino acids having hydropathic indices within ±2 are substituted for amino acids having hydropathic indices within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0054] Similar amino acid substitutions can also be made on the basis of hydrophilicity. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and particularly an immunologically equivalent, polypeptide. In making such changes, amino acids having hydrophilicity values within ±2 are substituted for amino acids having hydrophilicity values within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0055] As outlined above, amino acid substitutions are typically based on the relative similarities of the side chains of amino acids, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take into account various of the above-described features are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Val), (Leu: He, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: He, Leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of polypeptides as shown above. In particular, embodiments of polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide of interest.

[0056] As used herein, the term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in the reference nucleic acid or amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. For purposes of determining percent sequence identity, the comparison of sequences can be accomplished using various methods known in the art, for example, using the computer software programs available publicly, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters to use for measuring the alignment include any set of

[0057] As used herein, the term "bind" with respect to the interaction of a binding protein and an antigen means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, a binding protein recognizes and binds to a specific antigenic structure rather than to antigens in general. For example, if a binding protein binds to epitope "A", then in a reaction containing labeled "A" and the binding protein, the presence of molecules containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the binding protein.

[0058] As used herein, the term "specifically binds" refers to the binding of an antibody to its cognate antigen (e.g., guanosine), and not to other antigens, to a significant extent. The specific binding of an antibody to a target under such conditions requires that the antibody be selected for its specificity for the target. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to 5 mol –1 (e.g., 10 6 mol –1 , 10 7 mol –1 , 10 8 mol –1 , 10 9 mol –1 , 10 10 mol –1 , 10 11 mol –1 , and 10 12 mol –1 or greater).

[0059] As used herein, the phrase "pharmaceutically acceptable" refers to those compositions, polymers and other materials and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0060] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulation material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient.

[0061] As used herein, the terms "carrier" or "excipient" refer to an organic or inorganic ingredient, natural or synthetic, in a formulation, combined with one or more active ingredients. As will be appreciated by those skilled in the art, a carrier or excipient can be naturally selected or synthesized to minimize any degradation of the active ingredients and to minimize any adverse side effects in a subject.

[0062] As used herein, the terms "individual," "subject," and "patient" are used interchangeably to refer to any individual who is the target of an administration or treatment. The subject can be a vertebrate, such as a mammal. Thus, the subject can be a human or veterinary patient. The term does not denote a particular age or sex.

[0063] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment directed to facilitating recovery from or compensation for the associated disease, pathological condition, or disorder.

[0064] The term "treatment of HCC" or "HCC treatment" means reducing, inhibiting, or alleviating one or more symptoms associated with HCC in a subject having HCC.

[0065] As used herein, the term "inhibit" or "reduce" means to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to a native or control level. Thus, a reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, reduction compared to a native or control level.

[0066] As used herein, the term "effective amount" means an amount of composition used that is sufficient to improve one or more causes or symptoms of a disease or condition. Such improvement requires only reduction or alteration, not necessarily elimination. The precise dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or condition being treated, and the route of administration and the pharmacokinetics of the administered agent. As used herein, the term "therapeutic effective amount" means an amount of therapeutic agent that, when incorporated into and / or onto the particles described herein, produces some desired effect at a reasonable benefit / risk ratio suitable for any medical treatment. Effective amounts can vary depending on factors such as the disease or condition being treated, the specific targeted construct being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular compound without excessive experimentation. In some embodiments, the term "effective amount" refers to an amount of therapeutic or preventative agent that reduces or alleviates symptoms of one or more diseases or conditions of the brain, such as reducing tumor size (e.g., tumor volume).

[0067] The term "about" is used to describe values ​​that are higher or lower than the stated value within a range of about + / - 10%; in other embodiments, these values ​​may be within a range of about + / - 5%; in other embodiments, these values ​​may be within a range of about + / - 2%; and in other embodiments, these values ​​may be within a range of about + / - 1%. The foregoing ranges are intended to be clear from the context and do not imply further limitations.

[0068] Unless otherwise stated herein, the description of numerical ranges herein is intended only to serve as a shorthand for each individual value falling within that range, and each individual value is incorporated into the specification as if it were described separately herein.

[0069] Unless otherwise stated or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise required, the use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the embodiments and does not constitute a limitation on the scope of the embodiments. No language in the specification should be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0070] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in the preparation of, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds can not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a list of components is disclosed and a number of these components are discussed, each individual component and the combinations of each of these components are specifically contemplated, even if not explicitly stated. Thus, for example, if a class of components A, B, and C are disclosed as well as a class of components D, E, and F and an example of a combination of components A-D is disclosed, then each and every combination and permutation of each of the individual components is specifically contemplated and should be considered disclosed herein, even if not explicitly stated in the disclosure. Thus, for example, a combination of A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are all specifically contemplated and should be considered disclosed herein. This

[0071] These concepts apply to all aspects of this application, including but not limited to steps in the methods for making and using the disclosed compositions. Thus, if there are a plurality of additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular embodiment or aspect of the disclosed methods, and each such combination is specifically contemplated and should be considered disclosed.

[0072] II. Compositions

[0073] Compositions of anti-LANCL1 monoclonal antibodies and antigen-binding fragments thereof are provided. The disclosed compositions are based in part on the discovery that anti-LANCL1 monoclonal antibodies can inhibit pro-tumorigenic features of hepatocellular carcinoma cells, such as spheroid formation Figure 2 ) and in vivo tumorigenicity Figure 3B and 3C ) Based on this discovery, anti-LANCL1 monoclonal antibodies (mAbs) were generated and sequenced, and heavy and light chain variable domains were identified.

[0074] An exemplary consensus amino acid sequence of human LANCL1 protein is represented by SEQ ID NO: 1.

[0075] Human LANCL1 protein (399 aa; UniProt ID: 043813 LANC1 HUMAN)

[0076] MAQRAFPNPYADYNKSLAEGYFDAAGRLTPEFSQRLTNKIRELLQQMERGLKSADPRDGTGYTGWAGIAVLYLHLYDVFGDPAYLQLAHGYVKQSLNCLTKRSITFLCGDAGPLAVAAVLYHKMNNEKQAEDCITRLIHLNKIDPHAPNEMLYGRIGYIYALLFVNKNFGVEKIPQSHIQQICETILTSGENLARKRNFTAKSPLMYEWYQEYYVGAAHGLAGIYYYLMQPSLQVSQGKLHSLVKPSVDYVCQLKFPSGNYPPCIGDNRDLLVHWCHGAPGVIYMLIQAYKVFREEKYLCDAYQCADVIWQYGLLKKGYGLCHGSAGNAYAFLTLYNLTQDMKYLYRACKFAEWCLEYGEHGCRTPDTPFSLFEGMAGTIYFLADLLVPTKARFPAFEL (SEQ ID NO: 1)

[0077] Antigens used to make the disclosed antibodies and antigen-binding fragments contain the 1-42 aa region specific for LANCL1 protein: MAQRAFPNPYADYNKSLAEGYFDAAGRLTPEFSQRLTNKIRE (SEQ ID NO: 2). In some forms, the antibodies bind to a fragment of human LANCL1, e.g., human LANCL1 having the sequence of SEQ ID NO: 2. In some forms, a Cys (C) residue is added to SEQ ID NO: 2 for the purpose of immunizing a rodent, e.g., a mouse, to allow KLH conjugation. Typically, this Cys amino acid residue is not naturally present in the corresponding LANCL1 full-length protein.

[0078] In some embodiments, the antibody binds to a variant having at least 60% up to 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2. For example, in some forms, the antibody binds to a human LANCL1 variant sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. Thus, in some forms, a variant of a human LANCL1 protein shares an amino acid sequence having one or more amino acids different from SEQ ID NO: 1 or SEQ ID NO: 2, such as one or more substitutions, deletions, or additions at any of the amino acid positions of SEQ ID NO: 1 or SEQ ID NO: 2.

[0079] As discussed herein, antibodies that bind to human LANCL1 protein are expressly provided, in the form of any and all antibodies, including but not limited to intact antibodies and antigen binding fragments in the form of mono-, bi- and higher order multi-specificities, optionally as humanized or chimeric forms thereof, having one or more of the relevant CDRs or variants thereof, and / or one or both of the relevant VH and VL sequences or variants thereof. Thus, in some embodiments, the antibody is or includes a fragment having antigen-binding ability (e.g., Fab', F(ab')2, Fab, Fv, and rlgG), a recombinant single-chain Fv fragment (scFv), and di- (di-scFv) and tri- (tri-scFV) valent forms thereof.

[0080] A. Sequences of Anti-LANCL1 Monoclonal Antibodies

[0081] 1. Heavy Chain Variable Region

[0082] In some forms, the amino acid sequence of the kappa heavy chain variable region (VH) of the anti-LANCL1 monoclonal antibody (4D9) has the following sequence:

[0083]

[0084] In some forms, the amino acid sequence of the kappa heavy chain variable region (VH) of the anti-LANCL1 monoclonal (19G4) antibody has the following sequence:

[0085]

[0086] The complementarity determining regions (CDRs) are shown in bold italic in SEQ ID NO: 3 and SEQ ID NO: 4, including first, second, and third CDR regions CDR-1, CDR-2, and CDR-3, respectively. An exemplary amino acid sequence of CDR-1 of the heavy chain variable region is DYYMN (SEQ ID NO: 5). Exemplary amino acid sequences of CDR-2 of the heavy chain variable region include DINPNNGGASYNQKFKG (SEQ ID NO: 6), and VINPYNGHTNYNQKFKG (SEQ ID NO: 7). Exemplary amino acid sequences of CDR-3 of the heavy chain variable region include S GDGYYFAS (SEQ ID NO: 8) and FPYYGSSYRVDY (SEQ ID NO: 9). In some forms, the first, second, and third CDR regions of the heavy chain variable region include variant amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, respectively.

[0087] The framework region (FR) regions are underlined in SEQ ID NO: 3 and SEQ ID NO: 4, and include first, second, third, and fourth FR regions FR1, FR2, FR3, and FR4, respectively. Exemplary amino acid sequences for heavy chain variable region FR1 include EVQLQQSGPELVKPGASVKISCKASGYTFT (SEQ ID NO: 10) and EVQLQQSGPVLVKPGASVKMSCKASGYTFT (SEQ ID NO: 11). An exemplary sequence for heavy chain variable region FR2 is WVKQSHGKSLEWIG (SEQ ID NO: 12). Exemplary sequences for heavy chain variable region FR3 include KATLTVDKSSSTAYMELRSLTSEDSAVYYCVR (SEQ ID NO: 13) and KATLTVDKSSNTAYMELNSLTSEDSAVYYCAR (SEQ ID NO: 14). Exemplary sequences for heavy chain variable region FR4 include WGQGTLLTVSA (SEQ ID NO: 15) and WGQGTTLTVSS (SEQ ID NO: 16). In some forms, the first, second, third, and fourth FR regions of the heavy chain variable region include variant amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and / or SEQ ID NO: 16, respectively.

[0088] A signal peptide for the heavy chain variable region is italicized in SEQ ID NO: 3 and SEQ ID NO: 4. Exemplary sequences for the signal peptide of the heavy chain variable region include MGWSWIFLFLLSGTAGVLS (SEQ ID NO: 17) and MGWSWIFLFLLSGTAGVHS (SEQ ID NO: 18). In some forms, the signal peptide of the heavy chain variable region includes a variant amino acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18.

[0089] In some forms, the constant region of the heavy chain variable region can comprise the amino acid sequence: AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 36). In some forms, the constant region of the heavy chain variable region comprises a variant amino acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 36.

[0090] 2. Light chain variable region

[0091] In some forms, the amino acid sequence of the kappa light chain variable region (VL) of the anti-LANCL1 monoclonal (4D9) antibody has the following sequence:

[0092]

[0093] In some forms, the amino acid sequence of the kappa light chain variable region (VL) of the anti-LANCL1 monoclonal (19G4) antibody has the following sequence:

[0094]

[0095] The complementarity determining regions (CDRs) are shown in bold italic in SEQ ID NO: 19 and SEQ ID NO: 20, including first, second, and third CDR regions CDR-1, CDR-2, and CDR-3, respectively. An exemplary sequence for CDR-1 of the light chain variable region includes RASKSVSTSGYSYMH (SEQ ID NO: 21) and RASQSISNNLH (SEQ ID NO: 22). An exemplary amino acid sequence for CDR-2 of the light chain variable region includes LVSNLES (SEQ ID NO: 23) and YASQSIS (SEQ ID NO: 73). An exemplary amino acid sequence for CDR-3 of the light chain variable region includes QHIRELT (SEQ ID NO: 24) and QQINSWPLT (SEQ ID NO: 25). In some forms, the first, second, and third CDR regions of the light chain variable region include variant amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 73, respectively.

[0096] The framework region (FR) regions of the light chain variable region are shown underlined in SEQ ID NO: 19 and SEQ ID NO: 20, including first, second, third, and fourth FR regions FR1, FR2, FR3, and FR4, respectively. Exemplary amino acid sequences for light chain variable region FR1 include DIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 26) and DIVLTQSPATLSVTPGDSVSLSC (SEQ ID NO: 27). Exemplary sequences for light chain variable region FR2 include WNQQKPGQPPRLLIY (SEQ ID NO: 28) and WYQQKSHESPRLLIK (SEQ ID NO: 29). Exemplary sequences for light chain variable region FR3 include GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 30) and GIPSRFSGSGSGTDFTLSINSVETEDFGMYFC (SEQ ID NO: 31). Exemplary sequences for light chain variable region FR4 include FGGGTKLEIK (SEQ ID NO: 32) and FGAGTKLELK (SEQ ID NO: 33). In some forms, the first, second, third, and fourth FR regions of the light chain variable region include variant amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and / or SEQ ID NO: 33, respectively.

[0097] The signal peptide of the light chain variable region is shown in italics in SEQ ID NO: 19 and SEQ ID NO: 20. Exemplary sequences for the signal peptide of the heavy chain variable region include MGTAALGSRFHW (SEQ ID NO: 34) and MVFTPQILGLMLFWISASRG (SEQ ID NO: 35). In some forms, the signal peptide of the light chain variable region includes a variant amino acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 34 or SEQ ID NO: 35.

[0098] In some forms, the constant region of the light chain variable region can comprise the amino acid sequence: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 37). In some forms, the constant region of the light chain variable region comprises a variant amino acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 37.

[0099] 3. Exemplary LANCL1 Monoclonal Antibodies

[0100] In some forms, the disclosed monoclonal antibodies have the following sequences, where the CDRs are in bold italic and the FRs are underlined:

[0101] (i) 4D9:

[0102] VH:

[0103] CDR-1 is DYYMN (SEQ ID NO: 5), CDR-2 is DINPNNGGASYNQKFKG (SEQ ID NO: 6), and CDR-3 is SGDGYYFAS (SEQ ID NO: 8). FR1 is EVQLQQSGPELVKPGASVKISCKASGYTFT (SEQ ID NO: 10); FR2 is WVKQSHGKSLEWIG (SEQ ID NO: 12), FR3 is KATLTVDKSSSTAYMELRSLTSEDSAVYYCVR (SEQ ID NO: 13), and FR4 is WGQGTLLTVSA (SEQ ID NO: 15).

[0104] VL:

[0105] CDR-1 is RASKSVSTSGYSYMH (SEQ ID NO: 21), CDR-2 is LVSNLES (SEQ ID NO: 23), and CDR-3 is QHIRELT (SEQ ID NO: 24). FR1 is DIVLTQSPASLAVSLGQRATISY (SEQ ID NO: 26); FR2 is WNQQKPGQPPRLLIY (SEQ ID NO: 28), FR3 is GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 30), and FR4 is FGGGTKLEIK (SEQ ID NO: 32).

[0106] (ii) 19G4:

[0107] VH: CDR-1 is DYYMN (SEQ ID NO: 5), CDR-2 is VINPYNGHTNYNQKFKG (SEQ ID NO: 7), and CDR-3 is FPYYGSSYRVDY (SEQ ID NO: 9). FR1 is EVQLQQSGPVLVKPGASVKMSCKASGYTFT (SEQ ID NO: 11), FR2 is WVKQSHGKSLEWIG (SEQ ID NO: 12), FR3 is KATLTVDKSSNTAYMELNSLTSEDSAVYYCAR (SEQ ID NO: 14), and FR4 is WGQGTTLTVSS (SEQ ID NO: 16).

[0108] VL:

[0109] CDR-1 is RASQSISNNLH (SEQ ID NO: 22), CDR-2 is YASQSIS (SEQ ID NO: 73), and CDR-3 is QQINSWPLT (SEQ ID NO: 25).

[0110] FR1 is DIVLTQSPATLSVTPGDSVSLSC (SEQ ID NO: 27), FR2 is WYQQKSHESPRLLIK (SEQ ID NO: 29), FR3 is GIPSRFSGSGSGTDFTLSINSVETEDFGMYFC (SEQ ID NO: 31), and FR4 is FGAGTKLELK (SEQ ID NO: 33).

[0111] B. Chimeric and Humanized Antibodies

[0112] In some forms, the anti-LANCL1 antibody can be a chimeric antibody or a humanized antibody. The constant region need not be present, but if it is, it is typically essentially identical to that of a human immunoglobulin, i.e., at least about 85-90%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are essentially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody having a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, for example, the entire variable region of a chimeric antibody is non-human. It is a statement that the donor antibody has been "humanized" by a "humanization" process, in that the resulting humanized antibody is expected to bind to the same antigen as the donor antibody that provided the CDRs.

[0113] In most instances, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, which, by virtue of the introduction of an amino acid residue substitution, deletion or addition (i.e., mutation) that alters the binding affinity of the human immunoglobulin to FcγRIIB polypeptide.See also, e.g., European Patent Nos. EP 239,400, EP 592,106, and EP 519,596; International Publication Nos. WO 91 / 09967 and WO 93 / 17105; U.S. Patent Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-1125; Caldas et al., 2000, Protein Eng. 13:353-360; Morea et al., 2000, Methods 20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-10684; Roguska et al., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol. 235:959-973; Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0114] DNA sequences encoding preferred human acceptor framework sequences include, but are not limited to, FR segments from human germline VH segment VH1-18 and JH6, and human germline VL segment VK-A26 and JK4. In particular embodiments, one or more CDRs are inserted into the framework regions using conventional recombinant DNA techniques. The framework regions can be naturally occurring or consensus framework regions, and are preferably human framework regions (see, e.g., Chothia et al., 1998, “Structural Determinants In The Sequences Of Immunoglobulin Variable Domain,” J. Mol. Biol. 278: 457-479 for a listing of human framework regions).

[0115] A humanized or chimeric antibody can include substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody), and all or substantially all of the framework regions are those of a human immunoglobulin.

[0116] In some forms, the heavy chain variable region (VH) CDR-1 of the chimeric and / or humanized antibody is DYYMN (SEQ ID NO: 5). In some forms, the VH CDR-2 of the chimeric or humanized antibody is DINPNNGGASYNQKFKG (SEQ ID NO: 6) or VINPYNGHTNYNQKFKG (SEQ ID NO: 7). In some forms, the VH CDR-3 of the chimeric or humanized antibody is SGDGYYFAS (SEQ ID NO: 8) or FPYYGSSYRVDY (SEQ ID NO: 9). In some forms, the first, second, and third VH CDR regions are variant amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0117] In some forms, the light chain variable region (VL) CDR-1 of the chimeric or humanized antibody is RASKSVSTSGYSYMH (SEQ ID NO: 21) or RASQSISNNLH (SEQ ID NO: 22). In some forms, the VL CDR-2 of the chimeric or humanized antibody is LVSNLES (SEQ ID NO: 23) or YASQSIS (SEQ ID NO: 73). In some forms, the VL CDR-3 of the chimeric or humanized antibody is QHIRELT (SEQ ID NO: 24) and QQINSWPLT (SEQ ID NO: 25). In some forms, the first, second, and third VL CDR regions of the chimeric or humanized antibody are variant amino acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 73, or SEQ ID NO: 25.

[0118] In some forms, the heavy chain variable region (VH) of the chimeric humanized anti-LANCL1 monoclonal antibody (19G4) has the sequence: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGHTNYNQKFKGKATLTVDKSSNTAYMELNSLTSEDSAVYYCARFPYYGSSYRVDYWGQGTTLTVSS (SEQ ID NO: 75). In some forms, the heavy chain variable region (VH) of the chimeric humanized anti-LANCL1 monoclonal antibody (19G4) is a variant amino acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 75. In some forms, the variant of SEQ ID NO: 75 has the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGVINPYNGHTNYNQKFKGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCARFPYYGSSYRVDYWGQGTTVTVSS (SEQ ID NO: 77) (VH1). In some forms, the variant of SEQ ID NO: 75 has the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWIGVINPYNGHTNYNQKFKGRATMTVDTSTSTVYMELSSLRSEDTAVYYCARFPYYGSSYRVDYWGQGTTVTVSS (SEQ ID NO: 78) (VH2). In some forms, the variant of SEQ ID NO: 75 has the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVKQAPGQGLEWIGVINPYNGHTNYNQKFKGRATMTVDKSTSTVYMELSSLRSEDTAVYYCARFPYYGSSYRVDYWGQGTTVTVSS (SEQ ID NO: 79) (VH3).

[0119] In some forms, the light chain variable region (VL) of the chimeric humanized anti-LANCL1 monoclonal antibody (19G4) has the sequence: DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQINSWPLTFGAGTKLELK (SEQ ID NO: 76). In some forms, the light chain variable region (VL) of the chimeric humanized anti-LANCL1 monoclonal antibody (19G4) is a variant amino acid sequence having approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, and approximately 99% sequence identity with SEQ ID NO: 76. In some forms, variants of SEQ ID NO: 76 have the sequence EIVMTQSPATLSVSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQINSWPLTFGGGTKLEIK (SEQ ID NO: 80) (VL1). In some forms, variants of SEQ ID NO: 76 have the sequence EIVMTQSPATLSVSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPSRFSGSGSGTEFTLTISSLQSEDFAVYYCQQINSWPLTFGGGTKLEIK (SEQ ID NO: 81) (VL2). In some forms, variants of SEQ ID NO: 76 have the sequence EIVMTQSPATLSVSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPSRFSGSGSGTEFTLTISSLQSEDFAVYFCQQINSWPLTFGGGTKLEIK (SEQ ID NO: 82) (VL3).

[0120] Preferably, the antibody further comprises at least a portion of an immunoglobulin constant region (Fc) (typically that of a human immunoglobulin). The constant domain of the antibody can be selected with respect to the proposed function of the antibody, particularly the effector functions that can be desired. In some embodiments, the constant domain of the antibody is (or includes) a human IgA, IgD, IgE, IgG, or IgM domain. In particular embodiments, when the humanized antibody is intended for therapeutic use and antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity are desired, a human IgG constant domain, particularly an IgG constant domain of the IgGl and IgG3 isotypes, is used. In alternative embodiments, when the antibody is intended for therapeutic purposes and antibody effector functions are not desired, IgG2 and IgG4 isotypes are used. The present disclosure encompasses Fc constant domains that include one or more amino acid modifications that alter antibody effector functions, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514.

[0121] In some embodiments, the antibody contains both a light chain and at least the variable domain of a heavy chain. In other embodiments, the antibody can further comprise one or more of the CHI, hinge, CH2, CH3, and CH4 regions of a heavy chain. The antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgGl, IgG2, IgG3, and IgG4. In some embodiments, the constant domain is a complement-fixing constant domain, where it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgGl. In other embodiments where such cytotoxic activity is not desired, the constant domain can be of the IgG2 class. The antibody can include sequences from more than one class or isotype, and the particular constant domain is selected to optimize the desired effector functions, which is within the ordinary skill in the art. In some embodiments, the antibody is not mouse IgGl or mouse IgG2a.

[0122] The framework and CDR regions of a humanized antibody need not correspond precisely to the parent sequences, e.g., a donor CDR or consensus framework can be mutagenized by substitution, insertion, or deletion of at least one residue such that the CDR or framework residue at that site does not correspond to the consensus or donor antibody. However, such mutations are preferably not extensive. Typically, at least 75% of the humanized antibody residues will correspond to those of the parent framework region (FR) and CDR sequences, more typically 90%, and most preferably greater than 95%.Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or reshaping (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed, e.g., in U.S. Patent Nos. 6,407,213, 5,766,886, 5,585,089, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol. 169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al., 2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272:10678-84, Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res. 55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J. Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596. Typically, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified, for example, by modeling of the interactions of CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at particular positions.(See, e.g., Queen et al., U.S. Patent No. 5,585,089; U.S. Publication Nos. 2004 / 0049014 and 2003 / 0229208; U.S. Patent Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332:323).

[0123] C. Derivatives and Conjugates

[0124] The present disclosure contemplates, inter alia, the production and use of derivatives of any of the above-described antibodies and antigen-binding fragments thereof. The term derivative encompasses an antibody or antigen-binding fragment thereof that specifically binds to an antigen, but which includes one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications (also referred to as variants) relative to the "parental" (or wild-type) molecule. Such amino acid substitutions or additions can introduce naturally-occurring (i.e., DNA-encoded) or non-naturally-occurring amino acid residues.

[0125] The term derivative also encompasses, for example, chimeric or humanized variants of any of the disclosed antibodies, as well as variants having altered CH1, hinge, CH2, CH3 or CH4 regions, thereby forming, for example, antibodies with variant Fc regions that exhibit enhanced or impaired effector or binding characteristics, etc.

[0126] The term derivative additionally encompasses amino acid modifications that are not glycosylated, e.g., can be acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modification modulates one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, facilitation of antibody assembly, conformational integrity, and antibody-mediated effector function. In particular embodiments, the altered carbohydrate modification enhances antibody-mediated effector function relative to an antibody lacking the carbohydrate modification. Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the art (e.g., see Shields, R.L. et al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,” J. Biol. Chem. 277(30): 26733-26740; Davies J. et al. (2001) “Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production Cell Line: Expression Of Antibodies With Altered Glycoforms Leads To An Increase In ADCC Through Higher Affinity For FC Gamma RIII,” Biotechnology & Bioengineering 74(4): 288-294).Methods of altering carbohydrate content are known to those of skill in the art, see, e.g., Wallick, S.C. et al. (1988) "Glycosylation Of A VH Residue Of A Monoclonal Antibody Against Alpha (1-6) Dextran Increases Its Affinity For Antigen," J. Exp. Med. 168(3): 1099-1109; Tao, M.H. et al. (1989) "Studies Of Aglycosylated Chimeric Mouse-Human IgG. Role Of Carbohydrate In The Structure And Effector Functions Mediated By The Human IgG Constant Region," J. Immunol. 143(8): 2595-2601; Routledge, E.G. et al. (1995) "The Effect Of Aglycosylation On The Immunogenicity Of A Humanized Therapeutic CD3 Monoclonal Antibody," Transplantation 60(8):847-53; Elliott, S. et al. (2003) "Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering," Nature Biotechnol. 21 :414-21; Shields, R.L. et al. (2002) "Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity.," J. Biol. Chem. 277(30): 26733-26740).

[0127] In some embodiments, the humanized antibody is a derivative. Such humanized antibodies include substitution, deletion, or addition of one or more amino acid residues in a non-human CDR. The humanized antibody derivative can have substantially the same binding, better binding, or worse binding when compared to a non-derivative humanized antibody. In particular embodiments, one, two, three, four, or five amino acid residues of a CDR have been substituted, deleted, or added (i.e., mutated).

[0128] Derivative antibodies or antibody fragments can be modified by chemical modification using techniques known to those of skill in the art, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In one embodiment, the antibody derivative will have similar or identical function as the parent antibody. In another embodiment, the antibody derivative will exhibit altered activity relative to the parent antibody. For example, the derivative antibody (or fragment thereof) can bind more tightly to its epitope or be more resistant to proteolysis than the parent antibody.

[0129] Derivatized antibodies can be used to alter the half-life (e.g., serum half-life) of the parent antibody in a mammal, preferably a human. Preferably, such alterations will result in a half-life greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. Increased half-life of a humanized antibody of the disclosure, or fragment thereof, in a mammal, preferably a human, results in a higher serum titer of the antibody or antibody fragment in the mammal, and thus reduces the frequency of administration of the antibody or antibody fragment and / or reduces the concentration of the antibody or antibody fragment to be administered. Antibodies or fragments thereof with increased half-life in vivo can be produced by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased half-life in vivo can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as participating in the interaction between the Fc domain and the FcRn receptor. Humanized antibodies can be engineered to increase biological half-life (see, e.g., U.S. Patent No. 6,277,375). For example, a humanized antibody can be engineered in the Fc hinge domain to have increased half-life in vivo or serum.

[0130] Antibodies or fragments thereof with increased half-life in vivo can be generated by attaching a polymer molecule, such as a high molecular weight polyethylene glycol (PEG) to the antibody or antibody fragment. PEG can be attached to the antibody or antibody fragment with or without a multifunctional linker, either through site-specific conjugation of PEG to the N- or C-terminus of the antibody or antibody fragment or via the epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biologic activity will be used. The extent of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from antibody-PEG conjugates by, for example, size exclusion or ion exchange chromatography.

[0131] Antibodies can also be modified by the methods and coupling agents described by Davis et al. (see U.S. Patent No. 4,179,337) to provide compositions that can be injected into the circulatory system of a mammal without a substantially immunogenic response.

[0132] One embodiment encompasses modification of framework residues of humanized anti-LANCL1 antibodies. Framework residues in the framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues contributing to antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No. 5,585,089; and Riechmann, L. et al. (1988) “Reshaping Human Antibodies For Therapy,” Nature 332:323-327).

[0133] Yet another embodiment encompasses recombinant fusion or chemical conjugation (including covalent and non-covalent conjugation) of anti-LANCL1 antibodies (and more preferably humanized antibodies) and antigen-binding fragments thereof to heterologous molecules (i.e., unrelated molecules). The fusion need not be direct, but can occur through a linker sequence.

[0134] In one embodiment, such heterologous molecules are polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. Alternatively, such heterologous molecules can be enzymes, hormones, cell surface receptors, drug moieties such as: toxins (such as abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), proteins (such as tumor necrosis factor, interferons (e.g., alpha-interferon, beta-interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, or an apoptotic agent (e.g., tumor necrosis factor-alpha, tumor necrosis factor-beta)), biological response modifiers (such as, for example, lymphokines (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”)), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or macrophage colony stimulating factor (“M-CSF”)), or growth factors (e.g., growth hormone (“GH”))), cytotoxins (e.g., cytostatic or cytocidal agents such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil and tioguanine), alkylating agents (e.g., nitrogen mustards, thioepa, melphalan, BiCNU® (carmustine; BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, decarabazine, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin)), bleomycin, mithramycin, and anthramycin (AMC)), or antimitotic agents (e.g., vincristine and vinblastine).

[0135] Techniques for conjugating such therapeutic moiety to antibodies are well known; see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev. 62:119-158.

[0136] In one embodiment, the anti-LANCL1 antibody or fusion molecule includes an Fc moiety. The Fc moiety of such molecules can vary by isotype or subtype, can be chimeric or heterozygous, and / or can be modified, for example, to improve effector function, control of half-life, tissue accessibility, enhance biophysical properties such as stability, and improve production efficiency (and at a lower cost). Many modifications and methods for preparing the disclosed fusion proteins are known in the art, see, for example, Mueller, JP et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells,” Mol. Immun. 34(6):441-452, Swann, PG (2008) “Considerations For The Development Of Therapeutic Monoclonal Antibodies,” Curr. Opin. Immun. 20:493-499 (2008), and Presta, LG (2008) “Molecular Engineering And Design Of Therapeutic Antibodies,” Curr. Opin. Immun. 20:460-470. In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, such as a chimera having a constant Fc region of IgG2 / IgG4. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fcγ receptors and complement, IgG1 modified to improve binding to one or more Fcγ receptors, IgG1 modified to minimize effector function (amino acid changes), IgG1 with altered / glycan-free properties (typically by altering the expression host), IgG1 with altered pH-dependent binding to FcRn, and IgG4 with a serine residue #228 in the hinge region changed to proline (S228P) to enhance stability. The Fc region may include the entire hinge region or less than the entire hinge region.

[0137] For example, the therapeutic outcome of treating patients with non-Hodgkin's lymphoma or Waldenstrom's macroglobulinemia with rituximab (a chimeric mouse / human IgGl monoclonal antibody directed against CD20) is correlated with the individual's intrinsic affinity of the Fc domain of human IgGl to the Fc receptors Receptor allelic variants are expressed. In another example, patients with high affinity alleles of the low affinity activating Fc receptor show a higher response rate and, in the case of non-Hodgkin's lymphoma, an improved progression free survival. In another example, the Fc domain can contain one or more amino acid insertions, deletions, or substitutions that reduce binding to the low affinity inhibitory Fc receptor and preserve or enhance binding to the low affinity activating Fc receptor

[0138] Another embodiment includes IgG 2-4 hybrids and IgG4 mutants that have reduced binding to FcRs, which increases their half-life. Representative IG 2-4 hybrids and IgG4 mutants are described in Angal, S. et al. (1993) "A Single Amino Acid Substitution Abolishes the Heterogeneity Of Chimeric Mouse / Human (IgG4) Antibody," Molec. Immunol. 30(1): 105-108; Mueller, J.P. et al. (1997) "Humanized Porcine VCAM-Specific Monoclonal Antibodies with Chimeric IgG2 / G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells," Mol. Immun. 34(6):441-452; and U.S. Patent No. 6,982,323. In some embodiments, the IgGl and / or IgG2 domains are deleted, for example, Angal, s. et al. describe IgGl and IgG2 with serine 241 replaced by proline.

[0139] ​Substitutions, additions, or deletions in derivatized antibodies can be made in the Fc region of the antibody, thereby modifying the antibody's binding affinity to one or more FcγRs. Methods for modifying antibodies that bind to one or more FcγRs are known in the art, see, for example, PCT publications WO 04 / 029207, WO 04 / 029092, WO 04 / 028564, WO 99 / 58572, WO 99 / 51642, WO 98 / 23289, WO 89 / 07142, WO 88 / 07089 and US patents 5,843,597 and 5,642,821. In one particular implementation, modification of the Fc region results in the antibody having altered antibody-mediated effector function, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity (CDC) activity, phagocytic activity, or any combination thereof.

[0140] In some forms, this disclosure covers antibodies whose Fc region is modified such that the molecule will exhibit altered Fc receptor (FcR) binding activity, for example, exhibiting reduced activity against activating receptors such as FcγRIIa or FcγRIIIa, or increased activity against inhibitory receptors such as FcγRIIb. Preferably, such antibodies will exhibit reduced antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity (relative to wild-type Fc receptors).

[0141] Modifications that affect Fc-mediated effector functions are well known in the art (see U.S. Patent No. 6,194,551, and WO 00 / 42072; Stavenhagen, J.B. et al. (2007) “Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors,” Cancer Res. 57(18):8882-8890; Shields, R.L. et al. (2001) “High Resolution Mapping of the Binding Site on Human IgG1 for FcgammaRI, FcgammaRII, FcgammaRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcgammaR,” J. Biol. Chem. 276(9):6591-6604). Exemplary variants of human IgGl Fc domains with reduced binding to FcgammaRIIa or FcgammaRIIIa but unaltered or enhanced binding to FcgammaRIIb include S239A, H268A, S267G, E269A, E293A, E293D, Y296F, R301A, V303A, A327G, K322A, E333A, K334A, K338A, A339A, D376A.

[0142] In some embodiments, the present disclosure encompasses antibodies (e.g., Fabs or F(ab)2s, etc.) whose Fc regions would have been deleted.

[0143] Any molecule of the present disclosure can be fused to a marker sequence, such as a peptide, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I.A. et al. (1984) “The Structure Of An Antigenic Determinant In A Protein,” Cell, 37:767-778), and the “flag” tag (Knappik, A. et al. (1994) “An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments,” Biotechniques 17(4):754-761).

[0144] The present disclosure also encompasses antibodies or antigen-binding fragments thereof conjugated to a diagnostic or therapeutic agent or any other molecule for which it is desirable to increase serum half-life. Antibodies can be used diagnostically (in vivo, in situ, or in vitro) to, for example, monitor the development or progression of a disease, disorder, or infection, as part of a clinical testing procedure, to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions. The detectable substance can be coupled to the antibody directly or indirectly via an intermediary such as, for example, a linker known in the art. For metal ions which can be conjugated to the antibody for use as a diagnostic agent according to the present disclosure, see, for example, U.S. Patent No. 4,741,900. Such diagnosis and detection can be accomplished by coupling the antibody to a detectable substance including, but not limited to, various enzymes, including but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to, bismuth (Bi), carbon (C), chromium (Cr), cobalt (Co), fluorine (F), gadolinium (Gd), gold (Au), iodine (I), iridium (Ir), neon (Ne), nitrogen (N), phosphorus (P), platinum (Pt), sulfur (S), technetium (Tc), and zinc (Zn); and paramagnetic metal ions, such as but not limited to, manganese (Mn), iron (Fe), cobalt (Co), platinum (Pt), gold (Au), and chromium (Cr). The antibody can be directly or indirectly conjugated to the detectable substance using techniques known in the art. 213 14 51 57 18 153 ​​​​​Gd, 159 Gd), gallium ( 68 Ga、 67 Ga), germanium ( 68 Ge), holmium ( 166 Ho), indium ( 115 In、 113 In、 112 In、 111 In), iodine 131 I, 125 I, 123 I, 121 I), lanthanum ( 140 La), Lutetium 177 Lu), manganese 54 Mn), molybdenum 99 Mo), palladium ( 103 Pd), phosphorus ( 32 P), Praseodymium ( 142 Pr), Promethium ( 149 Pm), rhenium ( 186 Re、 188 Re), rhodium 105 Rh), Ruthenium ( 97 Ru, samarium 153 Sm), Scandium ( 47 Sc), selenium ( 75 Se), Strontium ( 85 Sr), sulfur ( 35 S), Technetium ( 99 Tc), thallium 201 Ti, tin 113 Sn、 117 Sn), tritium ( 3 H), Xenon 133 Xe), Ytterbium ( 169 Yb、 175 Yb), Yttrium 90 Y), Zinc 65 Zn); positron-emitting metallic and non-radioactive paramagnetic metal ions obtained using various positron emission tomography (PET) scans.

[0145] The molecules disclosed herein can be conjugated with a second antibody to form a heteroconjugate, as described by Segal in US Patent No. 4,676,980. Such heteroconjugated antibodies can additionally bind to haptens (such as fluorescein), or to cell markers, or to cytokines or chemokines (such as CCL21), etc.

[0146] The molecules of the present disclosure can be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens or other molecules capable of binding to target antigens, which are immobilized to the support by binding to the antibodies of the present disclosure or antigen-binding fragments thereof. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0147] D.CAR protein

[0148] Also provided are chimeric antigen receptor (CAR) proteins comprising the disclosed anti-LANCL1 proteins as antigen binding domains and cells expressing the same. Typically, the CAR further comprises a transmembrane domain and one or more intracellular / cytoplasmic domains.

[0149] CARs are engineered receptors with antigen-binding function and T cell activation function. Immunotherapy using T cells genetically engineered to express CARs is rapidly emerging as a promising new treatment for hematologic and non-hematologic malignancies. Based on the location of the CAR in the cell membrane, CARs can be divided into three major different domains, including an extracellular antigen-binding domain, followed by a spacer, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain is most often derived from the variable region of an immunoglobulin, often containing a VH chain and a VL chain connected by a linker to form a so-called "scFv". The segment inserted between the antigen-binding domain (e.g., scFv) and the transmembrane domain is the "spacer domain". The spacer domain can include a constant IgGl hinge-CH2-CH3 Fc domain. In some cases, the spacer domain and the transmembrane domain are derived from CD8. The intracellular signaling domain that mediates T cell activation can include a CD3 zeta co-receptor signaling domain derived from the C region of the TCR alpha and beta chains and one or more costimulatory domains.

[0150] In the disclosed CARs, the antigen binding domain is typically a disclosed anti-LANCL1 antibody. In some forms, the antigen binding domain is derived from an antibody, such as a disclosed anti-LANCL1 antibody. As noted above, the term antibody herein refers to a natural or synthetic polypeptide that binds a target antigen, and such antibodies can form part or all of the antigen binding domain of a CAR. The term includes polyclonal and monoclonal antibodies, including intact antibodies and functional (e.g., antigen binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD. The antigen binding domain of a CAR can contain a complementarity determining region (CDR) of an antibody, a variable region of an antibody, and / or an antigen binding fragment thereof. For example, the antigen binding domain of a LANCL1 CAR can be derived from a disclosed anti-LANCL1 antibody as described above. In some forms, the antigen binding domain can include a F(ab')2, Fab', Fab, Fv, or scFv.

[0151] In some forms, the CAR includes one or more spacer domains (also referred to as hinge domains) between the extracellular antigen binding domain and the transmembrane domain. A spacer domain is an amino acid segment that is typically found between two domains of a protein and can allow flexibility of the protein and movement of one or both domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain can be used. The spacer domain can be a naturally occurring protein spacer domain or hinge domain. In some forms, the hinge domain is derived from CD8a, such as a portion of the hinge domain of CD8a, e.g., a fragment containing at least 5 (e.g., 5, 10, 15, 20, 25, 30, 35, or 40) contiguous amino acids of the hinge domain of CD8a. Hinge domains of antibodies (e.g., IgG, IgA, IgM, IgE, or IgD antibodies) can also be used. In some forms, the hinge domain is the hinge domain that links the constant CH1 domain and CH2 domain of an antibody. Non-naturally occurring peptides can also be used as spacer domains. For example, the spacer domain can be a peptide linker, such as a (GxS)n linker, where x and n independently can be an integer of 3 or greater, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater.

[0152] In some forms, the CAR includes a transmembrane domain, which can be fused directly or indirectly to the antigen binding domain. The transmembrane domain can be derived from a natural source or synthetic source. In some forms, the transmembrane domain of the CAR includes a transmembrane domain of an alpha, beta, or zeta chain of a T cell receptor, CD8, CD4, CD28, CD137, CD80, CD86, CD152 (CTLA-4), or PD1, or a portion thereof. The transmembrane domain can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some forms, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some forms, the protein segment is at least about 15 amino acids, such as at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, such as in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776.

[0153] The intracellular signaling domain is responsible for activating at least one normal effector function of the immune effector cell expressing the CAR. The term effector function refers to a specialized function of a cell. For example, an effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. In some forms, the intracellular signaling domain includes a zeta chain of a T cell receptor or any homolog thereof (e.g., eta, delta, gamma, or epsilon), an MB1 chain, B29, Fc RIII, Fc Rl, and combinations of signaling molecules, such as CD3 zeta and CD28, 4-1BB, OX40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the activating protein family such as Fc gamma RIII and Fc epsilon RI can be used.

[0154] In addition to the stimulation of the antigen-specific signal, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation, and survival, as well as activation of the effector function of the cell. Thus, in some forms, the CAR includes at least one co-stimulatory signaling domain. The term co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response, such as an effector function. The co-stimulatory signaling domain can be a cytoplasmic signaling domain from a co-stimulatory protein that transduces a signal and modulates a response mediated by an immune cell, such as a T cell, NK cell, macrophage, neutrophil, or eosinophil. In some forms, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

[0155] CARs can be used to generate immune response cells (such as T cells) specific for a selected target (such as a malignant cell) with a wide variety of receptor chimeric constructs that have been described (see U.S. Patent Nos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and PCT Publication WO 9215322, each of which is specifically incorporated by reference herein in its entirety). Alternative CAR constructs can be characterized as belonging to successive generations. First generation CARs typically include a single chain variable fragment of an antibody specific for an antigen, for example including a VL linked to a VH of a specific antibody, linked by a flexible linker (for example by a CD8a hinge domain and CD8a transmembrane domain) to a transmembrane and intracellular signaling domain of CD3 zeta or FcR gamma (scFv-CD3 zeta or scFv-FcR gamma; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936, each of which is specifically incorporated by reference herein in its entirety). Second generation CARs incorporate one or more intracellular domains of costimulatory molecules such as CD28, OX40 (CD134) or 4-1BB (CD137) (for example scFv-CD28 / OX40 / 4-1BB-CD3 zeta; see U.S. Patent Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761, each of which is specifically incorporated by reference herein in its entirety). Third generation CARs include combinations of costimulatory intracellular domains, for example CD3 zeta chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3 zeta or scFv-CD28-OX40-CD3 zeta; see U.S. Patent No. 8,906,682; U.S. Patent No. 8,399,645; U.S. Patent No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000, each of which is specifically incorporated by reference herein in its entirety). Alternatively, costimulation can be programmed by expressing a CAR in an antigen-specific T cell selected to be activated and expanded upon engagement of its native alpha beta TCR with concomitant costimulation by antigen on a professional antigen presenting cell. Any of the first, second or third generation CARs described above can be used in accordance with the disclosed compositions and methods.

[0156] E. Nucleic Acids Encoding Anti-LANCL1 Antibodies and Antigen Binding Fragments

[0157] Also provided are isolated nucleic acids and vectors that encode or express anti-LANCL1 monoclonal antibodies. As used herein, an “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules present in the mammalian genome, including the nucleic acids normally located on either side or both sides of the nucleic acid in the mammalian genome.

[0158] An isolated nucleic acid can be, for example, a DNA molecule, provided that one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as a recombinant DNA incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpesvirus), or incorporated into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid, such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid that is present within hundreds to millions of other nucleic acids in, for example, a cDNA library or a genomic library, or a gel slice containing a restriction digest of genomic DNA, is not considered to be an isolated nucleic acid.

[0159] A nucleic acid can be single-stranded or double-stranded, and can be in the sense or antisense orientation, or can be complementary to a reference sequence. A nucleic acid can be DNA, RNA, or a nucleic acid analog. A nucleic acid analog can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modifications can improve, for example, the stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine for deoxy cytidine. Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxy phosphate backbone is replaced with a pseudopeptide backbone and four base moieties are retained. See, e.g., Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoramidite or alkylphosphonate triester backbone.

[0160] 1. A nucleic acid sequence encoding a VH

[0161] A nucleic acid sequence encoding the heavy chain variable region (VH) of the anti-LANCL1 monoclonal antibody is:

[0162]

[0163] TGA represents a stop codon.

[0164] A second nucleic acid sequence encoding the VH of the anti-LANCL1 monoclonal antibody is:

[0165]

[0166] TGA represents a stop codon.

[0167] The complementarity determining regions (CDRs) are shown in bold italic in SEQ ID NO: 38 and SEQ ID NO: 39, including the first, second, and third CDR regions CDR-1, CDR-2, and CDR-3, respectively. An exemplary nucleic acid sequence of CDR-1 of the heavy chain variable region includes GACTACTACATGAAC (SEQ ID NO: 40) and GACTACTATATGAAC (SEQ ID NO: 41). An exemplary nucleic acid sequence of CDR-2 of the heavy chain variable region includes GATATTAATCCTAACAATGGTGGTGCTAGCTACAACCAGAAGTTCAAGGGC (SEQ ID NO: 42), and GTTATTAATCCTTACAACGGTCATACTAACTACAACCAGAAGTTCAAGGGC (SEQ ID NO: 43). An exemplary nucleic acid sequence of CDR-3 of the heavy chain variable region includes TCGGGCGATGGTTACTACTTTGCTTCC (SEQ ID NO: 44) and TTCCCTTACTACGGTAGTAGCTATAGGGTTGACTAC (SEQ ID NO: 45).

[0168] In some forms, the first, second, and third CDR regions of the heavy chain variable region include a variant nucleic acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45.

[0169] The framework region (FR) regions are underlined in SEQ ID NO: 38 and SEQ ID NO: 39, and include first, second, third, and fourth FR regions FR1, FR2, FR3, and FR4, respectively. An exemplary nucleic acid sequence for FR1 of the heavy chain variable region includes GAGGTCCAGTTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACT (SEQ ID NO: 46) and GAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACT (SEQ ID NO: 47). An exemplary sequence for FR2 of the heavy chain variable region is TGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGA (SEQ ID NO: 48). Exemplary nucleic acid sequences for FR3 of the heavy chain variable region include AAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGA (SEQ ID NO: 49) and AAGGCCACATTGACTGTTGACAAGTCCTCCAACACAGCCTACATGGAGCTCAACAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGA (SEQ ID NO: 50). An exemplary sequence for FR4 of the heavy chain variable region includes TGGGGCCAAGGGACTCTGCTCACTGTCTCTGCA (SEQ ID NO: 51) and TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 52). In some forms, the first, second, third, and fourth FR regions of the heavy chain variable region include a variant nucleic acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and / or SEQ ID NO: 52.

[0170] The nucleic acid sequence encoding the VH signal peptide is shown in italic in SEQ ID NO: 38 and SEQ ID NO: 39. Exemplary nucleic acid sequences encoding the VH signal peptide include ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCTCTCT (SEQ ID NO: 53) and ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCT (SEQ ID NO: 54).

[0171] In some forms, the nucleic acid sequence encoding the VH signal peptide includes a variant nucleic acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 53 or SEQ ID NO: 54.

[0172] 2. Nucleic acid encoding VL

[0173] A nucleic acid sequence encoding the light chain variable region (VL) of the anti-LANCL1 monoclonal antibody is:

[0174]

[0175] TAG represents a stop codon.

[0176] A second nucleic acid sequence encoding the light chain variable region (VL) of the anti-LANCL1 monoclonal antibody is:

[0177]

[0178] TAG represents a stop codon.

[0179] The complementarity determining regions (CDRs) are shown in bold italic in SEQ ID NO: 55 and SEQ ID NO: 56, including first, second, and third CDR regions CDR-1, CDR-2, and CDR-3, respectively. An exemplary nucleic acid sequence for CDR-1 of the light chain variable region includes: AGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCAC (SEQ ID NO: 57) and AGGGCCAGCCAAAGTATTAGCAACAACCTACA (SEQ ID NO: 58). An exemplary nucleic acid sequence for CDR-2 of the light chain variable region includes: CTTGTATCCAACCTAGAATCT (SEQ ID NO: 59), and TATGCTTCCCAGTCCATCTCT (SEQ ID NO: 60). An exemplary nucleic acid sequence for CDR-3 of the light chain variable region includes: CAGCACATTAGGGAGCTTACACG (SEQ ID NO: 61) and CAACAAATTAACAGCTGGCCTCTCACG (SEQ ID NO: 62).

[0180] In some forms, the first, second, and third CDR regions of the light chain variable region include variant nucleic acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NOs: 57-62.

[0181] The framework regions (FR) regions are shown underlined in SEQ ID NO: 55 and SEQ ID NO: 56, including first, second, third, and fourth FR regions FR1, FR2, FR3, and FR4, respectively. An exemplary nucleic acid sequence for FR1 of the light chain variable region includes: GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATAC (SEQ ID NO: 63) and GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGC (SEQ ID NO: 64). An exemplary nucleic acid sequence for FR2 of the light chain variable region includes: TGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTAT (SEQ ID NO: 65) and CTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAG (SEQ ID NO: 66).

[0182] An exemplary nucleic acid sequence for FR3 of the heavy chain variable region includes GGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGT (SEQ ID NO: 61) and GGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGT (SEQ ID NO: 62). An exemplary nucleic acid sequence for FR4 of the heavy chain variable region includes TTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 63) and TTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 64). In some forms, the first, second, third, and fourth FR regions of the heavy chain variable region include variant nucleic acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NOs: 61-64. (SEQ ID NO: 67) and GGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGT (SEQ ID NO: 68). An exemplary sequence for FR4 of the light chain variable region includes TTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 69) and TTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 70). In some forms, the first, second, third, and fourth FR regions of the light chain variable region include variant nucleic acid sequences having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NOs: 63-70.

[0183] An exemplary nucleic acid sequence encoding a VL signal peptide is shown in italic in SEQ ID NO: 55 and SEQ ID NO: 56. Exemplary nucleic acid sequences encoding a VH signal peptide include ATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT (SEQ ID NO: 71) and ATGGTTTTCACACCTCAGATACTTGGACTTATGCTTTTTTGGATTTCAGCCTCCAGAGGT (SEQ ID NO: 72). In some forms, the nucleic acid sequence encoding a VL signal peptide includes a variant nucleic acid sequence having about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 72.

[0184] Nucleic acids, such as those described above, can be inserted into a vector for expression in a cell. As used herein, a “vector” is a replicon, such as a plasmid, bacteriophage, or cosmid, into which another DNA segment can be inserted and thereby be replicated. A vector can be an expression vector. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0185] The nucleic acid in the vector can be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct in a manner which enables the expression control sequence to control expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that is comprised of a region of a DNA molecule, usually within 100 nucleotides upstream of the transcription initiation point (usually near the start site for RNA polymerase II). In order for a coding sequence to be under the control of a promoter, the translational start site of the polypeptide's translation reading frame must be positioned between one and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity with respect to time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcriptional start site. Enhancers can also be located downstream from the transcriptional start site. A coding sequence is "operably linked" and "under the control" of an expression control sequence in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which can then be translated into a protein encoded by the coding sequence, when the RNA polymerase is able to transcribe the coding sequence into mRNA.

[0186] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived, for example, from bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0187] Expression vectors can include a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxy- or amino-terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein, and protein A.

[0188] A vector containing a nucleic acid to be expressed can be transferred into a host cell. The term "host cell" is intended to include prokaryotic and eukaryotic cells, which are capable of incorporating and reproducing the recombinant expression vector. As used herein, "transformed" and "transfected" encompass the introduction of nucleic acid molecules (e.g., vectors) into a cell by a variety of techniques, not limited to particular techniques. While not limited to a particular technique, many of these techniques are well established in the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride-mediated transformation. Mammalian cells can be transfected with nucleic acids by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., prokaryotic or eukaryotic cells such as CHO cells) can be used, for example, to produce anti-LANCL1 polypeptides described herein.

[0189] F. Formulations

[0190] The disclosed compositions can be formulated as part of a pharmaceutical formulation for in vivo administration to a subject. The disclosed compositions containing anti-LANCL1 antibodies and / or antigen binding fragments can be formulated as part of a pharmaceutical formulation for administration to a subject in need thereof. Generally, a pharmaceutical formulation contains an anti-LANCL1 antibody or antigen binding fragment thereof, a pharmaceutically acceptable excipient, and / or a pharmaceutically acceptable carrier, which is suitable for administration into the body of a subject (e.g., a human patient).

[0191] In some forms, the disclosed formulations can be used in monotherapy or combination therapy. Combination therapy can include administration of effective amounts of anti-LANCL1 antibodies together in the same mixture or in separate formulations. In some embodiments, the pharmaceutical formulation can include one or more additional active agents. Thus, in some embodiments, the pharmaceutical formulation comprises two, three, or more active agents.

[0192] Pharmaceutical formulations can be formulated to be a pharmaceutical dosage unit, referred to as unit dosage form. The phrase "dosage unit form" refers to physically discrete units suitable for unitary dosing to the patient to be treated. It will be understood, however, that the total daily usage of the compositions will be decided by the attending physician within the scope of sound medical judgment. The therapeutically effective dose is initially estimated in cell culture assays or in animal models, usually mice, rats, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information is then used to determine useful doses and routes of administration in humans.

[0193] 1. Delivery Vehicles

[0194] Suitable delivery vehicles for the disclosed anti-LANCL1 antibodies are known in the art. For example, in some embodiments, the active agent is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the composition can be incorporated into a vehicle such as a polymeric microparticle, which provides for controlled release of the active agent. In some embodiments, release of the drug is controlled by diffusion of the active agent from the microparticle and / or by degradation of the polymeric particle by hydrolysis and / or enzymatic degradation. Suitable polymers include ethyl cellulose and other natural or synthetic cellulose derivatives. Polymers that slowly dissolve and form gels in aqueous environments, such as hydroxypropyl methylcellulose or polyethylene oxide, can also be suitable as materials for drug-containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids such as polylactic acid (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0195] 2. Exemplary Formulations

[0196] Pharmaceutical compositions can be administered by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), enteral, or transmucosal routes of administration or using a bioerodible insert, and can be formulated in dosage forms appropriate for each route of administration.

[0197] In certain embodiments, the composition is administered topically, e.g., by direct injection into the site to be treated (e.g., injection into a tumor). In some embodiments, the composition is injected or otherwise administered directly into the vasculature, onto vascular tissue at or proximate to the intended treatment site (e.g., adjacent to a tumor). Typically, topical administration results in increased local concentrations of the composition, which are greater than those achievable by systemic administration. Targeting of the molecule or formulation can be used to achieve more selective delivery.

[0198] a. Formulations for parenteral administration

[0199] Anti-LANCL1 antibodies and antigen-binding fragments can be formulated for parenteral administration. For example, parenteral administration can include administration to a patient intravenously, intradermally, intraperitoneally, intramuscularly, subcutaneously, by injection, by infusion, etc.

[0200] In some forms, the anti-LANCL1 antibodies and / or antigen binding fragments can be administered in aqueous solution by parenteral injection. The formulations can also be in the form of suspensions or emulsions. Typically, the pharmaceutical compositions include an effective amount of the anti-LANCL1 antibodies and / or antigen binding fragments, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluent sterile water, various buffers containing amounts (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength of buffer saline; and optionally, additives such as detergents and solubilizers (e.g., TWEEN® 20, TWEEN® 80 also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Formulations can be lyophilized and reconstituted prior to use. The formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0201] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, e.g., solutions or suspensions; solid forms suitable for reconstitution into solutions or suspensions after addition of a reconstitution medium prior to injection; emulsions, e.g., water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions and microemulsions thereof, liposomes or emulsomes.

[0202] The carrier(s) can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, and the like), and combinations thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0203] Solutions and dispersions of the active compounds as free acids or bases or their pharmacologically acceptable salts can be prepared in water or another solvent or dispersion medium, as appropriate, in admixture with one or more pharmaceutically acceptable excipients, including but not limited to surfactants, dispersants, emulsifiers, pH adjusters, viscosity adjusters, and combinations thereof.

[0204] Suitable surfactants can be anionic, cationic, amphoteric, or non-ionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium salts of long chain alkyl sulfonates and alkyl aryl sulfonates, such as sodium dodecylbenzene sulfonate; dialkyl sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sulfosuccinates, such as bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimide, stearyl dimethyl benzyl ammonium chloride, polyoxyethylene and cocamine. Examples of non-ionic surfactants include ethylene glycol monostearate, myristyl propyl glycolate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 distearate, PEG-400 monododecanoate, polyoxyethylene monododecanoate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alaninate, sodium N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sultaine.

[0205] The formulation can contain a preservative to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation can also contain an antioxidant to prevent degradation of the active agent.

[0206] The formulation is typically buffered to a pH of 3-8 for parenteral administration after reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0207] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0208] A sterile injectable solution can be prepared by incorporating the active compound in the required amount in an appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powder can be prepared in a porous nature in the granules, which can increase the dissolution of the granules. Methods of preparing porous granules are well known in the art.

[0209] The parenteral formulations described herein can be formulated for controlled release, including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.

[0210] b. Enteric formulations

[0211] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be prepared using compression or molding techniques which are well known in the art. Gelatin or non-gelatin capsules can be prepared using well-known techniques which can encapsulate liquid, solid, and semi-solid fill materials.

[0212] The formulations can be prepared using a pharmaceutically acceptable carrier. As used herein generally, a "carrier" includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrants, bulking agents, stabilizers, and combinations thereof.

[0213] The carrier also includes all components of a coating composition, which can include plasticizers, pigments, colorants, stabilizers, and glidants. Delayed release dosage formulations can be prepared as described in standard references. These references provide information on carriers, materials, equipment, and methods for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0214] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as ethyl phthalate cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate; polyvinyl acetate phthalate, acrylic polymers and copolymers, and the methacrylic acid resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0215] Additionally, the coating material can contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilizers, pore formers, and surfactants.

[0216] Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also known as “fillers,” are generally necessary to increase the bulk of the solid dosage form, such that practical dimensions are provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dibasic calcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate, and powdered sugar.

[0217] Binders serve to impart cohesiveness to the solid dosage form, thereby ensuring that the tablet or bead or granule remains intact after formation of the dosage form. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, celluloses (including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose), and veegum, and synthetic polymers such as acrylic and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / poly methacrylic acid, and polyvinylpyrrolidone.

[0218] Lubricants serve to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

[0219] Disintegrants serve to facilitate disintegration or “decomposition” of the dosage form after administration, and generally include, but are not limited to, starches, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums, or cross-linked polymers, for example, cross-linked PVP (Polyplasdone® XL from GAF Chemical).

[0220] Stabilizers serve to inhibit or retard decomposition reactions of the drug, which include, for example, oxidation reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; vitamin E, tocopherols and salts thereof; sulfites such as sodium metabisulfite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0221] Oral dosage forms, such as capsules, tablets, solutions, and suspensions, can be formulated for controlled release. For example, one or more compounds and optionally one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in soft or hard gelatin or non-gelatin capsules or dispersed in a dispersion medium to form an oral suspension or syrup. The particles can be formed from the drug and a controlled release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled release coatings prior to incorporation into the finished dosage form.

[0222] In another embodiment, one or more compounds and optionally one or more additional active agents are dispersed in a matrix material that forms a gel or emulsion upon contact with an aqueous medium, such as physiological fluids. In the case of a gel, the matrix swells entrapping the active agent, which is released slowly over time by diffusion and / or degradation through the matrix material. Such matrices can be formulated into tablets or as a filling material for hard and soft capsules.

[0223] In yet another embodiment, one or more antibodies or antigen-binding fragments thereof and optionally one or more additional active agents are formulated into a marketed oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, for example, a delayed release coating or extended release coating. The one or more coatings can also contain the antibody or antigen-binding fragment thereof and / or additional active agents.

[0224] Extended release formulations are typically prepared as either diffusion or osmotic systems known in the art. Diffusion systems are generally comprised of two types of devices, reservoirs and matrices, and are well known and described in the art. Matrix devices are typically prepared by compressing the drug with a slowly dissolving polymeric carrier into tablet form. The three main types of materials used to prepare matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, polymethyl methacrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and AVICEL® 934, polyethylene oxides, and mixtures thereof. Fatty compounds include, but are not limited to, various waxes, such as carnauba wax and glycerol tri-stearate, and wax-like substances, including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.

[0225] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semipermeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low and high permeable coating materials in appropriate proportions.

[0226] Devices with the different drug release mechanisms described above can be combined into a final dosage form comprising a single or multiple units. Examples of multiple units include, but are not limited to, multi-layer tablets and capsules containing tablets, beads, or granules. Immediate release portions can be added to extended release systems by applying an immediate release layer on top of an extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

[0227] Extended release tablets containing hydrophilic polymers are prepared by techniques generally known in the art, such as direct compression, wet granulation or dry granulation processes. Their formulation typically incorporates a polymer, diluent, binder and lubricant, as well as the active pharmaceutical ingredient. Typical diluents include inert, powdered substances such as starch, powdered cellulose, particularly crystalline and microcrystalline cellulose, sugars such as dextrose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium or sodium phosphates, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose and dextrose. Natural and synthetic gums, including acacia, alginates, methylcellulose and polyvinylpyrrolidone can also be used. Polyethylene glycols, hydrophilic polymers, ethyl cellulose and waxes can also act as binders. Lubricants are necessary in tablet formulations to prevent sticking of the tablet and punch to the dies. Lubricants are selected from smooth solids such as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oil.

[0228] Extended release tablets containing waxy materials are typically prepared using methods known in the art such as direct blending, coacervation and water dispersion methods. In the coacervation method, the drug is mixed with the waxy material and spray coacervated or coacervated and sieved and processed.

[0229] Delayed release formulations can be produced by coating the solid dosage form with a polymeric film that is insoluble in the acidic environment of the stomach and soluble in the neutral environment of the small intestine.

[0230] Delayed release dosage units can be prepared, for example, by coating the drug or drug-containing composition with a selected coating material. The drug-containing composition can be, for example, a tablet for incorporation into a capsule, a tablet used as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, granules or granulates for incorporation into a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolysable, gradually water-soluble and / or enzymatically degradable polymers, and can be conventional "enteric" polymers. As will be appreciated by those skilled in the art, enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract, or erode slowly as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly the colon. Suitable coating materials for achieving delayed release include, but are not limited to, cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and sodium carboxymethyl cellulose; acrylic polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and other methacrylic acid resins commercially available under the trade name Eudragit® (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 and above), EUDRAGIT® S (soluble at pH 7.0 and above due to higher esterification), and EUDRAGITS® NE, RL and RS (water-insoluble polymers with varying degrees of permeability and swellability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonate copolymer and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials can also be used. Multi-layer coatings using different polymers can also be applied.

[0231] The preferred coating weight of a particular coating material can be readily determined by one skilled in the art by evaluating the individual release profiles of tablets, beads and granules prepared with varying amounts of each coating material. It is the combination of material, method and application form that produces the desired release profile, which can only be determined from clinical studies.

[0232] The coating composition can include conventional additives such as plasticizers, pigments, colorants, stabilizers, flow aids, and the like. Plasticizers are generally present to reduce the brittleness of the coating and typically comprise about 10 wt.% to 50 wt.% of the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil, and acetylated monoglycerides. Stabilizers are preferably used to stabilize the particles in the dispersion. Typical stabilizers are non-ionic emulsifiers such as sorbitan esters, polysorbates, and polyvinylpyrrolidone. Flow aids are recommended to reduce the sticking effect during film formation and drying and will typically comprise about 25 wt.% to 100 wt.% of the weight of the polymer in the coating solution. One effective flow aid is talc. Other flow aids such as magnesium stearate and glycerol monostearate can also be used. Pigments such as titanium dioxide can also be used. Small amounts of antifoaming agents, such as silicones (e.g., dimethyl silicone), can also be added to the coating composition.

[0233] Preferably, the aqueous solution is water, a physiologically acceptable aqueous solution containing salts and / or buffers (e.g., phosphate buffered saline (PBS)), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to those skilled in the art and include, but are not limited to, distilled water, deionized water, purified water or ultra-pure water, saline, phosphate buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions can include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, and gum tragacanth, as well as wetting agents such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0234] In another embodiment, solvents such as ethanol, acetone, ethyl acetate, tetrahydrofuran, diethyl ether, and propanol, as low toxicity organic (i.e., non-aqueous) Class 3 residual solvents, can be used in the formulation. The solvent is selected based on its ability to easily nebulize the formulation. The solvent should not react deleteriously with the composition. An appropriate solvent that dissolves the compound or forms a suspension of the compound should be used. The solvent should be volatile enough to enable an aerosol of the solution or suspension to be formed. Additional solvents or nebulizing agents such as freon can be added as needed to increase the volatility of the solution or suspension.

[0235] Due to its hydrophobic nature, the dried lipid powder can be directly dispersed in ethanol. For lipids stored in organic solvents such as chloroform, a desired amount of the solution is placed in a vial and the chloroform is evaporated under a stream of nitrogen to form a dried thin film on the surface of the glass vial. The film readily swells when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Non-aqueous suspensions of lipids can also be prepared in anhydrous ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0236] III. Methods of use

[0237] Methods of treatment are also provided and can be used alone or in combination with other methods disclosed herein, such as the disclosed methods of detection, diagnosis, prognosis, and treatment monitoring. The methods generally include administering to a subject in need thereof an effective amount of a disclosed composition, e.g., a humanized anti-LANCL1 monoclonal antibody, to treat the subject. This is particularly true where the disease or condition is characterized by increased expression or presence of LANCL1 protein.

[0238] A. Effective amount

[0239] An effective amount or therapeutically effective amount of a pharmaceutical composition including, e.g., an anti-LANCL1 antibody, an anti-LANCL1 antigen binding fragment, or a cell thereof (such as a therapeutic T cell) can be a dose sufficient to treat, inhibit or alleviate one or more symptoms of a disease or disorder, such as hepatocellular carcinoma, or otherwise provide a desired pharmacologic and / or physiologic effect, e.g., reduce, inhibit or reverse one or more underlying pathophysiological mechanisms underlying a disease or disorder, e.g., a cancer or autoimmune disease. Any LANCL1 -positive liver cancer can be treated using the disclosed compositions and formulations thereof. Exemplary cancers that can be treated include, but are not limited to, fibrolamellar hepatocellular carcinoma, bile duct cancer, and cholangiocarcinoma.

[0240] In some forms, when a pharmaceutical composition is administered, the amount administered can be expressed as an amount effective to achieve a desired anti-cancer effect in a recipient. For example, in some forms, the amount of a pharmaceutical composition is effective to inhibit the viability or proliferation of hepatocellular carcinoma cells in a recipient. In some forms, the amount of a pharmaceutical composition comprising an anti-LANCL1 antibody and a cell modified thereof, such as a therapeutic T cell, is effective to reduce tumor burden or reduce the total number of cancer cells in a recipient, and combinations thereof. In other forms, the amount of a pharmaceutical composition comprising an anti-LANCL1 antibody is effective to reduce one or more symptoms or signs of HCC in a cancer patient, or signs of a liver tumor in a patient having a liver cancer.

[0241] The effective amount of a pharmaceutical composition will vary from subject to subject depending on the species, age, weight, and general condition of the subject, the severity of the condition being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for each pharmaceutical composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the pharmaceutical compositions can be empirically determined. In some forms, the dosage range for administering the compositions is large enough to effect a reduction in cancer cell proliferation or viability, or for example, a reduction in tumor burden.

[0242] Preferably, the dosage is not so large as to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, and the like. Generally, the dosage will vary with the age, condition, and sex of the patient, the route of administration, and the other drugs being administered in combination or alternation, as well as the type, stage and location of the disease to be treated. The dosage can be adjusted by the individual physician in the event of any counter-indications. It should also be understood that the effective dosage of the composition can increase or decrease over the course of treatment. Changes in dosage can result and become apparent from diagnostic assays.

[0243] The dosage can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. The optimum dosage regimen can be calculated from measurements of the accumulation of drug in the body of the subject or patient. The optimum dosage can vary depending on the relative potency of individual pharmaceutical compositions, and can generally be estimated based on EC 50 values found to be effective in in vitro and in vivo animal models.

[0244] For example, in some embodiments, the antibody is packaged in an air-tight sealed container (e.g., an ampule or a pouch) that indicates the amount of antibody. In some embodiments, the antibody is provided as a dry sterile lyophilized powder or an anhydrous concentrate in an air-tight sealed container, and can be reconstituted, e.g., with water or saline, to an appropriate concentration for administration to a subject. Preferably, the antibody of the application is provided as a dry sterile lyophilized powder in an air-tight sealed container in a unit dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized antibody can be stored in its original container between 2°C and 8°C, and the antibody can be administered within 12 hours, preferably within 6 hours, 5 hours, 3 hours, or 1 hour, after reconstitution. In alternative embodiments, the antibody can be provided in a liquid form in an air-tight sealed container that indicates the amount and concentration of the antibody, fusion protein, or conjugated molecule. Preferably, the antibody in liquid form is provided in an air-tight sealed container at a concentration of at least 1 mg / ml, more preferably at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml of antibody.

[0245] In some formulations, the dose of anti-LANCL1 antibody administered to the patient ranges from about 0.01 mg / kg to 100 mg / kg of patient body weight. Preferably, the dose administered to the patient is 0.01 mg / kg to 20 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 5 mg / kg, 0.01 mg / kg to 2 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.01 mg / kg to 0.75 mg / kg, 0.01 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.25 mg / kg, 0.01 to 0.15 mg / kg, 0.01 to 0.10 mg / kg, 0.01 to 0.05 mg / kg, or 0.01 to 0.025 mg / kg of patient body weight. Specifically, the present invention considers doses administered to patients of 0.2 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg. Doses as low as 0.01 mg / kg can exhibit considerable pharmacodynamic effects. A dose level of 0.10–1 mg / kg is expected to be most suitable. Higher doses (e.g., 1 mg / kg to 60 mg / kg) have also been considered. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to exogenous peptides. Therefore, lower doses and lower frequencies of administration of human antibodies are generally possible. Furthermore, the dosage and frequency of antibody administration can be reduced by modifications (such as lipolysis) to enhance antibody uptake and tissue penetration.

[0246] Generally speaking, it can be said that a pharmaceutical composition containing CAR cells described in this article can achieve 10 4 Up to 10 9 Cells / kg body weight, preferably 10 5 Up to 10 7 Administered at a dose of cells / kg body weight (including all integer values ​​within those ranges). In some forms, patients may be treated by infusion of the disclosed pharmaceutical composition containing approximately 10... 4 Up to 10 12 One or more cells per square meter of body surface (cells / m²) 2 CAR-expressing cells (e.g., T cells) within the range of )

[0247] Injections and infusions of the disclosed compositions can be repeated as frequently and as many times as can be tolerated by the patient until the desired response is achieved. Thus, the antibody and CAR cell compositions can also be administered at these dosages once or more times. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be determined by those of skill in the medical art by monitoring the patient's signs of disease and adjusting the treatment accordingly. In some forms, the unit dosage is a unit dosage form for intravenous injection. In some forms, the unit dosage is a unit dosage form for oral administration. In some forms, the unit dosage is a unit dosage form for inhalation. In some forms, the unit dosage is a unit dosage form for intratumoral injection.

[0248] Treatment can continue for an amount of time sufficient to achieve one or more desired therapeutic goals (e.g., reduction in the amount of cancer cells relative to the amount at the start of treatment or complete absence of cancer cells in the recipient). Treatment can continue for a desired period of time and the progress of the treatment can be monitored using any means known for monitoring the progress of anti-cancer treatment in a patient. In some forms, administration occurs on each day of treatment, or each week, or each portion of a week. In some forms, the treatment regimen occurs over the course of up to two, three, four, or five days, weeks, or months, or for up to 6 months, or for more than 6 months, e.g., for up to one, two, three, or up to five years.

[0249] The efficacy of administering a particular dosage of a pharmaceutical composition comprising modified cells, such as therapeutic T cells, according to the methods described herein can be determined by evaluating the medical history, signs, symptoms, and objective laboratory tests known to be useful in assessing the status of a subject in need of treatment for a cancer or other disease and / or condition. These signs, symptoms, and objective laboratory tests will change depending on the particular disease or condition being treated or prevented, as is well known to any clinician treating such patients or researcher conducting experiments in the field. For example, a particular treatment regimen will be considered effective if, based on comparisons to appropriate control groups and / or knowledge of the normal progression of the disease in the general population or in a particular individual: (1) the subject's physical condition shows improvement (e.g., a tumor has partially or completely regressed), (2) the progression of the disease or condition shows stabilization or slowing or reversal, or (3) the need for other medications to treat the disease or condition is reduced or eliminated. In some forms, efficacy is assessed as a measure of reduction in tumor volume and / or tumor mass at a particular time point after treatment (e.g., 1-5 days, weeks, or months).

[0250] B. Mode of Administration

[0251] Any of the disclosed compositions can be combined with a pharmaceutically acceptable carrier for therapeutic use. The compositions described herein can be conveniently formulated into pharmaceutical compositions, consisting of one or more compounds and a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, which discloses typical carriers and conventional methodology for the preparation of pharmaceutical compositions, which can be used in conjunction with the preparation of formulations of the therapeutic agents described herein, and which is hereby incorporated by reference. Most typically, these will be standard carriers for the administration of compositions to humans. In one aspect, these include solutions, such as sterile water, saline, and buffered solutions at physiological pH, for both humans and non-human animals. Other therapeutics can be administered according to standard procedures used by those of skill in the art.

[0252] The pharmaceutical compositions described herein, including antibodies or modified cells such as therapeutic T cells, can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like, in addition to the therapeutic(s) selected.

[0253] Pharmaceutical compositions containing antibodies or modified cells such as therapeutic T cells and optionally one or more additional therapeutic agents can be administered to a subject in a variety of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Thus, for example, the pharmaceutical composition can be administered intravenously, or directly injected into a specific site, for example into or around a tumor. In addition, the pharmaceutical composition can be administered orally or parenterally, for example, by subcutaneous, intramuscular, intraperitoneal, intra-arterial (such as by hepatic arterial infusion or trans-arterial chemoembolization), intralymphatic, intravenous, intrathecal routes. In some forms, the composition is administered directly into a tumor or tissue, for example stereotactically.

[0254] In some forms, pharmaceutical compositions containing LANCL1-specific antibodies can contact healthy cells and / or tumor cells or cancer cells of a subject. In some embodiments, LANCL1-specific antibodies can contact cells in the liver, including cancerous hepatocellular carcinoma cells. In some embodiments, humanized LANCL1-specific antibodies can inhibit hepatocellular carcinoma and other solid tumors. LANCL1-specific antibodies can bind to proteins on the surface of subject cells, including, for example, cancer cells. In some embodiments, LANCL1-specific antibodies can inhibit liver tumor-initiating cell (LTIC) characteristics, i.e., and not limited to, in vitro spheroidization, in vivo tumorigenicity, chemoresistance to chemotherapeutic agents (including but not limited to cisplatin, 5-fluorouracil, etc.), and expression of cancer stemness-related genes. In some embodiments, LANCL1-specific antibodies can inhibit tumor cell growth. LANCL1-specific antibodies can inhibit tumor cell growth by modulating LANCL1 expression and / or LANCL1 function. In some embodiments, LANCL1-specific antibodies can modify intracellular ROS levels in cells, particularly HCC cells. For example, LANCL1 on the cell surface can suppress intracellular ROS levels; therefore, LANCL1-specific antibodies can block this function, leading to an increase in intracellular ROS levels, which may be lethal or damaging to HCC cells.

[0255] Parenteral administration (if used) is typically characterized by injection. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, preferably in solid form as solutions or suspensions in liquids prior to injection, or as emulsions. Recently revised methods of parenteral administration involve the use of slow-release or sustained-release systems to maintain a constant dose. See, for example, US Patent No. 3,610,795, which is incorporated herein by reference. Suitable routes of parenteral administration include intravascular administration (e.g., intravenous bolus, intravenous infusion, intra-arterial bolus, intra-arterial infusion, and catheter infusion into the vasculature); peritissue and intratissue injection (e.g., intraocular, intraretinal, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as via an osmotic pump); and direct application via catheters or other placement devices (e.g., implants comprising porous, non-porous, or gel-like materials).

[0256] Formulations for parenteral use are those that are sterile and fluid, and include aqueous or non-aqueous solutions, suspensions, and emulsions, which can also contain buffers, diluents, and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcohol / water solutions, emulsions or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluids and nutrient supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents, and inert gases can also be present.

[0257] Administration of the pharmaceutical compositions containing antibodies or genetically modified cells (e.g., CAR cells) can be local (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic.

[0258] C. Combination therapy

[0259] HCC can be effectively treated by local ablation, surgical resection, or liver transplantation. Treatment options depend on tumor characteristics, severity of underlying liver dysfunction, age, other medical comorbidities, and available medical resources and local expertise. Catheter-based local therapy is used for patients with intermediate-stage cancer. Kinase and immune checkpoint inhibitors have proven to be effective treatment options for patients with advanced HCC.

[0260] The disclosed compositions can be administered to a subject in need thereof alone or in combination with one or more additional therapies or procedures, or can be an adjunct to one or more primary therapies or procedures. The additional therapy or procedure can be concurrent or sequential with the combination therapy. In some forms, the additional therapy is performed between cycles of the drug or during a drug holiday that is part of the combination therapy dosage regimen. In preferred forms, the additional therapy is a conventional treatment for cancer, more preferably a conventional treatment for liver cancer. For example, in some forms, the additional therapy or procedure is surgery, transplant surgery, radiation therapy, or chemotherapy.

[0261] Exemplary additional therapeutic agents include conventional cancer therapies, such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy. Most chemotherapeutic drugs can be classified into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antineoplastic agents. These drugs affect cell division or DNA synthesis and function in some way. Additional therapies include monoclonal antibodies and tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC® or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors). In particular forms, the combination therapy is used concurrently or sequentially with a regimen of a chemotherapeutic agent, such as gemcitabine (Gemzar), oxaliplatin (Eloxatin), cisplatin, doxorubicin (Doxil), capecitabine (Xeloda), mitoxantrone (Novantrone), docetaxel, or cabazitaxel. In some forms, the adjunct or additional therapy is part of a combination therapy.

[0262] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxins, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, innotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pegaspargase, pentostatin, plicamycin, procarbazine, raltitrexed, streptozocin, tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, uracil mustard, vinblastine, vincristine, vindesine, and vinorelbine.daunorubicin), lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and derivatives thereof, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0263] Representative anti-angiogenic agents include, but are not limited to, antibodies against vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and other anti-VEGF compounds, including aflibercept (EYLEA®); MACUGEN® (pegaptanim sodium, an anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigment epithelium-derived factor (PEDF); COX-2 inhibitors, such as celecoxib (CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12 (IL-12); thalidomide (THALOMID®) and its derivatives such as lenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozyme inhibitors, such as ANGIOZYME® (Sirna Therapeutics); multi-functional anti-angiogenic agents, such as NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors, such as sunitinib (SUTENT®); tyrosine kinase inhibitors, such as sorafenib (Nexavar®) and erlotinib (Tarceva®); antibodies against epidermal growth factor receptor, such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®), and other anti-angiogenic agents known in the art.

[0264] In some forms, the compositions and methods are used prior to or in conjunction with surgical resection of a tumor, for example, to prevent metastasis of a primary tumor. In some forms, the compositions and methods are used to enhance the body's own anti-tumor immune function. In some forms, the additional therapy or procedure can be simultaneous or sequential to the therapy or combination therapy. In some forms, the additional therapy is performed between cycles of the drug or during a drug holiday as part of the dosage regimen. For example, in some forms, the additional therapy or procedure is surgery.

[0265] IV. Kits

[0266] Compositions, reagents, and other materials for use in connection with the disclosed compounds and cells can be packaged together in any suitable combination, as a kit for performing or aiding in the performance of the methods. This is useful if the components in a given kit are designed and adapted to be used together in the methods. For example, a kit having one or more components for administration to a subject can include a pre-measured dose of the composition in a sterile needle, ampule, tube, container, or other suitable container. The kit can include instructions for the dosage and dosing regimen.

[0267] The kit can include printed instructions for administering the compound in the uses as described above. The instructional materials can include publications, records, charts, or any other medium of expression which can convey the usefulness of the compositions and methods of the kit. The compositions can include antibodies in solid (i.e., dry powder or lyophilized) form or as a solution, such as an aqueous solution.

[0268] In one exemplary form, the kit contains:

[0269] (a) one or more single unit dose compositions comprising a disclosed LANCL1 antibody and / or antigen binding fragment thereof, pharmaceutical formulation thereof, and

[0270] (b) instructions for how to administer the dose to reduce one or more symptoms of HCC and / or reduce proliferation of HCC in a subject, e.g., a human patient.

[0271] The disclosed compositions and methods can be further understood by the following numbered paragraphs:

[0272] 1. An antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein:

[0273] (i) the three heavy chain variable region CDRs comprise, respectively, an amino acid sequence selected from the group consisting of DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), S GDGYYFAS (SEQ ID NO: 8), FPYYGSSYRVDY (SEQ ID NO: 9), or a functional variant thereof;

[0274] (ii) the three light chain variable region CDRs comprise the amino acid sequences RASKSVSTSGYSYMH (SEQ ID NO: 21), RASQSISNNLH (SEQ ID NO: 22), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), QQINSWPLT (SEQ ID NO: 25), YASQSIS (SEQ ID NO: 73), or functional variants thereof, respectively; and

[0275] wherein the antibody or antigen-binding fragment thereof binds to a LanC-like glutathione S-transferase 1 (LANCL1) protein.

[0276] 2. The antibody or antigen-binding fragment thereof of paragraph 1, wherein:

[0277] (i) the three heavy chain variable region CDRs comprise the amino acid sequences DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), SGDGYYFAS (SEQ ID NO: 8), or comprise the amino acids DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9), respectively, or functional variants thereof; and

[0278] (ii) the three light chain variable region CDRs comprise the amino acids RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), or comprise the amino acids RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25), respectively, or functional variants thereof.

[0279] 3. The antibody or antigen-binding fragment thereof of paragraph 1 or 2, wherein:

[0280] (i) the three heavy chain variable region CDRs comprise the amino acid sequences, respectively: DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), S GDGYYFAS (SEQ ID NO: 8), or functional variants thereof, and the three light chain variable region CDRs comprise the amino acid sequences, respectively: RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), or functional variants thereof; or

[0281] (ii) the three heavy chain variable region CDRs comprise the amino acid sequences, respectively: DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9), or functional variants thereof, and the three light chain variable region CDRs comprise the amino acid sequences, respectively: RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25), or functional variants thereof.

[0282] 4. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, wherein the three heavy chain variable region CDRs comprise the amino acids SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or functional variants thereof, and the three light chain variable region CDRs comprise the amino acids SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or functional variants thereof.

[0283] 5. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, wherein the three heavy chain variable region CDRs comprise the amino acids SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or functional variants thereof, and the three light chain variable region CDRs comprise the amino acids SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25, or functional variants thereof.

[0284] 6. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, wherein the three heavy chain variable region CDRs comprise the amino acids of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a functional variant thereof, respectively, and the three light chain variable region CDRs comprise the amino acids of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or a functional variant thereof, respectively.

[0285] 7. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, wherein the three heavy chain variable region CDRs comprise the amino acids of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or a functional variant thereof, respectively, and the three light chain variable region CDRs comprise the amino acids of SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25, or a functional variant thereof, respectively.

[0286] 8. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-7, wherein the functional variant has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 22, SEQ ID NO: 73, or SEQ ID NO: 25.

[0287] 9. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

[0288] 10. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0289] 11. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0290] 12. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

[0291] 13. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-12, comprising one or more constant domains from an immunoglobulin constant region (Fc).

[0292] 14. The antibody or antigen-binding fragment thereof of paragraph 13, wherein the constant domain is a human constant domain selected from the group consisting of IgA, IgD, IgE, IgG, or IgM.

[0293] 15. The antibody or antigen-binding fragment thereof of paragraph 14, wherein the human IgG constant domain is selected from the group consisting of IgGl, IgG2, IgG3, or IgG4.

[0294] 16. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-15, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody.

[0295] 17. A humanized antibody comprising one or more human IgG constant domains, a heavy chain variable region comprising three complementarity determining regions (CDRs), and a light chain variable region comprising three CDRs, wherein:

[0296] (i) the three heavy chain variable region CDRs comprise an amino acid sequence selected from the group consisting of DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), S GDGYYFAS (SEQ ID NO: 8), FPYYGSSYRVDY (SEQ ID NO: 9), or a functional variant thereof;

[0297] (ii) the three light chain variable region CDRs comprise an amino acid sequence selected from the group consisting of RASKSVSTSGYSYMH (SEQ ID NO: 21), RASQSISNNLH (SEQ ID NO: 22), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25), or a functional variant thereof; and

[0298] wherein the humanized antibody or antigen-binding fragment thereof binds to a human LanC-like glutathione S-transferase 1 (LANCL1) protein.

[0299] 18. The humanized antibody of paragraph 17, wherein:

[0300] (i) the three heavy chain variable region CDRs comprise the amino acid sequences DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), Sgdgyyfas (SEQ ID NO: 8), respectively, or the amino acid sequences DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9), respectively, or functional variants thereof; and

[0301] (ii) the three light chain variable region CDRs comprise the amino acid sequences RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), respectively, or the amino acid sequences RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25), respectively, or functional variants thereof.

[0302] 19. The humanized antibody of paragraph 17 or 18, wherein:

[0303] (i) the three heavy chain variable region CDRs comprise the amino acid sequences DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), Sgdgyyfas (SEQ ID NO: 8), respectively, or functional variants thereof, and the three light chain variable region CDRs comprise the amino acid sequences RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), respectively, or functional variants thereof; or

[0304] (ii) the three heavy chain variable region CDRs comprise the amino acid sequences, respectively: DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9), or functional variants thereof, and the three light chain variable region CDRs comprise the amino acid sequences, respectively: RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25), or functional variants thereof.

[0305] 20. The humanized antibody of any one of paragraphs 17-19, wherein the three heavy chain variable region CDRs comprise SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or functional fragments thereof, respectively, and the three light chain variable region CDRs comprise SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or functional fragments thereof, respectively.

[0306] 21. The humanized antibody of any one of paragraphs 17-19, wherein the three heavy chain variable region CDRs comprise SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or functional fragments thereof, respectively, and the three light chain variable region CDRs comprise SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25, or functional fragments thereof, respectively.

[0307] 22. The humanized antibody of any one of paragraphs 17-19, wherein the three heavy chain variable region CDRs comprise SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or functional fragments thereof, respectively, and the three light chain variable region CDRs comprise SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25, or functional fragments thereof, respectively.

[0308] 23. The humanized antibody of any one of paragraphs 17-19, wherein the three heavy chain variable region CDRs comprise SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or functional fragments thereof, respectively, and the three light chain variable region CDRs comprise SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, or functional fragments thereof, respectively.

[0309] 24. A nucleic acid encoding the antibody or antigen binding fragment of any one of paragraphs 1-16 or the humanized antibody of any one of paragraphs 17-23.

[0310] 25. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of paragraphs 1-16 or the humanized antibody of any one of paragraphs 17-23, and one or more pharmaceutically acceptable carriers and / or excipients.

[0311] 26. The pharmaceutical composition of paragraph 25 for use in a method of treating or preventing liver cancer in a subject.

[0312] 27. The pharmaceutical composition of paragraph 25 or 26, wherein the subject has hepatocellular carcinoma.

[0313] 28. The pharmaceutical composition of any one of paragraphs 25-27, wherein the subject is at risk of developing hepatocellular carcinoma.

[0314] 29. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of paragraphs 25-28.

[0315] 30. The method of paragraph 29, wherein the subject has a disease or disorder caused by or characterized by increased presence of a LanC-like glutathione S-transferase 1 (LANCL1) protein or fragment thereof.

[0316] 31. The method of paragraph 29 or 30, wherein the subject has liver cancer or is at risk of developing liver cancer.

[0317] 32. The method of any one of paragraphs 29-31, wherein the liver cancer is hepatocellular carcinoma.

[0318] 33. The method of any one of paragraphs 29-32, wherein the pharmaceutical composition is delivered via injection or infusion.

[0319] 34. The method of any one of paragraphs 29-33, wherein the pharmaceutical composition is effective to reduce proliferation of tumor initiating cells, spheroid formation of hepatocellular carcinoma cells, and / or block or reduce activity of LANCL1.

[0320] 35. The method of any one of paragraphs 29-34, wherein the administration is repeated every 4 hours, every 6 hours, every 12 hours, daily, optionally for a period of 1 day to 1 month, 1 day to 2 weeks, 1 day to 1 week, or 1 day to 3 days.

[0321] 36. The method of any one of paragraphs 29-35, further comprising administering to the subject one or more chemotherapeutic agents.

[0322] EXAMPLE

[0323] Example 1. Generation and screening of LANCL1 monoclonal antibodies

[0324] To generate specific antibodies targeting the N-terminal extracellular region of human LANCL1, a specific peptide corresponding to the N-terminal of the human LANCL1 protein was used as immunogen. Since human LANCL1 shares high homology with the LANCL2 protein in the middle region, the 1-42 aa region specific to LANCL1 protein was chosen. This 1-42 aa peptide (MAQRAFPNPYADYNKSLAEGYFDAAGRLTPEFSQRLTNKIRE) (SEQ ID NO: 2) was coupled to KLH via the cysteine residue at the C-terminus. The peptide was synthesized by GenScript (Piscataway, NJ) and QC checked by the company using MS and HPLC to have at least 85% purity. It was used to immunize three C57BL / 6 mice by the MonoBoost immunization strategy by GenScript company. Successful immunization was evaluated by indirect ELISA. Subsequently, fresh spleen cells from successfully immunized mice were subjected to cell fusion to obtain hybridoma clonal cells. Supernatants from hybridoma clonal cells were subjected to indirect ELISA to screen for potential positive clones. Twenty positive clones were identified, 1F1, 3B5, 4D9, 4E4, 4H8, 7G2, 8H9, 9H10, 13C12, 13F3, 13G10, 14G4, 16B6, 16F12, 17F1, 18B8, 18H10, 19G4, 20A8, 20F10, respectively. Figure 1A ) All supernatants from positive clones could detect denatured and non-denatured human LANCL1 1-42 aa peptide overexpressed in E. coli B21 lysate with GST tag by western blotting ( Figure 1B and 1C ), as summarized in Figure 1D .

[0325] To further screen for target monoclonal antibodies, antibodies were tested on non-denatured human full-length LANCL1 protein overexpressed in PLC / PRF / 5 lysate with His tag by western blotting ( Figure 1E ). It was found that both 4D9 and 19G4 could detect non-denatured human full-length LANCL1 protein in western blotting, with 19G4 showing stronger intensity of detected band ( Figure 1E ). It was found that 4D9 could only weakly detect non-denatured human LANCL1 protein but not denatured human LANCL1 protein; while 19G4 could strongly detect both non-denatured human LANCL1 protein and denatured human LANCL1 protein ( Figure 1F). Based on this, 4D9 and 19G4 hybridoma cells were subjected to further subcloning and expansion by GenScript company for purification of the corresponding 4D9 and 19G4 monoclonal antibodies. For this, 4D9 and 19G4 were identified as target monoclonal antibodies for further functional validation experiments.

[0326] Example 2. Functional validation of LANCL1 monoclonal antibodies 4D9 and 19G4

[0327] LANCL1 monoclonal antibodies 4D9 and 19G4 were tested on spheroid formation assay, which is the gold standard in vitro test for liver tumor initiating cell (LTIC) function. Both 4D9 and 19G4 significantly inhibited the spheroid formation ability of PLC / PRF / 5 cells at 3200 ng / 100 µl dose in 96-well plates seeded with 700 PLC / PRF / 5 cells Figure 2 ). PLC / PRF / 5 is an Alexander cell line isolated from the liver of a donor with hepatocellular carcinoma positive for hepatitis B virus. Higher doses of 6400 ng and 12800 ng / 100 µl resulted in more severe inhibition of spheroid formation compared to the corresponding IgG control.

[0328] 4D9 and 19G4 were tested for their inhibitory effect on tumor growth in vivo. PLC / PRF / 5 HCC cells in 0.5X growth factor-reduced Matrigel were injected subcutaneously at 5e5 cells / site on the single flank of each NOD-SCID mouse. When palpable tumors were observed at day 9 post PLC / PRF / 5 cell injection, a first dose of monoclonal antibodies and corresponding IgG control (ctrl) was injected intraperitoneally to groups of mice at 12 mg / kg. Two doses were administered per week for two weeks. Mouse body weight was not affected by the administration of 4D9 and 19G4 monoclonal antibodies compared to the IgG control (ctrl) Figure 3A ). 19G4 significantly inhibited tumor size compared to the IgG control as observed at day 19 post PLC / PRF / 5 cell injection, while 4D9 also showed a trend of inhibition, but not with sufficient statistical significance Figure 3B ). Tumors collected at endpoint (day 21 post PLC / PRF / 5 cell injection) showed reduced tumor size for 4D9 and 19G4 monoclonal antibody treatment compared to the IgG control (ctrl) Figure 3C ). Overall, 4D9 and 19G4 monoclonal antibodies inhibited the pro-tumorigenic features of HCC cells, with the latter 19G4 showing a more pronounced effect.

[0329] Example 3. Hybridoma sequencing for amino acid sequences of LANCL1 monoclonal antibodies 4D9 and 19G4

[0330] To obtain the DNA sequences encoding the respective amino acid sequences of each of the human LANCL monoclonal antibodies 4D9 and 19G4, the following method was used, using the protocol of Meyer et al. (5) and adopted by GenScript Corporation. Briefly, total RNA was isolated from hybridoma clone cells according to the technical manual of RNA Isolation Kit (Vazyme, Cat# RC112-01). Then, total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers according to the technical manual of SMARTScribe Reverse Transcriptase (TaKaRa, Cat# 639536). The antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) of cDNA ends rapid amplification (RACE) of GenScript. The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen the clones with inserts of correct size. Not less than five colonies of each fragment with inserts of correct size were sequenced. The sequences of different clones were aligned, and the consensus sequences of these clones were provided as SEQ ID NOs: 3, 4, 19, and 20.

[0331] It was found that the heavy (H) chains of 4D9 and 19G4 were mainly different in CDR2 and CDR3 regions, with some minor differences in FR3 and FR4 regions (see SEQ ID NOs: 3 and 4). For the light (L) chains of 4D9 and 19G4, there were differences between them in signal peptide, CDR2, FR3, and CDR3 regions; while there were minor differences between them in FR1, CDR1, FR2, and FR4 regions (see SEQ ID NOs: 19 and 20).

[0332] Example 4. LANCL1 monoclonal antibody 19G4 inhibits orthotopic HCC growth and lung metastasis in a mouse HCC model.

[0333] Further, LANCL1 monoclonal antibody 19G4 was used for in vivo functional testing in a mouse orthotopic HCC model, and LANCL1 monoclonal antibody 19G4 was used for in vitro functional testing on HCC cells.

[0334] For orthotopic experiments, 1 x 10 6Luciferase-labeled PLC / PRF / 5 HCC cells were injected into the left lobe of the liver of each NOD-SCID mouse. When tumor formation was observed (by luciferase signal imaging 2 weeks after orthotopic injection), mice were randomized into groups to receive 19G4 monoclonal antibody, corresponding mouse IgG control, or no treatment. Antibodies were injected intraperitoneally at 60 mg / kg. Two doses were administered per week for two weeks. Liver tumor size was indicated by luciferase signal imaging of the mice and extracted livers, respectively, at the endpoint. Lung metastasis of PLC / PRF / 5 HCC cells was assessed by luciferase signal in extracted lung tissue. These studies demonstrated that 19G4 monoclonal antibody significantly inhibited liver tumor growth and lung metastasis compared to IgG control Figures 4A-4C

[0335] Example 5. LANCL1 mAb 19G4 promotes cytotoxicity of HCC cells in vitro and inhibits migration and invasion of HCC cells.

[0336] Cytotoxicity assays were used to determine the IC50 of 19G4 monoclonal antibody relative to IgG control, and flow cytometry using annexin V was used to assess the effect of the antibody on HCC cell apoptosis to evaluate the cytotoxic activity of 19G4 monoclonal antibody on hepatocellular carcinoma cells. For the cytotoxicity assay, 2,000 PLC / PRF / 5 cells were plated per well on a 96-well plate, and different wells of PLC / PRF / 5 cells were treated with eight different indicated concentrations of 19G4 monoclonal antibody or respective IgG control. After 24 hours, cell viability was assessed using DAPI staining followed by cell counting. The IC50 value for 19G4 monoclonal antibody was determined to be 35.29 µg / ml, while the IgG control failed to inhibit the growth of HCC cells Figure 5A For the annexin V / PI staining assay, cells were plated at 10,000 cells / well on a 24-well plate with four wells per condition, and the wells of cells were treated with 128 µg / ml of 19G4 monoclonal antibody or IgG control, respectively, immediately after cell plating. A total of 1 × 10 5 Forty-eight hours of treatment with 19G4 monoclonal antibody resulted in increased apoptosis when compared to IgG control, which was not statistically significant Figure 5B

[0337] In addition to cytotoxicity, the effect of LANCL1 19G4 antibody on the migration and invasion capacity of HCC cells was investigated by using transwell migration and invasion assays, respectively. In the transwell migration assay, 1 × 10 5 ​​PLC / PRF / 5 cells were seeded in the upper chamber, and 128 µg / mL of 19G4 monoclonal antibody was applied to both the upper and lower chambers. The lower chamber contained 10% FBS as a chemical inducer, while the upper chamber contained serum-free medium. After incubation at 37°C for 6 hours, the number of cells that had migrated across the membrane to the bottom surface in the transwell was stained and counted in random views. Compared with the IgG control, the 19G4 antibody significantly reduced HCC cell migration (…). Figure 6A In the transwell invasion assay, after inoculation with 5 × 10⁻⁶ cells / day, the viral load was 100 μL / day. 5 Before culturing PLC / PRF / 5 cells, the upper chamber was pre-coated with 3 mg / ml matrix gel. All other conditions were the same as for the migration assay, except for an extended incubation period of 72 hours. Compared with the IgG control, the 19G4 antibody significantly reduced HCC cell invasion (…). Figure 6B Three independent tests were conducted for all measurements.

[0338] By employing a revised staining protocol (where cells were incubated with LANCL1 19G4 antibody at 4°C for 30 min, fixed with 4% paraformaldehyde for 10 min, and subsequently incubated with secondary antibody at 4°C for 30 min), we were able to use LANCL1 19G4 antibody for flow cytometry analysis of LANC1 cell surface expression. Interestingly, we found that over a 9-day period, the mean fluorescence intensity (MFI) of the total cell population for spherical cultures (but not two-dimensional adherent cultures) of PLC / PRF / 5 cells was significantly higher. Figure 7A ) and the percentage of cells that were positive for LANCL1 expression ( Figure 7B An increase was observed in the LANCL1 concentration. This may suggest an enrichment of LANCL1-positive cells in spherical cultures.

[0339] Based on the reliable functional data above regarding the inhibitory effect of the LANCL1 19G4 monoclonal antibody on HCC, subsequent studies proceeded to the humanization of the antibody.

[0340] Example 6. Humanization of LANCL1 monoclonal antibody 19G4.

[0341] Humanized antibodies were designed using CDR transplantation of human IgG4. Figure 8A In short, homology modeling of the parental 19G4 antibody Fv fragment was performed by searching the IgBLAST database to identify the optimal template for the Fv fragment sharing the highest sequence identity with the parental 19G4 antibody, for constructing the domain interface. The identified homologous antibody sequence was selected as the human recipient for transplanting the parental 19G4 antibody Fv fragment. Figure 8BNext, by comparing the sequences of the transplanted antibody and the parent antibody near the CDR, canonical residues, loop interactions, and basal core, different key residues in the sequences of the transplanted antibody and the parent antibody were identified as putative reversion mutation sites. Thus, variants of the humanized antibody could be designed by progressively incorporating one or more reversion mutations into the transplanted antibody sequence. In this way, three different variants of each of the heavy and light chains were obtained. When combined, these heavy and light chain variants resulted in nine different combinations (i.e., variants) of the humanized Ab. Figure 8C These humanized antibody variants were synthesized and purified, and subjected to affinity measurements using Biacore. Figure 9A The analytes, ligands, and capture molecules used in the Biacore measurement were LANCL1 1-42aa peptide, humanized 19G4 antibody variant, and protein A, respectively. Figure 9B All data was processed using Biacore 8K evaluation software version 4.0. Combined with sensor images as follows... Figure 9C As shown, and in conjunction with measurement data as follows Figure 9D As shown. All antibody variants exhibit the following affinities, ranging from 0.5 to 1.0 relative to the chimeric humanized 19G4 antibody VH+VL, with the lowest being VH3+VL3 ( Figure 9E The amino acid sequences of the heavy chain and variable regions of the light chain of chimeric humanized 19G4 antibody 9 and various humanized antibody variants 9 are provided herein (below).

[0342] The H chain variable region of the chimeric humanized antibody: EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGHTNYNQKFKGKATLTVDKSSNTAYMELNSLTSEDSAVYYCARFPYYGSSYRVDYWGQGTTLTVSS (SEQ ID NO: 75);

[0343] L-chain variable region of chimeric humanized antibody:

[0344] H-chain variable region of humanized antibody variant: VH1:

[0345] VH2:

[0346]

[0347] VH3:

[0348]

[0349] L chain variable region of the humanized antibody variants:

[0350] VL1:

[0351] VL2:

[0352] VL3:

[0353]

[0354] The risk of post-translational modifications (PTM) was determined for the variable regions of the heavy chain ( Figure 10A ) and light chain ( Figure 10B ) of the chimeric humanized 19G4 antibody and each humanized antibody variant. Except for a medium risk for the "NG" residues on the heavy chain of the humanized antibody variants, which is still acceptable, most sequences have a low PTM risk. Using the data from the above computer analysis, the monoclonal antibody variants were further tested for their functional effects on HCC cells. The data show that the humanized antibody variants VH2+VL2 and VH3+VL2 significantly inhibit spheroid formation, similar to the chimeric humanized antibody VH+VL, as indicated by the reduced number of spheroids formed at day 8 ( Figure 11A ) and day 11 ( Figure 11B ).

[0355] Summary / conclusion

[0356] The LANCL1 monoclonal antibodies 4D9 and 19G4 were identified to have an inhibitory effect on HCC cells and the DNA sequences encoding the amino acid sequences of each monoclonal antibody from the respective hybridoma clones were determined. The DNA and amino acid sequences of the monoclonal antibodies formed the basis for further engineering and improvement of the antibodies for transformative applications as a means to treat hepatocellular carcinoma (HCC) to target LANCL1 in liver tumor initiating cells.

[0357] The studies herein further functionally verified that the 19G4 monoclonal antibody significantly inhibits orthotopic HCC growth and lung metastasis, promotes cytotoxicity and apoptosis of HCC cells, and inhibits migration and invasion of HCC cells. By using the antibody, enrichment of LANCL1 positive cells in HCC spheroid cultures was observed compared to two-dimensional adherent cultures. The 19G4 antibody was then subjected to humanization and nine humanized antibody variants were designed, of which VH2+VL2 and VH3+VL2 showed a significant inhibitory effect on HCC cell spheroid formation.

[0358] References

[0359] 1. Yi SY, Hao YB, Nan KJ, and Fan TL. Cancer stem cells niche: a target for novel cancer therapeutics. Cancer Treat Rev. 2013;39:290-296.

[0360] 2. Visvader JE, and Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008;8:755-768.

[0361] 3. Tirino V, Desiderio V, Paino F, De Rosa A, Papaccio F, La Noce M, Laino L, et al. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013;27:13-24.

[0362] 4. Huang HY*, Tsui YM*, Ho DW, Chung CY, Sze KM, Lee E, Cheung GC, Zhang VX, Wang X, Lyu XY, Ng IO. LANCL1, a cell-surface protein, promotes liver tumor initiation via FAM49B-Rac1 axis to suppress oxidative stress. Hepatol. 2023;79(2):323-340.

[0363] 5. Meyer L, López T, Espinosa R, Arias CF, Vollmers C, DuBois RM. A simplified workflow for monoclonal antibody sequencing. PLoS One. 2019;14(6):e0218717.

[0364] It is to be understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims.

[0365] Using only routine experimentation, those of ordinary skill in the art will readily recognize or be able to ascertain many equivalents of the specific embodiments of the methods and compositions described herein. Such equivalents are considered to be within the scope of the claims.

Claims

1. An antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region including three complementarity-determining regions (CDRs) and a light chain variable region including three CDRs, wherein: (i) The three heavy chain variable regions (CDRs) each contain an amino acid sequence selected from the group consisting of: DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), SGDGYYFAS (SEQ ID NO: 8), FPYYGSSYRVDY (SEQ ID NO: 9), or a functional variant thereof; (ii) The three light chain variable regions (CDRs) respectively comprise amino acid sequences selected from the group consisting of: RASKSVSTSGYSYMH (SEQ ID NO: 21), RASQSISNNLH (SEQ ID NO: 22), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), QQINSWPLT (SEQ ID NO: 25), YASQSIS (SEQ ID NO: 73), or functional variants thereof; and The antibody or its antigen-binding fragment binds to the LanC-like glutathione S-transferase 1 (LANCL1) protein.

2. The antibody or its antigen-binding fragment according to claim 1, wherein: (i) The three heavy chain variable regions (CDRs) each contain the amino acid sequence: DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), SGDGYYFAS (SEQ ID NO: 8) or a functional variant thereof, and the three light chain variable regions (CDRs) each contain the amino acid sequence: RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24) or a functional variant thereof; or (ii) The three heavy chain variable regions CDRs each contain the amino acid sequence: DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9) or a functional variant thereof, and the three light chain variable regions CDRs each contain the amino acid sequence: RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25) or a functional variant thereof.

3. The antibody or its antigen-binding fragment according to claim 1, wherein: (a) The three heavy chain variable regions CDRs respectively contain amino acid sequences SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or their functional variants, and the three light chain variable regions CDRs respectively contain amino acid sequences SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or their functional variants; (b) The three heavy chain variable regions CDRs respectively contain amino acid sequences SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or their functional variants, and the three light chain variable regions CDRs respectively contain amino acids SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25 or their functional variants; (c) The three heavy chain variable regions (CDRs) respectively contain amino acid sequences SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or functional variants thereof, and the three light chain variable regions (CDRs) respectively contain amino acid sequences SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or functional variants thereof; or (d) The three heavy chain variable regions (CDRs) respectively contain amino acid sequences SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or their functional variants, and the three light chain variable regions (CDRs) respectively contain amino acid sequences SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25 or their functional variants.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the functional variant has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:22, SEQ ID NO:73, or SEQ ID NO:

25.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region including the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region including the amino acid sequence of SEQ ID NO:

3.

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising (a) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; or (c) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

3.

7. The antibody or antigen-binding fragment thereof according to claim 1, comprising a humanized light chain variable region including the amino acid sequence of SEQ ID NO: 76 or a functional variant thereof, and a humanized heavy chain variable region including the amino acid sequence of SEQ ID NO: 75 or a functional variant thereof.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region variant comprises: (a) the amino acid sequence of SEQ ID NO: 80, 81 or 82 and / or (b) the heavy chain variable region variant comprises the amino acid sequence of SEQ ID NO: 77, 78 or 79.

9. The antibody or antigen-binding fragment thereof according to claim 1, comprising one or more constant domains from the immunoglobulin constant region (Fc), wherein the constant domain is a human constant domain selected from the group consisting of IgA, IgD, IgE, IgG or IgM, optionally wherein the human IgG constant domain is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody.

11. A humanized antibody or its antigen-binding fragment, comprising one or more human IgG constant domains, a heavy chain variable region comprising three complementarity-determining regions (CDRs), and a light chain variable region comprising three CDRs. The humanized antibody or its antigen-binding fragment comprises (a) a humanized light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 76, 80, 81 or 82 and a humanized heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 75, 77, 78 or 79. in: (i) Each heavy chain variable region comprises three heavy chain variable region CDRs, the three heavy chain variable region CDRs comprising amino acid sequences selected from the group consisting of: DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), SGDGYYFAS (SEQ ID NO: 8), FPYYGSSYRVDY (SEQ ID NO: 9) or functional variants thereof; (ii) Each light chain variable region comprises three light chain variable region CDRs, the three light chain variable region CDRs comprising amino acid sequences selected from the group consisting of: RASKSVSTSGYSYMH (SEQ ID NO: 21), RASQSISNNLH (SEQ ID NO: 22), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25) or functional variants thereof; and The humanized antibody or its antigen-binding fragment binds to human LanC-like glutathione S-transferase 1 (LANCL1) protein.

12. The humanized antibody or its antigen-binding fragment according to claim 11, wherein: (i) The three heavy chain variable regions (CDRs) respectively comprise the amino acid sequences DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), and SGDGYYFAS (SEQ ID NO: 8), or respectively comprise the amino acid sequences DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), and FPYYGSSYRVDY (SEQ ID NO: 9), or functional variants thereof; and (ii) The three light chain variable regions (CDRs) respectively contain the amino acid sequences: RASKSVSTSGYSYMH (SEQ ID NO:21), LVSNLES (SEQ ID NO:23), QHIRELT (SEQ ID NO:24), or respectively contain RASQSISNNLH (SEQ ID NO:22), YASQSIS (SEQ ID NO:73), QQINSWPLT (SEQ ID NO:25), or their functional variants.

13. The humanized antibody or its antigen-binding fragment according to claim 12, wherein: (i) The three heavy chain variable regions (CDRs) each contain the amino acid sequence: DYYMN (SEQ ID NO: 5), DINPNNGGASYNQKFKG (SEQ ID NO: 6), SGDGYYFAS (SEQ ID NO: 8) or a functional variant thereof, and the three light chain variable regions (CDRs) each contain the amino acid sequence: RASKSVSTSGYSYMH (SEQ ID NO: 21), LVSNLES (SEQ ID NO: 23), QHIRELT (SEQ ID NO: 24) or a functional variant thereof; or (ii) The three heavy chain variable regions CDRs each contain the amino acid sequence: DYYMN (SEQ ID NO: 5), VINPYNGHTNYNQKFKG (SEQ ID NO: 7), FPYYGSSYRVDY (SEQ ID NO: 9) or a functional variant thereof, and the three light chain variable regions CDRs each contain the amino acid sequence: RASQSISNNLH (SEQ ID NO: 22), YASQSIS (SEQ ID NO: 73), QQINSWPLT (SEQ ID NO: 25) or a functional variant thereof.

14. The humanized antibody or its antigen-binding fragment according to claim 12, wherein: (a) The three heavy chain variable regions CDRs respectively contain SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or functional fragments thereof, and the three light chain variable regions CDRs respectively contain SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or functional fragments thereof; (b) The three heavy chain variable region CDRs respectively contain SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or functional fragments thereof, and the three light chain variable region CDRs respectively contain SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25 or functional fragments thereof; or (c) The three heavy chain variable regions CDRs respectively contain SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or their functional fragments, and the three light chain variable regions CDRs respectively contain SEQ ID NO: 22, SEQ ID NO: 73, SEQ ID NO: 25 or their functional fragments.

15. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-10 or a humanized antibody or antigen-binding fragment thereof according to any one of claims 11-14.

16. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-10, and one or more pharmaceutically acceptable carriers and / or excipients, wherein optionally the antibody or antigen-binding fragment thereof is a humanized antibody.

17. A method of treating a subject in need of treatment, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 16, optionally, the subject suffering from a disease or condition caused by or characterized by the presence of an increased amount of LanC-like glutathione S-transferase 1 (LANCL1) protein or a fragment thereof, wherein the pharmaceutical composition effectively reduces the proliferation of tumor-initiating cells, the formation of spheroids in hepatocellular carcinoma cells, and / or blocks or reduces the activity of LANCL1.

18. The method of claim 17, wherein the subject has liver cancer or is at risk of developing liver cancer.

19. The method of claim 18, wherein the liver cancer is hepatocellular carcinoma.

20. The method according to any one of claims 17-19, wherein the pharmaceutical composition is delivered by injection or infusion.

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