WO2025163490A1

WO2025163490A1 - Growth factor receptor agonist/antagonist

Info

Publication number: WO2025163490A1
Application number: PCT/IB2025/050927
Authority: WO
Inventors: Abdolreza Zarnegar
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 2024-01-30
Filing date: 2025-01-28
Publication date: 2025-08-07
Anticipated expiration: 2026-07-30

Abstract

Provided herein are antibodies against hepatocyte growth factor (HGF) receptor and methods of using such antibodies.

Description

GROWTH FACTOR RECEPTOR AGONIST/ANTAGONIST

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to United States Provisional Patent Application No. 63/626,769, the disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with government support under CA203985 and DK108891 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE STATEMENT

[0003] The Sequence Listing associated with this application is filed in electronic format and is hereby incorporated by reference into the specification in its entirety. The name of the file containing the Sequence Listing is 2408140. xml. The size of the file is 95,755 bytes, and the file was created on January 27, 2025.

BACKGROUND

Field of the Invention

[0004] The present invention relates to antibodies and antibody-based drugs, and methods of treatment of diseases mediated by the lack of proper MET tyrosine kinase receptor function. Such as organ failure (e.g., liver and kidney failure/disease), various forms of hepatitis (e.g., steatohepatitis, e.g., nonalcoholic steatohepatitis (NASH), alcohol hepatitis, viral hepatitis, autoimmune hepatitis, drug induced hepatitis such as Tylenol poisoning), and type 2 diabetes and its associated pathologies like retinopathy and macular degeneration.

Description of Related Art

[0005] Hepatocyte Growth Factor (HGF) is an important inducer of tissue growth and regeneration for various tissues and organs including skin, liver, lung, kidney, pancreas, as well as growth and survival of various stem cells. It inhibits cell death, tissue necrosis and degeneration. Interestingly HGF also is essential for glucose and fat metabolism and homeostasis. HGF exerts its effects by binding to and activating its specific cell surface tyrosine kinase receptor known as MET. MET, also known as hepatocyte growth factor receptor or HGF receptor, is a cell surface tyrosine kinase. HGF has poor tissue distribution when injected into experimental animals as it binds to heparin very avidly and is rapidly cleared by the liver. This property of HGF has hampered its clinical utility. Accordingly, new compounds are needed.

SUMMARY OF THE INVENTION

[0006] Provided herein is an antibody that binds to a hepatocyte growth factor (HGF) receptor, including a heavy chain variable region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 20, SEQ ID NO: 32, and SEQ ID NO: 33, and a light chain variable region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 21 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , and SEQ ID NO: 42.

[0007] Also provided herein is an antibody that binds HGF receptor including a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 2 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

[0008] Also provided herein is an antibody that binds HGF receptor including a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 6 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

[0009] Also provided herein is an antibody that binds HGF receptor including a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 9 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

[0010] Also provided herein is an antibody that binds HGF receptor including a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 1 1 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 13.

[0011] Also provided herein is an antibody that binds HGF receptor including a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 15 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 17.

[0012] Also provided herein is an antibody that binds HGF receptor including a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 19 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 21 .

[0013] Also provided herein is an scFv having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 22.

[0014] Also provided herein is a composition including an antibody as described herein and a pharmaceutically-acceptable excipient.

[0015] Also provided herein is a method of treating a patient having a disease responsive to a MET agonist, including administering to the patient an amount of a MET agonist antibody as described herein effective to treat the disease in the patient. [0016] Also provided herein is a method of treating a cancer in a patient having a cancer in which MET/HGF are overexpressed, including administering to the patient an amount of a MET antagonist (scFv) antibody compound as described herein.

[0017] Further non-limiting embodiments are set forth in the following numbered clauses:

[0018] 1. An antibody that binds to a hepatocyte growth factor (HGF) receptor, comprising:

[0019] a heavy chain variable region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 20, SEQ ID NO: 32, and SEQ ID NO: 33; and a light chain variable region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 21 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , and SEQ ID NO: 42.

[0020] 2. The antibody of clause 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 20; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 21 .

[0021] 3. The antibody of clause 1 or clause 2, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

[0022] 4. The antibody of any of clauses 1 -3, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

[0023] 5. The antibody of any of clauses 1 -4, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 37.

[0024] 6. The antibody of any of clauses 1 -5, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 37.

[0025] 7. The antibody of any of clauses 1 -6, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 39. [0026] 8. The antibody of any of clauses 1 -7, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 40.

[0027] 9. The antibody of any of clauses 1 -8, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 41 .

[0028] 10. The antibody of any of clauses 1 -9, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 42.

[0029] 1 1 . The antibody of any of clauses 1 -10, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 37.

[0030] 12. The antibody of any of clauses 1 -11 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 38.

[0031] 13. The antibody of any of clauses 1 -12, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 39.

[0032] 14. The antibody of any of clauses 1 -13, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 40.

[0033] 15. The antibody of any of clauses 1 -14, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 41 .

[0034] 16. The antibody of any of clauses 1 -15, wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 42.

[0035] 17. The antibody of any of clauses 1 -16, wherein the amino acid sequence comprises a complementarity-determining region (CDR) having the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.

[0036] 18. The antibody of any of clauses 1 -17, wherein the CDR comprises one or more mutation selected from S31 N, F33Y, S35G, Y59E, Y60E, P61 S, P61 Q, P61 L, P61 T, S103I, K187I, S190G, and S226D.

[0037] 19. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 2 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

[0038] 20. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 6 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

[0039] 21 . An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 9 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

[0040] 22. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 11 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 13.

[0041] 23. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 15 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 17.

[0042] 24. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 19 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 21 .

[0043] 25. An scFv having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 22.

[0044] 26. A composition comprising an antibody of any of clauses 1 -24 and a pharmaceutically-acceptable excipient.

[0045] 27. A method of treating a patient having a disease responsive to a MET agonist, comprising administering to the patient an amount of a MET agonist antibody compound of any of clauses 1 -24 effective to treat the disease in the patient.

[0046] 28. The method of claim 27, wherein the disease is one or more of: organ failure such as liver and kidney failure/disease, a hepatitis (e.g., steatohepatitis, alcohol hepatitis, viral hepatitis, autoimmune hepatitis, or drug induced hepatitis, such as Tylenol poisoning), or type 2 diabetes and/or its associated pathologies such as retinopathy and macular degeneration. [0047] 29. A method of treating a cancer in a patient having a cancer in which MET/HGF are overexpressed, e.g., highly overexpressed, such as a breast, colon, glioma, or thyroid cancer, comprising administering to the patient an amount of a MET antagonist (scFv) antibody compound of any of clauses 1 -24 to the patient effective to treat the cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 shows mutation sites of META4 antibody clones according to nonlimiting embodiments as described herein;

[0049] FIG. 2 shows PCR amplification of 1 B2-HC gene Notes: Lane M: DL2000 DNA Marker(2000,1000,750,500,250,1 OObp) Lane 1 -15: PCR products with different degenerate primers;

[0050] FIG. 3 shows PCR amplification of 1 C2-HC gene Notes: Lane M: DL2000 DNA Marker(2000,1000,750,500,250,1 OObp) Lane 1 -15: PCR products with different degenerate primers;

[0051] FIG. 4 shows PCR amplification of 1 C3-HC gene Notes: Lane M: DL2000 DNA Marker(2000,1000,750,500,250,1 OObp) Lane 1 -9: PCR products with different degenerate primers;

[0052] FIG. 5 shows PCR amplification of 1 B2-KC gene Notes: Lane M: DL2000 DNA Marker (2000,1000,750,500,250,1 OObp) Lane 1 -14: PCR products with different degenerate primer pairs;

[0053] FIG. 6 shows PCR amplification of 1 C2-KC gene Note: Lane M: DL2000 DNA Marker (2000,1000,750,500,250,1 OObp) Lane 1 -4: PCR products with different degenerate primer pairs;

[0054] FIG. 7 shows PCR amplification of 1 C3-KC gene Notes: Lane M: DL2000 DNA Marker (2000,1000,750,500,250,1 OObp) Lane 1 -14: PCR products with different degenerate primer pairs;

[0055] FIG. 8 is a map of plgG-mKC;

[0056] FIG. 9 is a map of plgG-mHC;

[0057] FIG. 10 shows Human VH framework acceptor. Result: the most appropriate human VH framework acceptor for parent J01 1819-ZYA VH region (SEQ ID NO: 20) is IGHV3-21 01 (SEQ ID NO: 56);

[0058] FIG. 11 shows Human VL framework acceptor. Result: the most appropriate human VL framework acceptor for parent J01 1819-ZYA VL region (SEQ ID NO: 21) is IGKV3-1 1 01 (SEQ ID NO: 57); [0059] FIG. 12 shows a schematic of pCantab 5E-Hu1 1 S-scFv construction;

[0060] FIG. 13 shows contiguous nucleotide sequences for primers for error-prone PCR described in Example 2 (SEQ ID NOS: 43 and 44);

[0061] FIG. 14 shows QC Gel electrophoresis of error-prone PCR of Hu11 S-scFv. Lane M: DL2000 Marker (2000, 1000, 750, 500, 250, 100 bp). Lane hu1 1 Smu: random mutated Hu11 S-scFv after error-prone PCR;

[0062] FIG. 15 shows PCR amplification of VH and VL fragments (wild-type and mutated). Lane M: DL2000 Marker (2000, 1000, 750, 500, 250, 100 bp). Lane VLwt: PCR amplification result of wild-type VL of Hu11 S-scFv. Lane VHwt: PCR amplification result of wild-type VH of Hul l S-scFv. Lane VLmul , VLmu2, VLmu3: PCR amplification result of mutated VL of Hul l S-scFv. Lane VHmul , VHmu2, VHmu3: PCR amplification result of mutated VH of Hu1 1 S-scFv;

[0063] FIG. 16 shows PCR assembling scFv. DNA marker: DL2000 Marker (2000, 1000, 750, 500, 250, 100 bp). VHmu-VLwt: assembling of mutated VH- wild-type VL scFv. VHwt-VLmu: assembling of wild-type VH- mutated VL scFv;

[0064] FIGS. 17A-17D show alignment of sequencing results from random Hotspot clones against SEQ ID NO: 22. Note: CDR regions (Kabat) are highlighted in gray frames;

[0065] FIG. 18 shows QC result of biotin-labeled antigen (B-Ag). Lane M: Protein Marker, Lane 1 : 5pg Streptavidin + PBS, Lane 2: 5pg Streptavidin + 2pg B-Ag, Lane 3: 2pg B-Ag + PBS. Conclusion: The biotin labeling efficiency was above 85%;

[0066] FIGS. 19A-19B show a summary of DNA sequencing, including (SEQ ID NO: 22, in FIG. 19A);

[0067] FIG. 20 shows PCR assembling VHm-VLm scFv. DNA marker: DL2000 Marker (2000, 1000, 750, 500, 250, 100 bp); R3VH: PCR amplification result of VHm from the round 3 output of VHm-VLwt Library; R3VL: PCR amplification result of VLm from the round 3 output of VHwt-VLm Library; OL: assembling of VHm-VLm scFv via overlap PCR;

[0068] FIGS. 21A-21 B show SDS-PAGE assay of the 9 clones in IgG format. Reduced SDS-PAGE in part A, Non-reduced SDS-PAGE in part B;

[0069] FIG. 22 shows a Western Blot of culture medium from hybridoma active clones using anti-mouse antibody as probe to detect secreted META4. Samples were run under reducing and non-reducing conditions as indicated which shows heavy and light chains (under reduced) and the whole molecule under non-reduced condition.;

[0070] FIGS. 23A-23C show activation of MET and its downstream mediators by META4 producing hybridoma clones made by immunizing mice with purified human MET extracellular domain. META4 is specific for human and primate MET but does not activate mouse MET;

[0071] FIG. 24 shows META4 produced by META4-producing hybridoma clones bind to MET on Hep-G2 cells;

[0072] FIGS. 25-26 show immunofluorescence staining in HepG2 cells with antihuman IgG antibodies;

[0073] FIG. 27 shows Human hepatocyte (HepG2) and Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium and then switched to serum free medium for overnight. They were treated with HGF or humanized META4 as indicated for 15 minutes, and cell extracts were subjected to western immunoblot using antibody to activated MET (phosphoMET) and then with antibody against activated AKT (phosphoAKT) which is a downstream effector of activated MET;

[0074] FIG. 28 shows Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium and then switched to serum free medium for overnight. They were treated with META4 as indicated for the indicated time and also with different concentrations of META4 (FOR 20 minutes) as shown in the figure. Cell extracts were subjected to western immunoblot using antibody to phosphorylated MET (pMET Tyr residue Y1003 which is essential for regulation of MET abundance after activation on 1234Y) and then with antibody against total MET as protein loading control;

[0075] FIG. 29 shows Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium and then switched to serum free medium for overnight. They were treated with HGF (100 ng/ml) or different concentrations of META4 (two different preparations) as indicated for 15 minutes. Cell extracts were subjected to western immunoblot using antibody to activated MET (phosphoMET);

[0076] FIG. 30 shows humanized META4 activates MET in Rhesus and Cynomolgus Monkey kidney cells and hepatocytes; [0077] FIG. 31 shows HGF and humanized META4 promote growth of human retinal pigmented epithelial cells (RPE cells) as determined by MTT assay;

[0078] FIG. 32 shows HGF and META4 protects human retinal pigmented epithelial cells (RPE cells) from oxidative damage and apoptosis induced by H202 and FasL as determined by CellTox assay;

[0079] FIG. 33 shows META4 protects human retinal pigmented epithelial cells (RPE cells) from apoptosis induced by FasL as determined by prevention of caspase- 3 activation;

[0080] FIGS. 34A-34B show Human Retinal Pigmented Epithelial (RPE-19, CRL- 2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (H,HGF) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA-sequencing for gene expression. Shown are volcano graphs depicting gene that are significantly changed by treatment vs. corresponding control; [0081] FIG. 35 shows Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (H,HGF) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA- sequencing for gene expression. Shown are heatmaps (co-clustering) of differentially expressed genes;

[0082] FIGS. 36A-36B show Human Retinal Pigmented Epithelial (RPE-19, CRL- 2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA- sequencing for gene expression. Shown are scatter plots depicting gene that are significantly changed(up or down) by treatment vs. corresponding control.

[0083] FIGS. 37A-37B show Human Retinal Pigmented Epithelial (RPE-19, CRL- 2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA- sequencing for gene expression. Shown are heatmaps (co-clustering) of differentially expressed genes META4 vs. control CNTRL;

[0084] FIG. 38 shows HGF injected systemically to db/db mice distributes to the liver;

[0085] FIG. 39 shows HGF injected systemically to db/db mice activates pERK in the liver;

[0086] FIG. 40 shows HGF therapy lowers hepatic lipids in lean mice;

[0087] FIG. 41 shows HGF therapy mobilizes hepatic lipids in db/db mice as evident by increased FFA (free fatty acids) due to lipolysis of hepatic fat;

[0088] FIG. 42 shows HGF Therapy [10 days a single daily injection] Ameliorates Liver Damage in db/db Mice;

[0089] FIG. 43 shows HIV+ patients have abundant pro-HGF (which is biologically inactive) and HGF antagonist NK1 in their plasma. These data indicate that there is a blockade of HGF action via 1 ) inhibition of pro-HGF activation which is mediated by HGF activator (HGFAC). Notably, pro-HGFAC is also abundant which itself needs to be cleaved and activated by serine protease like thrombin, kallikrein-related peptidase KLK4. HGF antagonist NK1 inhibits HGF action. Thus, the cascade of HGF axis is blocked at several tiers;

[0090] FIG. 44 shows Anti-Retroviral Therapy [ART] drugs like Efa (Efavarenz) and Mara (Maraviroc) inhibit processing (activation) of HGFAC. Human hepatocyte cell line (Hep3B) were treated as indicated for 48hrs and culture medium were analyzed by western blot. HGFAC is produced and secreted by hepatocytes as inactive pro- HGFAC and requires proteolytic cleavage. These data indicate that ARTs inhibit HGFAC activation;

[0091] FIG. 45 shows META4 protects hepatocytes from ART-induced cytotoxicity (MTT assay) ART [Rai]. Anti-Retroviral Therapy [ART] induces hepatotoxicity which is prevented by META4. Human hepatocyte cell line (HepG2) were treated without or with ART called Rai (Raltegravir) and with HGF and META4 as indicated for 48 hrs and processed for cell viability by MTT assay;

[0092] FIGS. 46A-46B show mice with humanized liver develop NAFLD if placed on an HFD.A, Images of liver sections from humanized liver stained with hematoxylin and eosin (H&E), Oil-Red-O, FAH, and TUNEL as indicated. Arrows points to fat-laden hepatocytes. B, Liver and serum triglyceride level. N = 4-6 mice per group. Bar graphs depict the relative expression. 001 and **P = .01 , respectively. C and D, FAH immunostain. FAH-positive human hepatocytes are marked by filled arrows and FAH- negative mouse hepatocytes are marked by unfilled arrows. In D, note the foci of inflammatory cells surrounding the human hepatocytes. E, TUNEL stain. Arrow points to the same region positive for FAH. Scale: 100 mm in panels A, C, E and 30 mm in panels B and D, respectively;

[0093] FIGS. 47A-47C show Humanized fatty liver phenocopies human NASH at the histologic, cellular, and biochemical levels. Results shown are from analyses performed side-by-side on the humanized (A) and human NASH livers (B), and nontransplanted livers for the indicated markers as determined by immunohistochemistry. Scale: 100 mm for left and 30 mm for right images in each column. C, Depicts higher magnification image of humanized liver stained with trichrome for collagen;

[0094] FIG. 48 shows Quantification of the results shown in FIGS. 47A-47C. Graphs in (A) and (B) depict indicated markers shown in FIGS. 47A-47C as determined by image analysis. C, Illustrates quantification of collagen content in the liver by measuring hydroxyproline a component of collagen. Nontransplanted FRGN and wild type CD1 mice are also included for comparison. Asterisks denote P < .05.

[0095] FIGS. 49A-49D show HGF antagonists NK1 and NK2 are expressed in human NASH liver. A, Results of RT-PCR (n = 3 cases per group); and B, Western immunoblot for HGF antagonist (n = 5 cases per group) using antibody to the N- terminal region of HGF. Bar graphs depict the relative expression. C, D, HGFAC expression is significantly reduced in the livers of humans with NASH. C, Shown is the relative abundance of HGF activator transcript in human liver as determined by RNA- seq. *P = .02. D, Depicted are the Western blot results for HGFAC in human normal and NASH livers (n = 5 and n = 6 cases per group as indicated);

[0096] FIG. 50 shows HGF antagonist is present in the plasma of patients with NASH. Shown are the results of Western immunoblot of plasma samples (3 microliters) using antibody to the N-terminal region of HGF. Coomassie blue stain of the gel is shown below the blots. Coomasie blue stain of gel is shown for equal loading of plasma samples. Bar graphs depicts the relative expression of NK1/NK2 signals. NASH (n = 10 different cases) and normal (n = 3 different cases);

[0097] FIG. 51 shows HGF expression is reduced in the liver of wild-type mice C57/BI6 fed a HFD whereas that of HGF antagonist is induced. A, Western blot data for HGF; and B, RT-PCR results for NK1 expression. Animals were culled at feed or after an overnight fast as indicated. Mice were fed on HFD for 3 months;

[0098] FIGS. 52A-52D show Robust and rapid activation of MET and MET signaling effectors by META4. A, Activation of MET in human hepatocyte cell line HepG2; shown is the Western blot for the indicated effectors. B, META4 does not activate rodent MET. Western blot data showing that META4 activates MET in human but not mouse hepatocytes (Hepa 1 -6 cell line). Cells were treated for 15 minutes and processed for MET activation (pMET 1234Y) and total MET as indicated. HGF was used as a positive control, which activates mouse and human hepatocytes. C, META4 activates MET in non-human primates Rhesus monkey kidney epithelial cell line LLC-MK2 and in human kidney epithelial cell line HEK-293. D, Production of active recombinant META4. HEK-293 ells were transfected with META4 heavy plus light chain expression vectors or by individual chains as indicated. Culture media were harvested 5 days post-transfection, and META4 was purified by protein-A chromatography. Activity was assessed by MET activation as in (A);

[0099] FIGS.53A-53B show META4 activates MET and MET in humanized mice liver. META4 was injected intraperitoneally at 1 mg/g, and livers were collected at 30 and 60 minutes and assessed for MET activation as indicated;

[00100] FIGS. 54A-54D show restoration of MET signaling by META4 therapy ameliorates liver inflammation and fibrosis in the humanized NASH and promotes expansion of the transplanted human hepatocytes. A, Shown are representative images of liver sections from humanized mice with NASH treated with META4 or with mlgG1 stained for the indicated markers. B-D, Confirmation of META4 effects at the protein level. A, Alpha smooth muscle actin (a-SMA); B, Vimentin; and C, IKBa. Livers from nontransplanted (non-TXP) FRGN and ob/ob mice are included for comparison (n = 4) for META4 and (n = 2) for and control;

[00101] FIGS. 55A-55B show META4 promotes survival and proliferation of human hepatocytes in humanized NASH model. Shown are representative images of liver sections stained for TUNEL (A) and Ki67 and FAH double staining as indicated. Scale: 100 mm in the left panel and 30 mm in the right panel, respectively. Black arrows point to FAH-positive and Ki67-negative, and white arrows point to hepatocytes positive for FAH and nuclear Ki67. Mice were on HFD for 6 weeks and then 4 weeks of META4 therapy (single intraperitoneal injection weekly). B, Results of Western blot for FAH indicating expansion (survival and proliferation) of human hepatocytes by META4; and [00102] FIGS. 56A-56B show META4 therapy ameliorates weight lost (A) and hepatomegaly (B) in mice with humanized liver. A, Bar graphs show gradual weight loss in control-treated mice after NTBC withdrawal. *P = .016. Significance was assessed by the Student t test (n = 7 per group). B, Shown are the gross appearance of livers and plots of liver to body ratios for META4- (n = 4) or mlgG1 (n = 4) treated mice as indicated. **P = .01 .

DETAILED DESCRIPTION

[00103] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases. As used herein "a" and "an" refer to one or more.

[00104] As used herein, the term "comprising" is open-ended and may be synonymous with "including", "containing", or "characterized by". As used herein, embodiments "comprising" one or more stated elements or steps also include but are not limited to embodiments "consisting essentially of" and "consisting of" these stated elements or steps.

[00105] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise indicated, polymer molecular weight is expressed as number-average molecular weight (Mri). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[00106] The term “contacting” refers to placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” In some cases, “contacting” includes transfecting, such as transfecting a nucleic acid molecule into a cell. In other examples, “contacting” refers to incubating a molecule (such as an antibody) with a biological sample.

[00107] An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, or particle) refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in the cell of the organism in which the component occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified”, thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. A preparation may be purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.

[00108] A nucleic acid molecule (a nucleic acid) refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

[00109] A first nucleic acid is said to be operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. Generally, operably linked DNA sequences are contiguous (e.g., in cis) and, where the sequences act to join two protein coding regions, in the same reading frame (e.g., open reading frame or ORF), for example to produce a fusion protein. Operably linked nucleic acids include a first nucleic acid contiguous with the 5' or 3' end of a second nucleic acid. In other examples, a second nucleic acid is operably linked to a first nucleic acid when it is embedded within the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid.

[00110] A “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species of group of species). For example, a nucleic acid sequence can be optimized for expression in yeast cells. Codon optimization does not alter the amino acid sequence of the encoded protein.

[00111] A conservative substitution is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, an antigen binding molecule or antibody polypeptide sequence may include one or more conservative substitutions (for example 1 -10, 2-5, or 10-20, or no more than 2, 5, 10, 20, 30, 40, or 50 substitutions) yet retains the affinity or avidity of a given antigen binding molecule such as those described herein for binding to META4. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site- directed mutagenesis or PCR. Methods are provided herein to ascertain proper expression of any sequence.

[00112] A polypeptide is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alphaamino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide”, “peptide”, or “protein” as used herein are intended to encompass any amino acid sequence and include proteins and modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are synthetically produced such as by recombinant or chemical synthesis methods. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[00113] Conservative amino acid substitutions are those substitutions that, when made, least or minimally interfere with the properties of the original protein, that is, in the context of the end-use, the structure and function of the protein is conserved and not significantly changed by such substitutions, and may be identified by use of matrices, such as the BLOSUM series of matrices, and other matrices. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine. In terms of antibody structure, conservative substitutions may be relatively freely made to framework amino acids and constant region amino acids, such as to humanize an antigen binding molecule.

[00114] As used herein, the term “epitope” refers to a physical structure or moiety of a molecule that interacts with an antibody or antibody binding reagent. In terms of proteins or polypeptides, the primary amino acid sequence can define an epitope, but secondary and tertiary protein structure, as well as post-translational modifications, can define an epitope. For example, META4 protein and protein fragments, including isoforms and post-transcriptionally-modified polypeptides for use in the immunodetection methods, devices, and kits may be produced in mammalian cells, such as HEK293 cells, to produce a protein with mammalian post-translational modifications. Portions of a natural protein can contain an epitope present in the complete natural protein and typically react to antibodies raised to the natural protein. [00115] A recombinant nucleic acid refers to a nucleic acid molecule (or protein or virus) that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids. The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein. [00116] “Sequence identity” refers to the similarity between nucleic acid or amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity may be measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, isoforms, or variants of a polypeptide often possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well- known in the art. Various programs and alignment algorithms are described in the art (see, e.g., Chao J, et al. Developments in Algorithms for Sequence Alignment: A Review. Biomolecules. 2022 Apr 6;12(4):546).

[00117] Once aligned, the number of matches may be determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity may be determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1 166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1 166-?1554*100=75.0). The percent sequence identity value may be rounded to the nearest tenth. For example, 75.1 1 , 75.12, 75.13, and 75.14 are rounded down to 75.1 , while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

[00118] Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants may typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[00119] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences may be entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example and without limitation, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithm of Needleman & Wunsch, by the search for similarity method of Pearson & Lipman, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by manual alignment and visual inspection. One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle. Using PILEUP, a reference sequence may be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package (see, e.g., Chao J, et al. Biomolecules. 2022 Apr 6;12(4):546).

[00120] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program may be used for nucleotide sequences. The BLASTP program may be used for amino acid sequences. [00121] As used herein, reference to “at least 70% identity” (or similar language) may refer to “at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. As used herein, reference to “at least 90% identity” (or similar language) may refer to “at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[00122] Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in polynucleotide strands that are typically in antiparallel orientation. Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. In RNA as opposed to DNA, uracil rather than thymine is the base that is complementary to adenosine. Two sequences comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w/v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1 X SSC (saline sodium citrate) to 10X SSC, where 1 X SSC is 0.15M NaCI and 0.015M sodium citrate in water. Hybridization of complementary sequences is dictated, e.g., by the nucleobase content of the strands, the presence of mismatches, the length of complementary sequences, salt concentration, temperature, with the melting temperature (Tm) lowering with shorter complementary sequences, increased mismatches, and increased stringency. Perfectly matched sequences are said to be “fully complementary”, though one sequence (e.g., a target sequence in an mRNA) may be longer than the other.

[00123] A vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. [00124] By “expression” or “gene expression,” it is meant the overall flow of information from a gene. A “gene” is a sequence of DNA or RNA which codes for a molecule, such as a protein or a functional RNA, such as an ncRNA that has a function. A “gene” is a functional genetic unit for producing its gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and generally comprising: a transcriptional control sequence, such as a promoter and other cisactin elements, such as transcriptional response elements (TREs) and/or enhancers; an expressed sequence that typically encodes a protein (referred to as an openreading frame or ORF) or functional/structural RNA; and a polyadenylation sequence). A gene produces a gene product (typically a protein, optionally post-translationally modified, or a functional/structural RNA) when transcribed. By “expression of genes under transcriptional control of,” or alternately “subject to control by” a designated sequence such as a promotor, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in cis) to the gene. A gene that is “under transcriptional control” of an inducible promotor or transcription control element, is a gene that is transcribed at detectably different levels in the presence of a transcription factor, e.g., in specific cell types or conditions. A “gene for expression of” a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environment, that is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression. In the case of a constitutive promoter “suitable conditions” means that the gene typically need only be introduced into a host cell. In the case of an inducible promoter, “suitable conditions” means when factors that regulate transcription, such as DNA-binding proteins, are present or absent, for example, an amount of the respective inducer is available to the expression system (e.g., cell), or factors causing suppression of a gene are unavailable or displaced - effective to cause expression of the gene.

[00125] In further detail, transcription is the process by which the DNA gene sequence is transcribed into RNA. The steps include transcript initiation, transcript elongation, and transcript termination. The molecular machinery of transcription includes but is not limited to: RNA polymerase, general transcription factors, enhancers, and promoter DNA, and RNA transcript. Transcription factors (TFs) are proteins that control the rate of transcription of genetic information from DNA to RNA, by binding to a specific DNA sequence (e.g., the promoter region). The function of TFs is to regulate genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. The promoter region of a gene is a region of DNA that initiates transcription of that particular gene. Promoters are located near the transcription start sites of genes, on the same strand, and often, but not exclusively, are upstream (towards the 5' region of the sense strand) on the DNA. Promoters can be about 100-1000 base pairs long. Additional sequences and non-coding elements can affect transcription rates. If the cell has a nucleus (eukaryotes), the RNA is further processed. This includes polyadenylation, capping, and splicing. Polyadenylation refers to the addition of a poly(A) tail to a messenger RNA. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. Capping refers to the process wherein the 5’ end of the pre-mRNA has a specially altered nucleotide. In eukaryotes, the 5’ cap (cap-0), found on the 5’ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5’ to 5’ triphosphate linkage. During RNA splicing, pre-mRNA is edited. Specifically, during this process introns are removed, and exons are joined together. The resultant product is known as mature mRNA. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.

[00126] Gene expression involves various steps, including transcription, post- transcriptional RNA modification, translation, and post-translational modification of a protein. Expression of a gene may also include reduction of the total amount of the protein product, such as by cleavage, sequestration, binding, or other means of decreasing the function or amount of a protein product.

[00127] Nucleic acids and vectors encoding the described fusion proteins may be provided. In some non-limiting examples, disclosed is a recombinant vector, such as a yeast plasmid, that expresses the disclosed fusion proteins. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence, but which encode the same protein sequence due to codon degeneracy. In some embodiments, the polynucleotide is codon-optimized for expression in mammalian cells.

[00128] Exemplary nucleic acids may be prepared by cloning techniques, e.g., as are broadly-known and implemented either commercially, or in the art. Multiple textbooks and reference manuals describe and provide examples of useful and appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through such techniques are known. Commercial and public product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), Addgene, and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources.

[00129] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

[00130] Provided herein are antigen binding molecules, e.g., antibody compounds comprising an antibody domain targeting MET proteins and epitopes, and methods of use of those compositions. The term “antigen binding molecule”, for ease of reference and unless otherwise specified, refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are typically covalently linked, as in immunoglobulin (see, e.g., Chailyan A, Marcatili P, Tramontane A. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 201 1 Aug;278(16):2858-66, and US Patent No. 1 1 ,578,428 B2, and US Patent Publication No. 2024/0158529, showing typical antibody structures, including humanized antibodies). As such, the antigen binding molecule operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein. Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen. The term “VH” refers to a heavy chain variable region of an antibody. The term “VL” refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced, and may be “humanized” to reduce immunogenicity, as is known in the related arts. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE. [00131] An antigen binding molecule or complexes thereof may be, for example and without limitation, a monoclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab', Fv fragments, single chain Fv (scFv) fragments, dsFv, Fabi fragments, F(ab')2 fragments, single domain antibodies, camelized (camelid) antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BiTE, e.g., a DbTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.

[00132] scFv molecules may be manufactured using any suitable technology. Typically, recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems. Huston J. S., M. Mudgett-Hunter, M. S. Tai et al., “Protein engineering of single-chain Fv analogs and fusion proteins, "Methods in Enzymology, vol. 203, pp. 46-88, 1991 ; Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250; Gqciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017 Dec 18;12(12):e0189964; Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci. 2020 Aug 31 ;21 (17):6324; Petrus MLC, Kiefer LA, Puri P, Heemskerk E, Seaman MS, Barouch DH, Arias S, van Wezel GP, Havenga M. A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol. 2019 Nov;103(21 -22):8875- 8888; and Toleikis L, Frenzel A. Cloning single-chain antibody fragments (ScFv) from hybridoma cells. Methods Mol Biol. 2012;907:59-71 ; see, also, www.kbdna.com/cloning-scfv

[00133] The antigen binding molecules described herein, comprise, at their core paratopes formed from VL and VH polypeptides, that are defined by three CDR’s (typically loops), CDR1 , CDR2, and CDR3, which for VH peptides may be termed HCDR1 , HCDR2, and HCDR3, respectively, and which for VL peptides may be termed LCDR1 , LCDR2, and LCDR3, respectively, each of which are flanked by, and separated by framework (e.g., joining or scaffold) amino acid sequences that space apart and support the CDRs, and which may differ from antibody-to-antibody, and which may be “humanized” to minimize antigenicity when administered to a human patient. In nature, HCDR3 and LCDR3 are typically the most variable of the CDRs, contributing significantly to antibody specificity. Various methods may be used to identify the precise limits of each CDR, but the sequences provided herein can be evaluated by any suitable method to determine the CDRs.

[00134] Exemplary sequences of antibody heavy and light chains are provided in the attached sequence listing, which is incorporated herein by reference in its entirety. Antibody constant and variable regions, including CDR sequences and framework sequences can be readily ascertained from the sequences provided in in the attached sequence listing. Reference to a CDR herein may refer to a Kabat CDR numbering scheme or any other applicable CDR numbering scheme, including but not limited to Chothia, Martin (enhanced Chothia), Gelfand, IMGT, Honneger, or any other numbering scheme (see, e.g., Dondelinger M, et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Front Immunol. 2018 Oct 16;9:2278). For example and without limitation, antibody sequence annotation, including definition of CDR sequences may be conducted using tools described and provided in abYsis (abysis.com), or Abnum (www.bioinf.org.uk/abs/abnum/). Other methods of CDR identification are known in the art (see, e.g., Kunik V, Ashkenazi S, Ofran Y. Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521 -4; Adolf- Bryfogle J, Xu Q, North B, Lehmann A, Dunbrack RL Jr. PylgClassify: a database of antibody CDR structural classifications. Nucleic Acids Res. 2015 Jan;43(Database issue):D432-8), and assorted online tools and applications as are broadly-available. As such, an amino acid sequence of a VH or VL may be provided or determined, comprising CDRs, and one of ordinary skill can determine the precise metes and bounds of CDRs within that antibody sequence without undue experimentation. Framework sequences may be optimized (see, e.g., Gopal R, Fitzpatrick E, Pentakota N, Jayaraman A, Tharakaraman K, Capila I. Optimizing Antibody Affinity and Developability Using a Framework-CDR Shuffling Approach-Application to an Anti- SARS-CoV-2 Antibody. Viruses. 2022 Nov 30;14(12):2694) and/or humanized based on knowledge of amino acid sequences of the CDRs, e.g., LCDR1 , LCDR2, LCDR3, HCDR1 , HCDR2, and HCDR3 of antibodies described herein.

[00135] Antibodies may be produced by any effective method, such as by hybridoma or it may be recombinantly or synthetically produced. In the context of the present disclosure and for ease of reference, “antibodies” or “antibody” may refer to both natural antibodies as well as protein antibody analogs, antibody fragments, and derivatives, any of which comprising VL and/or VH sequences and/or CDRs (e.g., all three CDRs of any VH or VL region, defining a paratope as described herein) according to any example, aspect, or embodiment described herein. The antibody or antibodies may be synthetic, in that they do not comprise a naturally-occurring sequence, such as certain antigen binding molecules, including engineered versions and derivatives thereof, such as scFv versions thereof, humanized versions thereof, BiTEs, and/or sequence derivatives thereof, including without limitation, proteins comprising the CDRs (e.g., one or more, or all three CDRs) of the antibodies provided herein.

[00136] Nanobodies, which may be referred to a VHH antibodies or single-domain antibodies, may be constructed using CDR sequences, such as CDRs of the antibodies described herein. Nanobodies may be created by grafting of the complementarity determining regions (CDRs) from already existing, non-camelid antibodies to VHH frameworks, followed by affinity maturation using synthetic phage libraries (see, e.g., Wagner HJ, Wehrle S, Weiss E, Cavallari M, Weber W. A Two- Step Approach for the Design and Generation of Nanobodies. Int J Mol Sci. 2018 Nov 2 ; 19(1 1 ):3444). A VH, alone, may be capable of defining an antigen-binding site (e.g., paratope) with sufficient strength to be pharmacologically-useful, and can be referred to as a nanobody (see, e.g., Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 2009 Aug;198(3):157-74, providing structure and sequences of various nanobodies and Bever CS, Dong JX, Vasylieva N, Barnych B, Cui Y, Xu ZL, Hammock BD, Gee SJ. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016 Sep;408(22):5985- 6002). Single-domain antigen binding molecules originally were camelid antibodies, which naturally comprise only heavy chains (see, e.g., Mitchell LS, Colwell LJ. Comparative analysis of nanobody sequence and structure data. Proteins. 2018 Jul;86(7):697-706). More recently other single-chain antigen-binding molecules have been developed. Construction and humanization of single-domain antigen binding molecules is broadly-known (see, e.g., Valdes-Tresanco MS, Molina-Zapata A, Pose AG, Moreno E. Structural Insights into the Design of Synthetic Nanobody Libraries. Molecules. 2022 Mar 28;27(7):2198; Wu Y, Jiang S, Ying T. Single-Domain Antibodies As Therapeutics against Human Viral Diseases. Front Immunol. 2017 Dec 13;8:1802; Hoey RJ, Eom H, Horn JR. Structure and development of single domain antibodies as modules for therapeutics and diagnostics. Exp Biol Med (Maywood). 2019 Dec;244(17):1568-1576; Rossotti MA, Belanger K, Henry KA, Tanha J. Immunogenicity and humanization of single-domain antibodies. FEBS J. 2022 Jul;289(14):4304-4327; and Khodabakhsh F, Behdani M, Rami A, Kazemi-Lomedasht F. Single-Domain Antibodies or Nanobodies: A Class of Next-Generation Antibodies. Int Rev Immunol. 2018;37(6):316-322). Multimerization methods are broadly-known, too (see, e.g., Miller A, Carr S, Rabbitts T, Ali H. Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats. MAbs. 2020 Jan-Dec;12(1 ):1752529), for example to produce bi-specific antibody binding molecules, such as bi-specific T-cell engagers (e.g., BiTEs), discussed in further detail, below. Nanobody construction has been commercialized, e.g. in Crescendo Biologies’ Humabody platform (see, Teng Y, et al., Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse. N Biotechnol. 2020 Mar 25;55:65-76, US 1 1 ,547,099 B2, and WO 2016/062988 for exemplary constructs, transgenic mice, and methods for producing VH nanobodies). [00137] An antibody-drug conjugate (ADC) may be provided. An antigen binding molecule according to any aspect, embodiment, or example provided described herein may be linked to a payload (e.g., a cargo or warhead) that causes a desired physiological effect, such as killing a cell expressing a binding partner to the antigen binding molecule. The antigen binding molecule, e.g. an antibody, can be effectively covalently-linked to other moieties, for example by their Fc sequences yet retain significant antigen-binding capacity. ADCs comprise an antigen binding moiety (a linked antigen-binding molecule), a linker that may be cleavable or non-cleavable, and the payload moiety. A “moiety” is a chemical group or entity, often functional, that forms part of a larger molecule. Linkers may be used to join a payload moiety to the antigen binding molecule, and choice of linkers can depend on how the APC is handled by the cell, and how the payload becomes effective on processing by a cell, or by release due to chemical lability of the linker. Non-cleavable linkers include, without limitation, alkyl moieties, and thioether moieties e.g., Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC)). Cleavable or labile linkers may include, for example and without limitation, acid-labile linkers (hydrolysable in lysosomes or endosomes), Lysosomal protease-sensitive linkers (e.g., peptide-based linkers), /3-glucuronide linkers, and glutathione-sensitive disulfide linkers, with examples including, without limitation: ester-, hydrazone-, Valine-citrulline (v-c)-, Valine-alanine (v-a)-, and phenylalanine-lysine (p-l)-containing linkers, (see, e.g., Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020 Jan;18(1 ):3-19, describing exemplary linking methods and suitable cytotoxic payloads or warheads). Examples of payloads include, without limitation: microtubule-disrupting agents, such as auristatin, maytansinoids, eribulin (e.g., eribulin mesylate), tubulysins, cryptophycins, and EG5 inhibitors; DNA-damaging agents, such as, without limitation calicheamicin, duocarmycins, doxorubicin, enediyne, topoisomerase I inhibitors, and Pyrrolo[2,1 - c][1 ,4] benzodiazepines; RNA-targeting payloads, such as thailanstatins and amatoxins; immune payloads, such as Toll-like receptor agonists, STING agonists, glucocorticoid receptor modulators; and other payloads, such as Bcl-xL inhibitors, NAMPT inhibitors, and proteasome inhibitors such as carmaphycins. Design and optimization considerations for production of ADCs are provided in Khongorzul P, et al. (Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020 Jan;18(1 ):3-19, describing exemplary linking methods and suitable cytotoxic payloads or warheads, and see, e.g., Gogia P, Ashraf H, Bhasin S, Xu Y. Antibody-Drug Conjugates: A Review of Approved Drugs and Their Clinical Level of Evidence. Cancers (Basel). 2023 Jul 30;15(15):3886; Baah S, Laws M, Rahman KM. Antibody-Drug Conjugates-A Tutorial Review. Molecules. 2021 May 15;26(10):2943; and Wang Z, Li H, Gou L, Li W, Wang Y. Antibody-drug conjugates: Recent advances in payloads. Acta Pharm Sin B. 2023 0ct;13(10):4025-4059). ADCs with multiple payloads, PROT AC-guided ADCs, ADCs with peptide-drug-conjugates, and ADCs with photo-reactive payloads also may be produced. As such a person of ordinary skill can produce, based on the teachings herein and without undue experimentation, an ADC comprising an antigen binding molecule as described herein linked to a cytotoxic payload via a chemical linker.

[00138] While specific types of antigen binding molecules are described specifically herein, any antigen binding molecule comprising CDR sequences as described herein for binding META4 contemplated. Such antigen binding molecules may be evaluated and used in an affinity assay, such as an ELISA assay, bilayer interferometry (e.g., BLItz, see, e.g., Muller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR- Cas Effector Complexes. Front Mol Biosci. 2020 May 27;7:98), single-molecule array (e.g., SIMOA), surface plasmon resonance (SPR), oblique-incidence reflectivity difference (OI-RD) binding affinity, or cell binding assay, are contemplated. Antigen binding molecules with high binding affinities may bind to their corresponding antigen with a KD of 1 pM or less, 500 nM or less, 100nM or less, 75nM or less, 50nM or less, or 25 nM or less. Such antigen binding molecules may find use in therapies for conditions related to (e.g., responsive to) MET agonists, and for diagnostic purposes. [00139] By “target-specific” or reference to the ability of one compound to bind another target compound specifically, it is meant that the compound binds to the target compound to the exclusion of others in a given reaction system, e.g., in vitro, or in vivo, to acceptable tolerances, permitting a sufficiently specific diagnostic or therapeutic effect according to the standards of a person of skill in the art, a medical community, and/or a regulatory authority, such as the U.S. Food and Drug Agency (FDA), in aspects, in the context of administering a reagent as described herein to a patient.

[00140] As used herein, the “treatment” or “treating” of a patient means administration to a patient by any suitable dosage regimen, procedure and/or administration route of a composition, device, or structure (e.g., an antigen binding molecule or antibody as described herein) with the object of achieving a desirable clinical/medical end-point, including but not limited to, any suitable treatment for a condition associated with and/or responsive to a MET agonist, and also includes monitoring the patient for development of a condition associated with and/or responsive to a MET agonist by any useful method, including by use of an antigen binding molecule described herein. Exemplary conditions associated with and/or responsive to a MET agonist include, without limitation, organ failure (such as liver and kidney failure/disease), organ fibrosis (such as lung and kidney fibrosis), a hepatitis (e.g., steatohepatitis, alcohol hepatitis, fatty liver disease, viral hepatitis, autoimmune hepatitis, or drug induced hepatitis, such as Tylenol poisoning), a biliary disease (such as primary cholangitis), inflammatory bowel disease, and/or type 2 diabetes, including restoring insulin responsiveness in insulin-resistance, and/or its associated pathologies such as retinopathy and macular degeneration. In non-limiting embodiments, the condition is HIV, AIDS, and/or a cancer in which MET/HGF are overexpressed, e.g., highly overexpressed, such as a breast, colon, glioma, or thyroid cancer.

[00141] A "therapeutically effective amount" refers to an amount of a drug product or active agent effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An “amount effective” for treatment of a condition is an amount of an active agent or dosage form, such as a single dose or multiple doses, effective to achieve a determinable end-point. The “amount effective” is preferably safe - at least to the extent the benefits of treatment outweighs the detriments, and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration. A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[00142] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate compositions, such as parenteral or inhaled compositions, in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[00143] An “effective amount” or “amount effective” to achieve a desirable therapeutic, pharmacological, medicinal, or physiological effect is any amount that achieves the stated purpose, for example, an amount of an active agent (antigen binding molecule or antibody) described herein effective to treat a condition associated with and/or responsive to a MET agonist. Based on the teachings provided herein, one of ordinary skill can readily ascertain effective amounts of the elements of the described dosage form and produce a safe and effective dosage form and drug product. Examples of an effective amount of an active agent compounded in a delivery vehicle includes from 100 pg per ml (picograms per milliliter) to 1 mg/ml (milligrams per milliliter) of solution, including any increment therebetween, such as from 1 ng/ml (nanogram/milliliter) to 1 mg/ml or from 1 ng/ml to 1 pg/ml (microgram/milliliter).

[00144] Drug products, or pharmaceutical compositions comprising an active agent (e.g., drug), may be prepared by any method known in the pharmaceutical arts, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s). As used herein, a “pharmaceutically acceptable excipient”, “carrier”, or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohol’s such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the active agent. In certain aspects, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used in delivery systems, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are broadly-known to those skilled in the art. The preferred form may depend on the intended mode of administration and therapeutic application, which will in turn dictate the types of carriers/excipients. Suitable forms include, but are not limited to, liquid, semi-solid, and solid dosage forms.

[00145] Pharmaceutical formulations adapted for oral administration may be presented, for example and without limitation, in capsules, tablets, oral solutions, or the like, and include suitable carriers and coatings as are broadly-known in the pharmaceutical arts.

[00146] Pharmaceutical formulations adapted for parenteral administration may be presented, for example and without limitation, in syringes, vials, bottles, IV/infusion bags, or the like, as are broadly-known to those of ordinary skill. Excipients include, for example and without limitation, water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable emulsifiers, lipids, surfactants, or the like may be utilized to maintain an active agent in solution.

[00147] Pharmaceutical formulations adapted for transdermal administration may be presented, for example and without limitation, as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time or electrodes for iontophoretic delivery.

[00148] Pharmaceutical formulations adapted for topical administration may be formulated, for example and without limitation, as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.

[00149] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. For example, sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with suitable carrier(s), followed by filter-sterilization. An appropriate fluidity of a solution can be maintained, for example, by the use of a rheology modifier. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[00150] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.

[00151] The therapeutic agents described herein can be administered by any effective route. Examples of delivery routes include, without limitation: topical, for example, epicutaneous, inhalational, enema, ocular, otic, and intranasal delivery; enteral, for example, orally, by gastric feeding tube, and rectally; and parenteral, such as, intravenous, intraarterial, intrathecally, intramuscular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal, with intrathecal and oral approaches being preferred in many instances. Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes, containing a composition comprising the therapeutic agent useful for treatment of graft rejection as described herein.

[00152] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the therapeutic agent may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate therapeutic agents in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms may be dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic agent for the treatment of sensitivity in individuals.

[00153] Provided herein are antibodies and antibody-based drugs, and methods of treatment of diseases mediated by the lack of proper MET tyrosine kinase receptor function. In certain disease, conditions, and/or disorders, HGF-MET function is hampered (reduced or lacking). Thus, treatment with MET agonistic antibody (referred to herein in some instances as META4) will overcome the blockage and restore MET function. On the other hand, in diseases in which HGF-MET is overactive, such as a variety of cancers including colon, breast, lung, liver, pancreas, prostate and ovarian cancer, employing an antagonistic MET antibody (ScFv-META4) will reduce or block the overactive MET in the cancer cells.

[00154] A potent MET agonist, referred to herein generally as META4 was generated by immunizing mice with the extracellular portion of human MET as antigen, producing hybridomas that produced META4. cDNAs encoding the heavy and light chains of META4 were successfully cloned from hybridoma-producing clones and cDNA expression vectors that encode active META4 were constructed. Since META4 is a mouse monoclonal lgG1 it was humanized so that it can be used in humans for therapeutic purposes. The humanized version is as active as the parental version. META4 is specific for human and monkey (Macaca Mullata, also known as Rhesus monkey). META4 reagents are expected to have great clinical utility in regenerative medicine and in settings like organ failure such as liver and kidney failure/disease, various forms of hepatitis (steatohepatitis, alcohol hepatitis, viral hepatitis, autoimmune hepatitis, drug induced hepatitis such as Tylenol poisoning), and type 2 diabetes and its associated pathologies like retinopathy and macular degeneration to name a few.

[00155] A version of META4 (a META4-scFv (single-chain Fragment variable)) was cloned. It may comprise the antigen binding region of META4’s heavy and light chains linked together by a linker region. Please see the exemplary sequence of META4 scFv (SEQ ID NO: 22), described in Example 2, FIGS. 14-15 and Table 4 as well as the binding data Tables 5-7, for instance). The single chain version retains binding activity for MET and cannot dimerize like the original IgG META4 because it lacks the constant regions of heavy and light chains. Hence, it should not be able to activate MET because MET needs dimerization to become activated. Thus META4-scFv may work as an antagonist of MET which may be used in cancer therapy in certain kinds of types in which MET/HGF are highly overexpressed like breast, colon, glioma, thyroid, and pancreas cancer to name a few. Examples of MET-binding scFvs are provided in Jin, H et al. MetMAb, the one-armed 5D5 anti-c-Met antibody, inhibits orthotopic pancreatic tumor growth and improves survival. Cancer Res. 2008 Jun 1 ;68(1 1 ):4360-8., Prat, M et al. Monoclonal Antibodies against the MET/HGF Receptor and Its Ligand: Multitask Tools with Applications from Basic Research to Therapy. Biomedicines. 2014 Dec 3;2(4):359-383., and Vafaei, R et al. Development of a MET-targeted single-chain antibody fragment as an anti-oncogene targeted therapy for breast cancer. Invest New Drugs. 2023 Apr;41 (2):226-239. Further tests are required to determine if META4- scFv acts as agonist or antagonist. In either case, it can be used as a therapeutic reagent.

[00156] META4 can also be converted to one-arm META4 lgG1 , which may act as an antagonist of MET and thus can be used in cancer therapy in cases in which HGF- MET action is dysregulated, as mentioned above.

[00157] Currently there are no effective therapies for degenerative diseases such as steatohepatitis (NASH and ASH) and others such age-related macular degeneration (AMD). We have generated data showing that META4 is a potent growth and survival factor various epithelia cells including hepatocytes and retinal pigmented epithelial cells. META4 is stable and bioavailable as opposed to HGF which has poor pharmacokinetics.

[00158] Provided herein are antibody compositions and methods of use of those compositions (e.g., as MET agonists). The term “antibody”, for ease of reference and unless otherwise specified, refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof. As such, the antibody operates as a ligand for its cognate antigen, which can be virtually any molecule. Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site capable of specifically binding an antigen (e.g., a paratope). The term “VH” refers to a heavy chain variable region of an antibody. The term “VL” refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE.

[00159] An antibody may be a monoclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab', Fv fragments, single chain Fv (scFv) fragments, dsFv, Fabi fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BiTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition.

[00160] Antibodies may be produced by any effective method, such as by hybridoma or it may be recombinantly or synthetically produced. In the context of the present disclosure and for ease of reference, “antibodies” or “antibody” may refer to both natural antibodies as well as protein antibody analogs, antibody fragments, and derivatives comprising VL and VH sequences and/or CDRs according to any example, aspect, or embodiment described herein. The antibody or antibodies may be synthetic, in that they do not comprise a naturally-occurring sequence, such as the mouse Hu-1 1 S monoclonal antibody derivatives, including engineered versions and derivatives thereof, such as scFv versions thereof, humanized versions thereof, and/or sequence derivatives thereof, including without limitation, proteins comprising the VH and/or VL, or CDRs thereof (e.g., all six CDRs of any VH or VL region), of Hu-1 1 S derivatives, such as clones 2, 5, 6, 12, 13, 15, 16, 17, or 18 of Example 2, FIG. 1 , or clones 5, 12, 15, 16, and 17 provided in Example 2, FIG. 1 and FIG. 2, e.g., of clone 16.

[00161] Unless indicated otherwise, nucleic acid sequences are provided in 5’ to 3’ orientation, and amino acid sequences are provided in an N to C-terminal orientation. Reference to a CDR herein may refer to a Kabat CDR numbering scheme (see, Example 2, FIG. 3, or any other applicable CDR numbering scheme, including but not limited to Chothia, Martin (enhanced Chothia), Gelfand, IMGT, Honneger, or any other numbering scheme (see, e.g., Dondelinger M, et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Front Immunol. 2018 Oct 16;9:2278). Reference to an antibody comprising CDRs of a specified clone, such as hu1 1 S, include all six CDRs; H1 , H2, H3, L1 , L2, and L3, or alternatively referred to as HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3. [00162] In non-limiting embodiments, an antibody as disclosed herein may bind to hepatocyte growth factor (HGF) receptor. In non-limiting embodiments the antibody may include a heavy chain variable region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 20, SEQ ID NO: 32, and/or SEQ ID NO: 33. In non-limiting embodiments, the antibody may have a light chain variable region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to a sequence selected from SEQ ID NO: 21 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , and/or SEQ ID NO: 42. In non-limiting embodiments, the antibody may have both a heavy chain variable region and a light chain variable region (e.g., the antibody may have a heavy chain variable region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 20, SEQ ID NO: 32, and/or SEQ ID NO: 33 and a light chain variable region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to a sequence selected from SEQ ID NO: 21 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , and/or SEQ ID NO: 42).

[00163] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 20; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 21 .

[00164] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 34, 35, and/or 36. [00165] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 34, 35, and/or 36.

[00166] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 37.

[00167] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 38.

[00168] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 39.

[00169] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 40.

[00170] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 41 .

[00171] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 32; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 42.

[00172] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 37.

[00173] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 38.

[00174] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 39.

[00175] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 40.

[00176] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 41 .

[00177] In non-limiting embodiments, the antibody may have a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 33; and a light chain variable region has an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 42.

[00178] In non-limiting embodiments, the antibody may include a complementaritydetermining region (CDR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55. In non-limiting embodiments, the antibody includes one or more substations and/or mutations in the CDR. In non-limiting embodiments, the one or more substitutions and/or mutations are one or more of S31 N, F33Y, S35G, Y59E, Y60E, P61 S, P61 Q, P61 L, P61 T, S103I, K187I, S190G, and S226D.

[00179] Also provided herein is an antibody that binds HGF and may have a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 2 and/or a light chain variable region and a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 4.

[00180] Also provided herein is an antibody that binds HGF and may have a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 6 and/or a light chain variable region and a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 4.

[00181] Also provided herein is an antibody that binds HGF and may have a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 9 and/or a light chain variable region and a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 4.

[00182] Also provided herein is an antibody that binds HGF and may have a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 1 1 and/or a light chain variable region and a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 13.

[00183] Also provided an antibody that binds HGF and may have a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 15 and/or a light chain variable region and a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 17.

[00184] Also provided herein is an antibody that binds HGF and may have a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 19 and/or a light chain variable region and a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 21. [00185] Also provided herein is an scFv having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, and/or 100% sequence identity (all values and subranges therebetween inclusive) to SEQ ID NO: 22.

[00186] Antibodies as disclosed herein may be included in pharmaceutical compositions, including with one or more pharmaceutically-acceptable carriers and/or excipients as are known to those of skill in the art, and which may include water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable antibodies and, optionally, pharmaceutically-acceptable carriers and/or excipients may be provided in a kit, optionally with a medical device such as a syringe, vial, bottle, IV/infusion bag, metered dose inhaler, wearable injection device, or the like, as are broadly-known to those of ordinary skill.

[00187] Also provided herein are methods of using antibodies as described herein for treating a condition, disease, and/or disorder and/or preventing a disease, condition, and/or disorder. In non-limiting embodiments, the condition, disease, and/or disorder is one that relates to an HGF receptor and/or is or may be responsive to a MET agonist. Without limitation, such conditions, diseases, and/or disorders may include organ failure (such as liver and kidney failure/disease, a hepatitis (e.g., steatohepatitis, alcohol hepatitis, viral hepatitis, autoimmune hepatitis, or drug induced hepatitis, such as Tylenol poisoning), and/or type 2 diabetes, including restoring insulin responsiveness in insulin-resistant diabetes, and/or its associated pathologies such as retinopathy and macular degeneration. In non-limiting embodiments, the condition, disease, and/or disorder is a cancer, for example a cancer in which MET/HGF receptor is over expressed, as are known in the art, and which may include breast, colon, glioma, and/or thyroid cancer. In non-limiting embodiments, the condition, disease, and/or disorder is HIV and/or AIDS. In non-limiting embodiments, the condition, disease, and/or disorder is one of affecting the eye. In treatment and/or prevention of any condition, disease, and/or disorder, the composition including at least the antibody as described herein may be administered in any useful dosing regimen, by any suitable route, in any suitable amount effective to prevent and/or treat the condition, disease, and/or disorder.

[00188] As may be appreciated, antibodies, such as those described herein, may be useful in assays, for example binding assays, such as immunoassays. Suitable reagents for such binding assays are known to those of skill in the art and may be included in a kit with an antibody as described herein. Examples:

[00189] Preparation of monoclonal antibody: As indicated above, a mouse monoclonal antibody hybridoma was prepared using standard methodology using the extracellular portion of human MET as antigen. The hybridoma produced a monoclonal lgG1 antibody with the exemplary META4 VH and VL sequences as follows (also referred to as Hu-11 s, below):

VH sequence (SEQ ID NO: 20)

EVRLVESGGGLVKPGGSLKLSCAASGFTFSSYFMSWVRQTPEKRLEWVATI SSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAIYYCARQDYR SPFYFDYWGQGTTLTVSS

VL sequence (SEQ ID NO: 21)

ENVLTQSPAIMSASPGERVTMTCSASSSVSYMHWCQQKSSSSPKLWIYDT SKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGSGT RLEIK

[00190] An exemplary scFv is provided below as Hu11 s-scFv having the following amino acid and nucleotide sequences:

Amino acid sequence of Hu11S-scFv (VH-Linker-VL) (SEQ ID NO: 22)

EVRLVESGGGLVKPGGSLRLSCAASGFTFSSYFMSWVRQAPEKRLEWVATI SSGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCARQDYR SPFYFDYWGQGTLLTVSSGGGGSGGGGSGGGGSENVLTQSPATLSASPG ERATMSCSASSSVSYMHWSQQKSSQAPRLLIYDTSKLASGVPARFSGSGS GNDYTLTISSLEPEDFAVYYCFQGSGYPLTFGQGTRLEIK

Nucleotide sequence of Hu11S-scFv (VH-Linker-VL) (SEQ ID NO: 23)

GAAGTGCGTCTGGTTGAAAGTGGTGGTGGTCTGGTTAAACCGGGCGGT AGTCTGCGTCTGAGCTGTGCGGCCAGCGGCTTCACCTTCAGCAGCTACT TCATGAGCTGGGTTCGCCAAGCCCCGGAAAAACGTCTGGAATGGGTTGC GACCATCAGCAGCGGCGGTAGCTACACGTACTACCCGGATAGCGTGAA AGGCCGTTTCACCATCAGCCGCGATAACGCGAAGAACAGCCTCTATCTG CAGATGAGCAGTCTGCGCGCGGAAGATACCGCGGTTTATTACTGTGCCC GCCAAGATTACCGCAGCCCGTTCTACTTCGACTATTGGGGCCAAGGCAC GCTGCTCACCGTTAGCAGTGGTGGAGGCGGTTCAGGCGGAGGTGGTTC TGGCGGTGGCGGATCGGAAAATGTTCTGACCCAGAGTCCAGCGACGCT GAGTGCGAGCCCGGGCGAACGCGCGACCATGAGCTGCAGCGCCAGCA GCAGCGTGAGCTACATGCACTGGAGCCAGCAGAAAAGTAGCCAAGCCC CGCGTCTGCTGATCTACGATACCAGCAAACTGGCCAGTGGCGTTCCGGC CCGCTTTAGTGGTAGCGGCAGTGGCAACGACTATACGCTGACCATCAGT AGCCTCGAACCAGAAGACTTCGCCGTGTACTACTGCTTCCAAGGCAGCG GTTATCCGCTGACCTTTGGTCAAGGCACCCGTCTGGAAATCAAG

[00191] Additional derivatives of the Hu-1 1 s sequences are described below.

Example 1 : Cloning and sequencing of Hu-11s

[00192] Three antibody genes were cloned using degenerate primers, the RT-PCR was conducted after isolation of total RNA. The RT-PCR results are shown in FIGS. 4-9. After that, the PCR products were ligated into T vectors for DNA sequencing locally. The antibody genes are listed below. After checking the sequences, three different cell lysates produce the same antibody genes. The sequence of the antibodies is set forth in SEQ ID NOS: 24-29).

[00193] The sequence of plgG-mKC is set forth in SEQ ID NO: 30, a map of which can be seen in FIG. 8.

[00194] The sequence of plgG-mHC is set forth in SEQ ID NO: 31 , a map of which can be seen in FIG. 9.

Example 2 - Humanization and Affinity Maturation of Hu-11S derivatives

Summary

[00195] Both amino acid sequence and DNA sequence of a mouse antibody (marked as J01 1819-ZYA) were used to perform humanization and affinity maturation of this antibody.

Humanization

[00196] Firstly, bioinformatics analysis and computational modeling were performed for the parent mouse antibody, the result is shown in FIG. 15. The sequences used appear below:

[00197] VH amino acid sequence of J01 1819-ZYA (SEQ ID NO: 20): EVRLVESGGGLVKPGGSLKLSCAASGFTFSSYFMSWVRQTPEKRLEWVATISSGGSYTYY PDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAIYYCARQDYRSPFYFDYWGQGTTLTVSS [00198] VL amino acid sequence of J01 1819-ZYA (SEQ ID NO: 21): ENVLTQSPAIMSASPGERVTMTCSASSSVSYMHWCQQKSSSSPKLWIYDTSKLASGVPGR FSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGSGTRLEIK

[00199] As shown in FIG. 10, the most appropriate human VH framework acceptor for parent J011819-ZYA VH region is IGHV3-21 01 , and FIG. 11 showed that the human VL framework acceptor for parent J01 1819-ZYA VL region is IGKV3-1 1 01 . By using those human VH and VL framework acceptor, in silico CDR-grafting was performed.

[00200] According to the Backmutation design rule, two VH candidates and three VL candidates were obtained and the sequence information was listed in Table 1 and Table 2. However, there is an extra Cystine in VL parental sequence (highlighted in yellow), which may cause extra developability issues during CMC. Therefore, Cys was replaced with Ser or Tyr if other features of the antibody are not be affected. As a result, 12 (2*6) humanized antibody candidates were obtained in total by using different VH and VL pair.

Table 1. Amino acid sequence of Humanized VH designs (SEQ ID NOS: 32 and 33)

Table 2. Amino acid sequence of Humanized VL designs (SEQ ID NOS: 34-36)

DomainGapAlign tool (www.imgt.org) was used to analyze the humanization percentage of all the designs. Based on the IMGT® humanization percentage data, huVHvI is the VH design with the highest humanization percentage (87.8%), and huVLvI is the VL design with the highest humanization percentage (76.8%). Compared to the parental mouse antibody, the humanization percentage of huVHv1 VLv1 is improved significantly and huVHv1 VLv1 was selected out as the top hit, whose VH is huVHvI and VL is huVLvI .

[00202] The T-cell epitope, B cell epitope, and MHC II epitope study of huVHvI VLv1 were conducted by using Protean 3D software. For huVHvI , the predicted B cell epitopes are R, EKRLEW (SEQ ID NO: 60), and A, the predicted T cell epitope is RLVE (SEQ ID NO: 61 ), the predicted MHC II epitopes are VRLVES (SEQ ID NO: 62) and MSSLRA (SEQ ID NO: 63), and the predicted antigenicity epitopes are APEKR (SEQ ID NO: 64 and QAPEKRLE (SEQ ID NO: 65); for huVLvI , the predicted antigenicity epitopes are QQKSSQA (SEQ ID NO: 66) and GSGSGND (SEQ ID NO: 67), the predicted B cell epitopes are N, A, CQQKSSQA (SEQ ID NO: 68) and SGSGSGN (SEQ ID NO: 69, the predicted MHC II epitope is LTQSPATLSAS (SEQ ID NO: 70), and the predicted T cell epitopes are SSQA (SEQ ID NO: 71 ) and RATMS (SEQ ID NO: 72). However, all the predicted epitopes contain backmutations and the epitopes were kept because removal of these backmutations may lead to loss of affinity and/or developability.

[00203] The in silica post-translational modifications (PTMs) assessment and in silica physical stability assessment of the parent mouse antibody and the humanized antibody, huVHvI VLv, were performed using BioLuminate of Schrodinger software.

[00204] After that, whole IgG expression was performed using the variable region sequence of WT and 12 humanized clones (shown in Table 3), and the constant region sequence of human IgG 1 kappa.

Table 3. Variable region sequence of clones for IgG expression

[00205] The expression vectors of the clones were transiently transfected and expressed in HEK293 cells, and Protein A affinity chromatography was used to purify the antibodies. Clone Hu-12Y and Hu-22Y showed a significant low expression yield, while the rest 11 clones (including WT) had normal expression yields. Then, the ELISA assay was performed for the 13 clones, hybridoma purified IgG as positive control. Except for Hu-12Y and Hu-22Y, the rest 10 humanized clones showed comparable or even higher binding affinity for the antigen when compared to the WT clone. As for the obvious difference of binding signal between WT clone and the positive control, hybridoma purified IgG provided, it may be caused by the different secondary antibodies and their different dilution ratio in the assay. To confirm the result, 4 humanized clones were randomly selected to repeat the ELISA assay and the WT clone and hybridoma purified IgG were also included in assay. The result was consistent with the first assay. In summary, clone Hu-1 1 S, Hu-12S, Hu-13S, Hu-1 1 Y, Hu-13Y, Hu-21 S, Hu-22S, Hu-23S, Hu-21 Y, Hu-23Y can be selected out as good humanized candidates, and the humanization process was successful.

[00206] Six clones, Hu-1 1 Y, Hu-23Y, Hu-1 1 S, Hu-12S, Hu-13Y, Hu-21 Y, were used to perform activity assay further and the six clones all showed activity.

[00207] Clone Hu-1 1 S and Hu-23Y were selected for further affinity detection, clone WT as control. A new batch of expression for clone Hu-1 1 S, Hu-23Y and WT was performed to generate enough amount for affinity detection. Then the concentrationdependent ELISA was conducted, and all the three clones had high binding affinity for both antigen and biotin labeled antigen, no obvious difference was found between the two antigen formats. Clone Hu-11 S had comparable binding signal with clone WT, and weaker binding signal was found in clone Hu-23Y. After that, affinity assessment via BLI was performed. The KD value of clone WT, Hu-1 1 S, Hu-23Y, was 8.00x10-1 1 , 1 .81 x10-10, 1 .71 x10-10, all beyond nM level, indicating all the three clones had high binding affinity for the antigen. However, the affinity of Hu-11 S and Hu-23Y were reduced when compared to the clone WT.

Affinity maturation

[00208] Based on the amino acid sequence information of parent antibody Hu-11 S, VH and VL mutated phage display scFv libraries were designed and constructed via error-prone PCR (marked as Hu11 S VHmu-VLwt library and VHwt-VLmu library). According to the sequence alignment analysis based on the antibody database and structural modeling analysis of Discovery Studio, a Hotspot mutation phage display scFv library was designed and constructed via NNK Primers (marked as Hotspot mutation library). All the procedure is detailed as below.

Hul ls VHmu-VLwt library and VHwt-VLmu library construction

[00209] Firstly, the scFv were designed as VH-(G4S)3-VL and optimized the nucleic acid sequences based on the E.coli and Homo sapiens expression system, simultaneously added the Sfi \ and Not I restriction enzyme cutting site. The sequence information is summarized in Table 4 and the codon optimization analysis result indicated the nucleic acid sequences of Hu11 S-scFv was suitable for both E.coli and Homo sapiens expression system.

Table 4. Sequence of Hu11 S-scFv

Note: The (G4S)3 is highlighted in italics. The VH sequence is marked in underlining and the VL sequence is marked in squiggly underlining.

[00210] Then, both scFv gene and pCantab 5E phagemid were digested by restriction enzymes and ligated together with T4 DNA ligase to generated Hu1 1 S- scFv-pCantab 5E (shown in FIG. 12). After electro-transformed into competent cells E.coli TG1 , monoclonal phage ELISA and soluble ELISA assay were conducted . As shown in Table 5 and Table 6, the result revealed that Hu1 1 S-scFv-pCantab 5E was constructed successfully, and the phage-display Hu1 1 S-scFv and soluble Hu1 1 s-scFv all showed appropriate binding affinity for the antigen. As shown in Table 7, the successful soluble expression of Hul l S-scFv was confirmed by coating the periplasmic extract of expression induced at 30 °C or 37 °C and detecting with the usage of anti-E tag mouse mAb-HRP.

Table 5. Monoclonal phage ELISA of Hu1 1 S-scFv Table 6. Soluble ELISA of Hu1 1 S-scFv

Table 7. QC analysis of Hu1 1 S-scFv soluble expression

[00211] Based on the error-prone PCR, 2 primers (FIG. 13) were designed to introduce random mutation into Hu1 1 s-scFv and the mutated fragments (marked as Hu1 1 Smu) were analyzed by Gel electrophoresis as shown in FIG. 14. The length of fragments is all about 750 bp, in line with expectations. Then, the Hu1 1 Smu was used as template to amplify the randomly mutated VL and VH fragments, respectively, named as VLmu and VHmu. Simultaneously, Hu1 1 S-scFv-pCantab 5E was used as template to amplify wild-type VL and VH fragments, respectively and the products marked as VLwt and VHwt. All the four kind of fragments were analyzed by Gel electrophoresis as shown in FIG. 15. The length of VLmu, VHmu, VLwt and VHwt fragments is 31 1 bp, 377bp, 398bp and 463bp, respectively, all in accordance with expectations. And then, the scFv cassettes of VHmu-VLwt and VHwt-VLmu were assembled by over-lapping PCR as shown in FIG. 16.

[00212] After that, both scFv genes and phagemid were digested by restriction enzymes and ligated together with T4 DNA ligase. The ligation mix was desalted, re suspended in distilled water, and then electro-transformed into competent cells E. coli TG1 to construct the library.

[00213] The final libraries were constructed. The capacity of the two libraries is 1 .1 x10^A10 (VHmu-VLwt library) and 9.7x10^A9 (VHwt-VLmu library), respectively. At last, the phages display scFv proteins were packaged with the aid of helper phage M13K07.

[00214] The quality of the libraries was tested by sequencing with S1 primer. 25 clones were picked up randomly from each library and were tested the positive insertion ratio. As shown in FIG. 3, the accuracy of Hu1 1 s VHmu-VLwt library is 68.2% (15/22*100%). Total number of amino acid mutation in 15 clones containing correct reading frame is 41 and the average mutation number in each is about 2.7. While the data is not shown, the accuracy of Hul l s VHwt-VLmu library is 83.3% (20/24*100%). Total number of amino acid mutation in 20 clones containing correct reading frame is 48 and the average mutation number in each is about 2.4. These results indicated that both libraries were constructed with high quality. Mutations of interest in the various clones include, relative to SEQ ID NO: 22, S7T, K13E, G15A, S17C and S17T, L18Q, F27Y, F29Y, S30C, Y32C and Y32H, F33S, S35R, Q39H, P41 Q and P41 L, E42G, K43R, S52R, G55D, T58S and T58P, P61 R, T69A, N77K, S78C, Y80H and Y80N, E89Q, T91 S, V93G, C96S, A97V, Y101 N, R102S, Y106C, D108G, W1 10C and W110R, and G101 D. Mutations of interest in the various clones, relative to SEQ ID NO: 55, C68A, G161A, A193T, A230G, G233A, G246A, A253T, C261 A, A271 G, T282A, G305C, T325A, and G331 T. Mutations of interest in the various clones include, relative to SEQ ID NO: 22, G132D, S147R, A148V, S157G, S159R and S159G, S161 C and S161 I, S163G, V164A, S165C and S165G, Y166C, H168R, W1 69R, S170G and S170R, a deletion of amino acid 171 , S174I, S175N and S175C, Q176H, P178T, L181 Q, S186N, K187I, L188M and L188Q, V192A and V192I, F196L, S197N, S199G and S199I, G200S, S201 R, D204E, T208A, S21 1 R, L212H, P214A, F217Y, Y221 C, F223I, Q224H, G227S, Y228F, and P229Q. Mutations of interest, relative to SEQ ID NO: 58, include C124T, G167C, G172A, C179G, A187G, C198G, A237G, A242G, G271 T, T293C, and T297A.

Hotspot mutation library construction

[00215] Based on the antibody database and Discovery Studio, the sequence alignment and structural modeling analysis of Hu1 1 S was conducted. According to the analysis results, 18 hotspots in the CDR regions of scFv-Hu1 1 S (the sequence is listed in Table 4) were selected. For heavy chain, the hotspots are F33, S35, T50, S52, S53, G54, Y57, Y59 and P61. For light chain, the hotspots are S163, V164, S165, D184, T185, K187, F223, G225 and L230. Then the different combination of those hotspots, were performed following by using NNK primers to introduce mutations into the hotpots of each combination, as a result, each one contained 2 to 7 amino acid mutations.

[00216] The final Hotspot mutation library was. The capacity of the library is 2.1 xl 0^A9, and the phages display scFv proteins were packaged with the aid of helper phage M13K07. 30 clones were selected randomly and sequenced with S1 primer to verify the quality of the library. As shown in FIGS. 17A-17D, the accuracy of Hul l s Hotspot mutation library is nearly 100% (27/27*100%) and the average mutation number in each is about 5-7. These results indicated that the Hotspot mutation library was constructed with high quality.

Library screening

[00217] Firstly, a new batch of biotin-labeled antigen (marked as B-Ag) was generated and a QC test was conducted. As show in FIG. 18, the biotin labeling efficiency was above 85%.

[00218] Then, four rounds of library screening based on the three mutant libraries were performed against the antigen. Obvious enriching effects were found for all the three libraries, Hu1 1 S VHmu-VLwt library, VHwt-VLmu library and Hotspot mutation library.

Phage ELISA

[00219] After that, 136 clones were randomly selected out from the 3rd round output of the three mutation Libraries to perform monoclonal phage ELISA, using Hu1 1 S parental phage as control. In detail, 46 clones were randomly selected out from VHmu- VLwt library (clone 1 -46) and VHwt-VLmu library (clone 47-92) respectively, and 43 clones were randomly selected out from Hotspot mutation library (clone 93-136). According to the result (not shown), 28 clones with the highest positive signal were selected out for sequencing.

[00220] Meanwhile, 136 clones were randomly selected from the 4th round output of the three mutation Libraries to perform monoclonal phage ELISA, using Hu1 1 S parental phage as control. In detail, 46 clones were randomly selected out from VHmu- VLwt library (clone 137-182) and VHwt-VLmu library (clone 183-228) respectively, and 43 clones were randomly selected out from Hotspot mutation library (clone 229-272). According to the result (not shown), 51 clones with the highest positive signal were selected out for sequencing.

Sequencing and Soluble ELISA

[00221] In total, 79 highly positive clones from phage ELISA were selected for DNA sequencing. 78 clones have been sequenced successfully and 1 clone failed to obtain the correct sequence. All the sequencing result was summarized in FIGS. 19A-19B, and 15 unique sequences were obtained. 8 clones without TAG codon were selected out from the 15 unique sequence for further soluble ELISA verification. Compared to the hul l s clone, 7 clones (2, 3, 6, 12, 13, 14, 15) showed higher binding affinity for antigen and only No. 8 clone had lower binding signal.

Sub-library Construction and Screening

[00222] The VHm (containing mutations in VH) sequences from the 3rd round output pool of VHm-VLwt library, and VLm (containing mutations in VL) sequences from the 3rd round output pool of VHwt-VLm library were amplified (FIG. 20), respectively, and randomly assembled to scFvs via overlap PCR (FIG. 20) to construct a sub-library. The end sub-library was constructed and the capacity of the library is 5.2x109. Then, two rounds of library screening based on the sub-library against the antigen were performed. Enriching effect was observed. After that, 94 clones (marked as clone 273- 366) were randomly selected out from the 2nd round output of the sub-library to perform monoclonal phage ELISA, using Hu11 S parental phage as control. Based on the monoclonal phage ELISA results, 25 clones with the highest positive signal were selected out for sequencing. According to the DNA sequencing result, 23 clones have been sequenced successfully and 2 clones (clone 321 and 357) failed to obtain the correct sequence. Among the 23 clones with sequencing results, clone 334 contains stop codon in its sequence, thus total 22 clones can be used for further analysis. All the sequencing result was summarized, and 3 unique sequences were obtained from the output of sub-library.

[00223] To sum up, total 18 unique sequences were obtained from the whole affinity maturation process, among which, 15 sequences were from the output of the three mutant libraries, and 3 sequences were from the output of sub-library. Based on the former ELISA results and the characteristic of sequence, we selected out 9 sequences for further IgG antibody expression in eukaryotic system and affinity evaluation. The mutation sites of the 9 clones were summarized in FIG. 1.

Candidates expression and binding affinity assessment [00224] The parent antibody Hu-1 1 S and the 9 candidates were expressed, purified and analyzed, as shown in FIGS. 21 A-21 B. The data showed that clone 5, 12, 13, 16, and 17 had higher expression yield than Hu-1 1 S.

[00225] Binding affinity assessment was performed, and clones 2, 6, 13, and 18 had similar KD value with Hu-1 1 s, while clones 5, 12, 15, 16, and 17 had enhanced affinity than Hu-1 1 S. Accordingly, clones 5, 12, 15, 16 and 17 were selected for BLI affinity assessment with 5 Ab concentrations. All the five clones had better affinity than Hul l s. It is notable that clone 16 had a value of 2.67x10-1 1 , which was about 1 1 .5-fold affinity enhancement, compared to Hu-1 1 s with the KD value of 3.06x10-10. In conclusion, the affinity maturation was successful. The sequence information of Hul l s and the top 5 clones in lgG1 format was summarized and is set forth in SEQ ID NOS: 1 -19.

Example 3 - Additional data

[00226] META4 producing hybridoma clones were made essentially according to standard methods by immunizing mice with purified human MET extracellular domain (FIG. 22). As can be seen in FIGS. 23A-23C, META4 activates MET and its downstream mediators as determined by western immunoblot using antibody to activated MET (phosphorylated-ME). MET4 was also found to be specific for human and primate MET but does not activate mouse MET. As can be seen in FIG. 24, META4 produced by META4-producing hybridoma clones binds to MET on Hep-G2 cells. META4 expression was checked in 293 cells using pVITRO1 -M80-F2-lgG1/k or pVITRO1 -Trastuzumab-lgG1/k vectors (vectors were provided by CreativeBiolabs) in which META4 heavy and light chains are cloned. Immunofluorescence Staining in HepG2 cells with anti-Human IgG antibodies in FIGS. 25-26 shows META4 binds to intact HepG2 cells. As can be seen in FIG. 27, humanized META4 potently activates MET and its MET’s downstream target AKT in human hepatocyte and human retinal pigmented epithelial cells. Clones Hu-1 1 & Hu-23 and hybridoma (Hy-B, Hy-E). Both Hu-1 1 and Hu-23 were seen to be very active, just like parental hybridoma clones.

Example 4

HGF and META4 potently activate MET in human retinal pigmented epithelial cells (RPE cells)

[00227] Human hepatocyte (HepG2) and Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium and then switched to serum free medium for overnight. They were treated with HGF or humanized META4 as indicated for 15 minutes, and cell extracts were subjected to western immunoblot using antibody to activated MET (phosphoMET) and then with antibody against activated AKT (phosphoAKT) which is a downstream effector of activated MET.

META4 like HGF rapidly activate MET in human retinal pigmented epithelial cells (RPE cells); Time point and Dose Response

[00228] Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium and then switched to serum free medium for overnight. They were treated with META4 as indicated for the indicated time and also with different concentrations of META4 (FOR 20 minutes) as shown in FIGS. 28 and

29, respectively. Cell extracts were subjected to western immunoblot using antibody to activated MET (phosphoMET) and then with antibody against total MET as protein loading control. As can be seen in FIG. 28. Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium and then switched to serum free medium for overnight. They were treated with HGF or different concentrations of META4 as indicated for 15 minutes. Cell extracts were subjected to western immunoblot using antibody to activated MET (phosphoMET)..

Humanized META4 activates MET in Rhesus and Cynomolgus Monkey kidney cells and hepatocytes

[00229] Rhesus monkey hepatocytes and kidney epithelial cells were grown in MEM medium and then switched to serum free medium for overnight. They were treated with HGF or humanized META4 (FIG. 39, top panel) and parental META4 clones (FIG.

30, lower panel) for 15 minutes. Cell extracts were subjected to western immunoblot using antibody to activated MET (phosphoMET) and then with antibody against total MET as loading protein control. As can be seen in FIG. 30, META4 and humanized version potently and rapidly activate MET in these cells just like HGF the natural ligand. HGF and humanized META4 promote growth of human retinal pigmented epithelial cells (RPE cells) as determined by MTT assay

[00230] Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium in 12 well assay plates and then switched to serum free medium for overnight. They were treated with HGF or humanized META4 for 48 hours and subjected to cell growth assay (MTT assay). Shown are the bar graphs of the mean values of triplicate wells for each treatment. As can be seen in FIG. 31 , META4 and it humanized versions stimulate significant cell growth akin to HGF. Note the higher the number the more cell growth/cell survival.

HGF and META4 protects human retinal pigmented epithelial cells (RPE cells) from oxidative damage and apoptosis induced by H202 and FasL as determined by CellTox assay

[00231] Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM medium in 12 well assay plates and then switched to serum free medium for overnight. Cells were treated with HGF or META4 for 48 hours in the presence of FasL or H202 which both induce cell death. Cells were subjected to cell death assay using CellTox reagent from Promega. Shown are the bar graphs of the mean values of triplicate wells for each treatment. As can be seen in FIG. 32, META4 and it humanized versions prevent cells death akin to HGF. Note the higher the number the more cell death.

META4 protects human retinal pigmented epithelial cells (RPE cells) from apoptosis induced by FasL as determined by prevention of caspase-3 activation

[00232] Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without Fas ligand (FasL) which induces cell death by apoptosis the hallmark of it is caspase 3 activation. Cells were also treated with SU1 14 a potent inhibitor of MET. Cell extracts were subjected to western blot using antibody against activated caspase-3. As can be seen in FIG. 33 META4 and it humanized versions prevent cells death by apoptosis which is induced by FasL-Caspase-3 activation, akin to HGF. Note that inhibition of MET by MET kinase inhibitor SU1 144 exacerbates FasL action showing MET function is essential for promotion of cell survival..

A Variety Of Important Genes And Cellular Pathways Are Affected By Hgf/Meta4 In Rpe Cells As Determined By Rna-Seg

[00233] RNA-seq was done by Arraystar company without knowing the identity of each sample or the experiments.

[00234] For FIGS. 34A-34B, Human Retinal Pigmented Epithelial (RPE-19, CRL- 2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (H,HGF) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA-sequencing for gene expression. Shown in FIGS. 34A-34B are volcano graphs depicting gene that are significantly changed by treatment vs. corresponding control.

[00235] For FIG. 35, Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (H,HGF) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA- sequencing for gene expression. Shown in FIG. 35 are heatmaps (co-clustering) of differentially expressed genes.

[00236] For FIG. 36, Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA- sequencing for gene expression. Shown in FIG. 36 are scatter plots depicting gene that are significantly changed(up or down) by treatment vs. corresponding control.

[00237] For FIG. 37, Human Retinal Pigmented Epithelial (RPE-19, CRL-2302 purchased from ATCC) cell lines were grown in MEM and then switched to serum free medium for overnight. They were treated with or without META4 and with or without HGF for 6 hours and 24 hours (C, control) (M,META4) in duplicate wells (N=2 independent samples). RNA was prepared at the indicated time and subjected to RNA- sequencing for gene expression. Shown in FIG. 37 are heatmaps (co-clustering) of differentially expressed genes META4 vs. control CNTRL.

[00238] Heat Maps (not shown) of the top 50 genes in META4 6Hrs_vs_CNTRL 6Hrs and META4 24Hrs_vs_CNTRL 24Hrs were generated to examine gene expression.

Example 5 - Regarding HGF action on hepatic lipid metabolism and reducing liver damage (caused by fatty liver) in insulin resistant diabetic mice

[00239] Based on its affinity for the HGF receptor, META4 may be suitable for treatment of diabetes, and may be suitable for restoring insulin responsiveness in insulin-resistance. See Fafalios et al., A hepatocyte growth factor receptor (Meginsulin receptor hybrid governs hepatic glucose metabolism, Nat. Med. 201 1 , 17(12): 1577-1584. To this end, FIG. 38 reveals that HGF injected systemically to obese diabetic mice (ob/ob mice) distributes to the liver. Shown is the time post human HGF injection. hHGF was detected by western blot using antibody specific for human HGF. FIG. 39 shows injected HGF activates its downstream effector ERK in the liver which is known to stimulate cell proliferation and activates a variety of important genes. FIG. 40 shows that HGF therapy (daily injection for 10 days) lowers hepatic lipids (triglycerides, TG). N=6 mice per group.

[00240] FIG. 41 depicts that HGF therapy mobilizes hepatic lipids in db/db mice as evident by increased FFA (free fatty acids in the plasma) due to lipolysis of hepatic fat. FIG. 42 shows that HGF Therapy [10 days a single daily injection] Ameliorates Liver Damage in db/db Mice as evident by reduction in ALT a marker of liver injury..

Example 6 - Blockage of HGF action in HIV+ patients and the toxic effects of HIV drugs (ARTs) antiretroviral therapy on human hepatocytes and its rescue by META4

[00241] HIV+ patients have abundant pro-HGF (which is biologically inactive) and HGF antagonist NK1 in their plasma. The data in FIG. 43 indicate that there is a blockade of HGF action via 1 ) inhibition of pro-HGF activation which is mediated by HGF activator (HGFAC). Notably, pro-HGFAC is also abundant which itself needs to be cleaved and activated by serine protease like thrombin, kallikrein-related peptidase KLK4. HGF antagonist NK1 inhibits HGF action. Thus, the cascade of HGF axis is blocked at several tiers.

[00242] Anti-Retroviral Therapy [ART] drugs like Efa (Efavarenz) and Mara (Maraviroc) inhibit processing (activation) of HGFAC. Human hepatocyte cell line (Hep3B) were treated as indicated for 48hrs and culture medium were analyzed by western blot. HGFAC is produced and secreted by hepatocytes as inactive pro- HGFAC and requires proteolytic cleavage. The data in FIG. 44 indicate that ARTs inhibit HGFAC activation.

[00243] As shown in FIG. 45, META4 protects hepatocytes from ART-induced cytotoxicity (MTT assay) ART [Rai]. Anti-Retroviral Therapy [ART] induces hepatotoxicity which is prevented by META4. Human hepatocyte cell line (HepG2) were treated without or with ART called Rai (Raltegravir) and with HGF and META4 as indicated for 48 hrs and processed for cell viability by MTT assay.

Example 7 - A META4 efficacy in treating NASH [00244] The therapeutic potential of a META4 antibody reagent was evaluated in a humanized model of NASH (mice with human hepatocytes) and the therapy successfully restored normal liver function, ameliorated NASH and its molecular hallmarks.

Results

Humanized Livers Develop Nonalcoholic Fatty Liver Disease

[00245] To generate a humanized NAFLD model, we took advantage of mice deficient in fumarylacetoacetate hydrolase (FAH), an enzyme responsible for catabolism of tyrosine known as FRGN, the livers of which can be repopulated with human hepatocytes. This humanized chimeric mouse model has been proposed to be an invaluable tool to study drug metabolism, excretion, and toxicity in a system more relevant to humans. In our studies, we used the humanized mice approximately 6 months after they were subjected to the transplantation protocol. We tested whether the transplanted mice (henceforth referred to as humanized mice) develop a fatty liver phenotype if fed a high-fat diet (HFD). Accordingly, these mice were randomly divided into HFD and regular diet (RD) groups. Nontransplanted FRGN mice were also used as an additional control cohort. Mice were then fed regular chow (RD) or Harlan Teklad TD.88137 “Western Diet” chow (HFD) for 6 weeks. During the experiment, mice were monitored for food intake and body weight. At the end of 6 weeks, they were culled, and their sera and livers were harvested for histologic, biochemical, and molecular studies. We found that the humanized livers became severely steatotic showing macrovesicular hepatocytic fatty change only if humanized mice were fed an HFD (FIGS. 46A-46B). Liver and serum triglycerides and cholesterol were also elevated in the humanized mice on HFD (FIG. 46A). To show that the transplanted human hepatocytes in fact accumulate fat, we performed immunohistostating for FAH, and the data revealed that the human hepatocytes become steatotic and that host mouse hepatocytes (which are deficient in FAH) exhibit little or no steatosis (FIG. 46B). Nontransplanted FRGN mice also had little or no steatosis on a HFD for 6 weeks. It should be noted that neither of the human hepatocyte donors had fatty liver at the time of harvest. Mice in general develop NAFLD only after prolonged feeding of a HFD depending on the genetic background (more than 15 weeks). The fat laden human hepatocytes succumbed to lipotoxicity as evidenced by marked inflammatory cell accumulation surrounding the FAH-positive hepatocytes inducing their death as evaluated by TUNEL (FIG. 46B). The results described in FIGS. 46A-46B were repeated in a separate set of experiments using FRGN mice transplanted with human hepatocytes from a different donor.

Humanized Liver Recapitulates Human Nonalcoholic Steatohepatitis

[00246] A prominent feature of NASH is liver fibrosis, which develops in the background of inflammatory cell infiltration of the hepatic parenchyma. Thus, we compared the humanized liver (FIG. 47 A) with human liver with clinically proven NASH side-by-side (FIG. 47B). We observed infiltration of inflammatory leukocytes, in particular macrophages and neutrophils, ballooning hepatocytes, stellate cell activation, and collagen deposition (FIG. 47A, C) in the livers of humanized mice exposed to a HFD akin to human NASH livers. Neither inflammatory cell infiltrate nor liver damage was detected in the humanized mice fed a RD or in the nontransplanted mice placed on a HFD (FIG. 47A). The data summarized in FIGS. 47A-47B and 48 overall show that the humanized mice fed a HFD develop a NASH phenotype like that seen in human NASH at the histologic, cellular, and biochemical levels.

[00247] We next carried out whole transcriptome analyses using RNA-Seq and, as a complementary approach, human-specific GeneChip microarray (human Affymetrix U133 Plus 2.0 Array, which has more than 54,000 probes encompassing the whole human encoding transcriptosome) to investigate whether the model genocopies human NASH. In parallel for comparison, we included human normal and NASH livers in our experiments. To avoid bias in data interpretation, samples were anonymized prior to analyses. RNA-seq reads were aligned to the human genome reference to assess the human-specific gene expression profile. The results showed that, in human NASH liver as compared with human normal liver, the expression of approximately 1280 genes were significantly upregulated, and 600 genes were down-regulated (P < .05 and at least 1.5-fold changes). About 10,900 genes remained unchanged. When humanized NASH livers were compared with humanized normal livers, close to 1800 genes were significantly induced, 923 genes were repressed, and 8650 genes remained unchanged. We also compared humanized NASH livers with normal human livers and found that the expression of 1 180 genes was induced, 1 150 genes repressed, and 10,100 genes remained unaffected. In concordance with these data, microarray results revealed the expression of about 1000 genes were upregulated and 600 genes were down-regulated in both human and humanized NASH livers compared with their normal counterpart. Comparison of the groups using bioinformatic tools including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis analyses revealed that the human and humanized NASH shared similarity in the most highly deregulated biological processes. The common down-regulated processes included: drug metabolism - cytochrome P450, metabolism of xenobiotics by cytochrome P450, and lipid and glutathione metabolism, to name a few and the upregulated processes were inflammatory response, NAFLD pathway, viral infection (ie, hepatitis C and B), degenerative diseases (like Alzheimer and Parkinson diseases), oxidative phosphorylation, and cell death pathways (such as necroptosis, apoptosis, and ferroptosis) (not shown). We performed principal component analysis and found that NASH livers co-cluster, and normal livers aggregate together (not shown).

[00248] We next tested the hypothesis that hepatocyte lipotoxicity generates cues that recruit innate immune inflammatory cells such as macrophages and neutrophils to the liver and induce their expansion promoting liver injury. Accordingly, we aligned the RNA-Seq data from humanized livers to the mouse genomic reference to gain insight into the modification of mouse-specific gene expression in the model. The results uncovered that cytokine and chemokine signaling pathways that activate macrophages and neutrophils and promote leukocyte transendothelial migration are significantly upregulated in humanized NASH liver as compared with humanized normal liver.

Expression of Hepatocyte Growth Factor Antagonist is Upregulated in Nonalcoholic Steatohepatitis

[00249] Alternative splicing of a given pre-m RNA transcript can generate mRNA variants yielding protein isoforms with distinct functions. This mode of mRNA generation plays a critical role in homeostasis and disease, and almost one-half of human genes are believed to undergo alternative splicing events. RNA-Seq and microarray mRNA expression profiling are reported to be powerful techniques to detect differentially expressed alternative splice variants. Our RNA-Seq analysis revealed that significant changes in splicing events happen in NASH livers as compared with the corresponding normal livers. We found that in human NASH versus human normal liver, 1647 splice variants of various transcripts were down-regulated and 2433 were upregulated. Similarly, in humanized NASH as compared with the humanized control counterpart, we uncovered that splice variants of 926 transcripts were upregulated and 869 were down-regulated. Most of the alternative splicing events were of skipped exon type as compared with other classes such as alternative 5' splice site, alternative 3' splice site, retained intron, and mutually excluded exons (not shown). These transcripts belong to a wide array of biological functions, such as growth and development, autophagy, and metabolism. Some representatives splice variants included: YAP1 , FGFR3, BMP1 , MAPK5, ATG13, Caspase 8, GSTM4, and SLC22A25 (a solute carrier), which underwent differential alternative splicing events in human and humanized NASH. Consistent with these observations, pathway analyses revealed that significant changes occur in the expression of the components of spliceosome machinery in human and humanized NASH (not shown). Importantly, we made the novel observation that the expression of the alternative splice variant of HGF, which generates HGF antagonists called NK1 and NK2, is significantly upregulated in human NASH liver. These isoforms only encode the N-terminal portion of HGF and lack kringles 3 and 4 as well as the entire beta chain of HGF. The NK1 isoform cDNA was first cloned from a human fibroblast cell line, and NK2 was cloned from human placenta. Structure-function studies have shown that the N-terminal region of HGF alpha chain is necessary and sufficient for binding to the HGF receptor (MET) but is unable to activate MET and that the beta chain which is in the C-terminal portion of HGF is required for receptor dimerization and activation. Our RNA-Seq and microarray data revealed that the mRNAs for the HGF antagonists NK1 and NK2 are expressed in normal human liver at low levels but are significantly upregulated in human NASH. To confirm this novel finding, we made reverse primers specific to the 3'-untranslated regions of human NK1 or NK2 and forward primers corresponding to human HGF’s N- terminal region. We subsequently performed reverse transcription polymerase chain reaction (PCR) on human normal and NASH liver, cloned the resulting cDNA and sequenced it. The results proved that NK1 and NK2 mRNAs are indeed expressed in human liver and are highly upregulated in human NASH liver (FIG. 49A). To extend this finding, we performed Western blot analyses using antibodies specific to the N- terminal region of HGF (which is present in NK1 and NK2). NK1 and NK2 proteins have a predicted Mr of about 25 to 32 kDa, whereas canonical HGF has an Mr of about 70 to 90 kDa (proteolytically cleaved or unprocessed HGF, respectively). Using Western blot analysis, we confirmed that NK1/NK2 proteins are significantly upregulated in human NASH liver and the plasma of patients with NASH (FIG. 49B and 50, respectively). HGF protein is produced and secreted as a single chain pro- HGF molecule. This precursor is biologically inactive and requires enzymatic cleavage by a specific serine protease called HGFAC, which is expressed by hepatocytes. Notably, our transcriptome and protein analyses revealed that HGFAC mRNA and protein abundance are significantly reduced in human NASH liver as compared with human normal liver (FIGS. 49C-49D). Another serine protease system, uPA (urokinase type plasminogen activator) and tPA (tissue type plasminogen activator), has also been shown to cleave pro-HGF to its active double chain form. Interestingly, our transcriptome analyses revealed that the expression of the gene Serpinel encoding plasminogen activator inhibitor-1 (PAI-1 ), a potent inhibitor of uPA and tPA, is significantly induced (by more than 4-fold) in human and humanized NASH liver. Others have also reported that PAI-1 is upregulated in human nonalcoholic and alcoholic fatty liver disease and that PAI-1 is an independent marker of poor prognosis in patients with NAFLD.

[00250] We next asked if HFD causes a change in hepatic HGF expression in wild type mice (C57BL/6). We discovered that HGF expression is reduced (FIG. 51 , panel A), whereas HGF antagonist NK1 is induced by HFD (FIG. 51 , panel B). To our knowledge, this is the first time that the HGF antagonists have been detected in the liver and, more importantly, the first time they are implicated in human disease like NASH. Collectively, our data reveal that HGF function is impaired in NASH liver at several levels via (1 ) increased expression of HGF antagonists and (2) blockage of pro-HGF activation via reduction in HGFAC and upregulation of PAI-1 .

Generation of META4, a Potent Agonist of MET, the Receptor for HGF

[00251] The HGF-MET axis governs key aspects of liver homeostasis by promoting the survival and proliferation of hepatocytes as well as liver regeneration. Moreover, we have shown that this ligand-receptor system is essential for hepatic glucose and fat metabolism in cooperation with insulin receptor signaling. We reported that systemic injection of HGF into diabetic insulin resistance ob/ob mice restores insulin sensitivity. All of the biological responses of HGF are elicited by its ability to bind to and activate MET, a transmembrane tyrosine kinase receptor. Several preclinical studies have suggested that HGF has therapeutic potential as a promoter of tissue regeneration and restoration of homeostasis of various organs including the liver. However, the clinical application of HGF has been hampered due to the fact that it binds avidly to heparin and heparan sulfate in the extracellular matrix and, because of this, HGF exhibits poor tissue distribution when injected intravenously, intraperitoneally, subcutaneously, or intramuscularly. HGF administered systemically is also unstable because it is rapidly cleared by the liver and does not reach other organs. Furthermore, as mentioned earlier, HGF is produced as an inactive pro-HGF precursor and requires protease cleavage to become bioactive: disruption of HGF activation renders it ineffective. In fact, in patients with fulminant hepatic failure and in patients with cirrhotic liver, plasma pro-HGF is elevated but it is not cleaved, and hence is inactive. These findings combined with our data that HGF action is compromised in NASH liver at multiple levels prompted us to therapeutically target the HGF-MET axis in NASH using the humanized NASH model we described herein. We reasoned that generation of an HGF-MET agonist with good pharmacokinetics and stability should overcome HGF’s blockage opening avenues for its therapeutic application for organ dysfunction including liver diseases such as NASH.

[00252] Monoclonal antibodies that bind to and activate specific growth factor receptors have recently been reported to be an effective way to modulate a given receptor in vitro and in vivo. Moreover, antibodies have good tissue distribution and more importantly long plasma half-life (more than 30 days for lgG1 ). For instance, monoclonal antibody to fibroblast growth factor receptor 1 (FGFR1 ) was shown to mimic FGF21 , activate FGFR1 in adipocytes, and ameliorate hyperglycemia in a mouse model of diabetes. Therefore, we generated mouse monoclonal antibodies against the extracellular domain of human MET and screened these antibodies for their ability to activate MET using cell-based assays. Akin to HGF, one clone, which we named META4 (pronounced metaphor), potently and rapidly (within minutes) activated MET and its downstream effectors, such as Gab-1 (an IRS family member), Akt, and Erk in human hepatocytic cell lines like HepG2 hepatocytes (FIG. 52A). Given, the fact that META4 was raised against human MET extracellular domain (also called the ectodomain), we wanted to explore if META4 activated rodent MET. We found that META4 is highly specific for human MET and does not stimulate mouse MET using mouse hepatocytes cultures (FIG. 52B). This finding led us to hypothesize that the epitope-binding site of META4 on human MET is not conserved in rodent MET. Sequence alignment analyses revealed that the amino acid sequence of the extracellular domain of MET is not fully conserved between human and rodents, but it is highly conserved between human and nonhuman primates like rhesus monkeys. We next tested if META4 activates MET in cells derived from nonhuman primates. We stimulated the normal kidney epithelial cell line LLC-MK2 from rhesus monkey with META4 and discovered that META4 efficiently activates MET in these cells like human kidney epithelial HEK-293 cell line (FIG. 52C). We cloned the META4 cDNAs (ie, light and heavy chains) from META4-producing hybridoma cells and expressed the cloned cDNAs in HEK293 cells, purified the recombinant META4 by protein-A chromatography and evaluated it for its ability to activate MET. FIG. 52D illustrates that purified recombinant META4 is a strong activator of MET in human hepatocytes. Finally, we tested whether META4 activates MET signaling in humanized mice. The results showed that indeed META4 potently induces MET and its down-stream effectors like IRS and glycogen synthase in the livers of humanized mice (FIGS. 53A- 53B).

META4 Therapy Ameliorates Nonalcoholic Steatohepatitis in a Humanized Model of Nonalcoholic Fatty Liver Disease

[00253] Given the above results showing that HGF-MET axis is compromised in NASH and that META4 protected hepatocytes against lipotoxicity by promoting hepatocyte homeostasis (by impacting metabolic processes as well as fostering hepatocyte survival and regeneration), we were prompted to test if META4 has therapeutic potential against NASH using the humanized model that we described above. Accordingly, we divided a cohort of humanized mice into experimental (injected with META4) and control (injected with isotype-matched mouse lgG1 ) groups (n = 7 per group). These mice were placed on HFD and then treated with META4 or isotype matched mlgG1 (control-treated). META4 therapy was administered for 4 weeks. During these experiments, we monitored the mice for food intake and body weight. At the end of the experiment, we collected their sera and livers for histologic, biochemical, and molecular studies as described for FIGS. 54A-55B. The results demonstrated that control (mlgG1 ) treated mice exhibited marked pericellular fibrosis, which was accompanied by pronounced macrophage and neutrophil infiltration. Notably, META4 treatment inhibited inflammatory cell infiltration, ameliorated fibrosis, halted hepatocyte death, and stimulated marked proliferation of human hepatocytes (costaining with Ki-67 and FAH) (FIGS. 55A-55B).

[00254] It is well-known that when the protective drug NTCB is withdrawn from FRGN mice and if they are not transplanted with FAH-proficient hepatocytes or the proliferation and survival of the transplanted hepatocytes is inhibited (in our case, due to lipotoxicity), the animals lose weight, become sick by 4 weeks, and die due to massive host hepatocyte death, liver failure, and its associated secondary pathologies. Therefore, to decipher the pro-growth, pro-regenerative activities of META4 on the homeostasis of the transplanted hepatocytes under the lipotoxic conditions, mice were subjected NTBC regimen consisting of 3 cycles of NTBC withdrawal lasting 2 weeks for each cycle. We found that the control (mlgG1 ) treated mice gradually lost weight and became moribund leading to the control mice dying by 4 weeks, whereas META4- treated mice survived, behaved normally, and did not lose weight (FIG. 56A). It should be noted that no major inflammatory cell infiltrate and no liver damage were detected in humanized mice on RD or in the non-transplanted mice placed on HFD or on RD with the same NTBC regimen we used for the humanized mice (see FIG. 58). One of the clinical hallmarks of NAFLD is hepatomegaly. Of note, we found that META4 therapy dampened this feature in humanized NASH. Specifically, the liver to body ratio in control-treated mice was 15%, and it was reduced significantly (P = .01 ) in META4- treated mice by 4 weeks of therapy (FIG. 56B).

META4 Therapy Corrects the Expression of Key Hepatic Genes That are Deregulated in NASH

[00255] To gain further insight into the molecular mechanisms by which the HGF- MET signaling axis in the liver maintains hepatic homeostasis (and ameliorates NASH), we carried out RNA-Seq on livers from humanized mice that were treated with META4 or control mlgG1 . The results provided a wealth of information revealing that the HGF-MET signaling axis in the liver governs key pathways that regulate hepatic homeostasis. In brief, RNA-Seq results revealed that the expression of approximately 1800 genes was significantly changed by META4 treatment as compared with the control treatment (mlgG1 ). About 11 12 genes were down regulated, 750 genes were induced, and 9300 genes remained unaffected. Bioinformatic analysis uncovered that the affected genes belong to various pathways such as metabolism, growth, cell survival, and cell death. Specifically, the MET signaling axis suppressed the pathways of NAFLD, oxidative stress, inflammation, cell death, NFkB, chemokine, and tumor necrosis factor-alpha (not shown). Pathways that were upregulated by META4 encompass those that are involved in glucose and fat metabolism, drug metabolism, insulin signaling, bile secretion, and antioxidation (not shown). Examples of genes upregulated by META4 include CYP3A4, CYP2E1 , and CYP3A7 (which are the key regulators of bile acid synthesis and xenobiotic metabolism), and antioxidant enzymes like catalase and glutathione S-transferase.

Discussion

[00256] The studies presented in this paper have several salient features. First, we developed a humanized model of NASH that recapitulates its human disease counterpart. Second, we made the major discovery that the HGF-MET system is compromised (blocked) in human NASH at various levels including upregulation of HGF antagonists NK1 and NK2, down-regulation of HGF activator enzyme called HGFAC, and upregulation of PAI1 , a potent inhibitor of uPA/tPA, enzymes that can activate HGF. To our knowledge, our findings are the first to show that the HGF-MET axis is blocked in human NASH and provide insight into molecular mechanisms involved in NASH pathogenesis. Lastly, we generated a potent stable agonist of MET (the receptor for HGF), which we have named META4 and used it not only to restore HGF-MET function and to combat NASH in this novel humanized animal model, but to also discover the genes regulated in hepatocytes by the HGF-MET axis.

[00257] It has been reported that fatty liver not only causes hepatocyte death (due to lipotoxicity, which promotes oxidative stress and inflammatory cytokine and chemokine induction) but also inhibits hepatocyte proliferation and liver regeneration. Specifically, it was shown that mice with diet-induced NAFLD exhibit diminished liver regeneration in response to partial hepatectomy. We found that HFD significantly (P = .002) represses HGF in wild-type mice and induces HGF antagonist expression. Notably, the HGF-MET axis has been shown to be essential for liver regeneration in experimental models. Our results showed that restoring HGF-MET function (by META4 therapy) in a humanized NASH model results in proliferation and expansion of the transplanted human hepatocytes in vivo under toxic insults such as those provoked by lipotoxicity. META4 therapy also completely abrogated inflammation and led to repair of the injured liver. Given the fact that META4 exclusively affects human hepatocytes (because it is specific for human MET and does not activate murine MET), the data indicate that the injured hepatocytes are the instigators of liver inflammation and damage by promoting the recruitment of inflammatory cells, for instance.

[00258] In the liver, specialized nonparenchymal cells known as hepatic stellate cells mainly express the HGF gene in the liver, and HGF expression becomes repressed in these cells as they undergo activation and de-differentiation into myofibroblastic cells. HGF antagonist isoforms NK1 and NK2 are produced by alternative splicing of the pre- mRNA for HGF, which yields truncated HGF versions that retain part of the N-terminal portion, which is responsible for MET binding but lack kringles 3 and 4 and the entire beta chain of HGF, which are essential for MET dimerization and activation. We found that the ratio of mRNA of HGF to that of HGF antagonists NK1 and NK2 is more than 10 to 1 in normal human liver. In NASH liver as compared with normal liver, the abundance of NK1 and NK2 transcripts increases significantly. We postulate that lipotoxicity alters HGF mRNA splicing resulting in an isoform switch from full length (canonical) HGF to truncated HGF antagonists. Future studies are warranted to decipher the molecular mechanisms involved in upregulation of NK1 and NK2 in the diseased liver setting (such as NASH) and identify the exact cellular origin of these antagonists in the liver (ie, hepatic stellate cells, fatty hepatocytes, Kupffer cells, and other inflammatory cells like neutophils).

[00259] Another important finding is that the innate immune cells like macrophages and neutrophils drive hepatic inflammation and injury in our humanized NASH model in the background of fatty human hepatocytes just like that seen in human NASH. Macrophages and neutrophils are well-known to be the major culprits inciting liver injury in human NASH liver contributing to the demise of hepatocytes. There is little or no infiltration of T and B lymphocytes in human NASH as opposed to viral hepatitis and autoimmune hepatitis. In fact, reports show that macrophages play a key role in NASH development in the diet-induced model in wild type mice. The authors demonstrated that elimination of hepatic macrophages by administration of the chemical cladronate diminished the NASH phenotype. And a role for chemokine/chemokine receptor was proposed in macrophage recruitment and accumulation in the liver. Other studies have shown that neutrophil and macrophage infiltration of the liver also plays a critical role in NASH promotion and that depletion of these cell types dampens NASH development. We discovered marked macrophage and neutrophil accumulation in our humanized NASH model closely mimicking the phenotype seen in human NASH and diet-induced NASH in murine models. Our data reveal that the culprits inciting liver inflammation in response to lipotoxicity are indeed the fat-laden human hepatocytes, which release monokines/cytokines and chemoattractants to recruit and activate host inflammatory host cells like macrophages and neutrophils. Through transcriptomic (RNA-seq and microarray) studies, we found that a variety of chemokine ligands and receptors such as CXCL2 and (a potent attractant for polymorphonuclear leukocytes), CCL20 (a neutrophil attractant thought to play an important role in NASH development and progression), and several cytokines/cytokine receptors (like TNFR1 , TNFR2, TRAIL, TWEAKR, Fas, and ICAM1 ) are upregulated in humanized NASH. Notably, we found that META4 therapy repressed the expression of some of these like TWEAKR, RIPK1 , and CCL20. [00260] An important corollary revealed by our work is that META4 not only has therapeutic applicability to the treatment of liver diseases in which hepatocytic damage and death prevail (like NASH and other forms of hepatitis) but also likely has therapeutic potential to promote repair of other damaged organs and tissues in which the HGF-MET axis is known to be functionally important. We believe that future studies that assess META4 efficacy for treating degenerative diseases using non-human primate models and humanization of META4 are warranted. Additionally, studies of its safety and potential undesirable side effects (such as fostering tumorigenesis) are also logical. We should emphasize that we did not detect any evidence of liver tumor development in our humanized mice treated with META4, including no evidence of human hepatocyte dysplasia and no increase in alpha-fetoprotein expression in the liver. In fact, expression of human albumin mRNA in the META4-treated humanized livers was even higher than normal human liver assayed side-by-side in RNA-seq analyses. We believe that the many benefits of restoring the HGF-MET axis by META4 treatment overcome concerns about its potential pro-tumorigenic effect. In fact, activation of the HGF-MET axis may even curtail tumorigenesis by promoting tissue repair and healing, as chronic tissue injury is thought to be a major driver of carcinogenesis. In support of this claim, some studies have shown that HGF offers protective properties against cancer. For example, it was reported that injection of HGF to rats suppresses carcinogen-induced hepatocyte transformation. Using genetic approaches like transgenic mice, others showed that the HGF-MET axis inhibits liver tumorigenesis in these experimental mouse models. Specifically, they reported that hepatocyte-specific elimination of MET in the liver in mice (ie, MET knock out mice) caused enhanced hepatocarcinogenesis, whereas overexpression of HGF in the liver in transgenic mice reduced liver tumorigenesis. Also, various factors that induce growth such as growth hormone, hematopoietic growth factors, and insulin (insulin receptors share close similarity to MET in signal transduction) have been safely administered to patients for decades. Future studies using nonhuman primate models could be helpful to assess the effectiveness and safety profile of META4 therapy in various degenerative models including NASH.

Methods

Generation of Mice With Humanized Liver and High-fat Diet Feeding

[00261] The Institutional Care and Use Committee of the University of Pittsburgh approved all mouse experiments. FRGN (Fah-/-; Rag2-/-; Interleukin 2 common Gamma chain-/-; Nod background) were used for generation of mice with humanized livers as described. In brief, recipient mice (males and females, 2-3 months old) were transplanted intrasplenically with one million freshly isolated human hepatocytes obtained from the Liver Tissue Cell Distribution System at the University of Pittsburgh. Human hepatocytes were derived from healthy liver tissue from patients undergoing surgical resection for biliary stricture and hepatolithiasis (gallstones) or benign liver tumor. One donor was a 43-year-old female with biliary stricture and hepatolithiasis, and the other 2 donors had benign liver tumors (a 29-year-old female and a 60-year- old male). None had evidence of fatty liver. All chimeric mice used in our NAFLD experiments had a similar level of human serum albumin of about 3 mg/mL and were used approximately 6 to 8 months post-transplantation. HFD (“Western diet”) was obtained from Harlan Laboratory. Mice were fed this diet or regular chow (RD) for a total of 6 to 10 weeks as indicated. Nontransplanted FRGN mice on the same regimen were also used as an additional control. For META4 therapy, mice were placed on HFD and then randomly divided to control (isotype matched mlgG1 ) or META4 treated groups (7 mice per group). META4 or isotype matched mlgG1 (control) were administered at 1 mg/kg body weight in sterile saline via weekly intraperitoneal injection. To decipher the pro-growth, pro-regenerative activities of META4 on the homeostasis of the transplanted hepatocytes under the lipotoxic conditions, mice placed on the same NTBC regimen consisting of 3 cycles of NTBC withdrawal lasting 2 weeks for each cycle.

Generation of Mice With Humanized Liver and High-fat Diet Feeding

[00262] The Institutional Care and Use Committee of the University of Pittsburgh approved all mouse experiments. FRGN (Fah-/-; Rag2-/-; Interleukin 2 common Gamma chain-/-; Nod background) were used for hepatocyte repopulation studies (Yecuris, Inc, Tualatin, OR). FRGN mice were housed in a specific-pathogen free facility and maintained on 8 mg/mL NTBC (Ark Pharm, Libertyville, IL) in the drinking water. Chimeric mice were generated essentially as described. In brief, recipient mice (males and females, 2-3 months old) were transplanted intrasplenically with one million freshly isolated human hepatocytes obtained from the Liver Tissue Cell Distribution System at the University of Pittsburgh. Human hepatocytes were derived from healthy liver tissue from patients undergoing surgical resection for biliary stricture and hepatolithiasis (gallstones) or benign liver tumor. One donor was a 43-year-old female with biliary stricture and hepatolithiasis, and the other 2 donors had benign liver tumors (a 29-year-old female and a 60-year-old male). None had evidence of fatty liver. Transplanted mice were maintained on 8 mg/mL NTBC for 4 days following transplantation, and NTBC was then removed to promote expansion of human hepatocytes. Mice were cycled off/on NTBC for 5 to 8 months to achieve a high-level human hepatocyte chimerism. The extent of human hepatocyte chimerism was assessed by measuring human albumin in the blood of repopulated mice (Human Albumin ELISA Quantitation Set, E80-129, Bethyl Laboratories).

[00263] All chimeric mice used in our NAFLD experiments had a similar level of human serum albumin of about 3 mg/mL and were used approximately 6 to 8 months post-transplantation. HFD (“Western diet”) was obtained from Harlan Laboratory. Mice were fed this diet or regular chow (RD) for a total of 6 to 10 weeks as indicated. Nontransplanted FRGN mice on the same regimen were also used as an additional control. For META4 therapy, mice were placed on HFD and then randomly divided to control (isotype matched mlgG1 ) or META4 treated groups (n = 4 per group). META4 or isotype matched mlgG1 (control) were administered at 1 mg/kg body weight in sterile saline via weekly intraperitoneal injection.

Human Liver Samples for Transcriptomic and Proteomic Analyses

[00264] Liver specimens were obtained from University of Pittsburgh Health Sciences Tissue Bank according to approved institutional review board protocol. The NASH samples were biopsy-confirmed cases (diagnosed by the Department of Pathology at our institution). Human plasma from normal and biopsy-proven NASH subjects was obtained from Discovery Life Sciences (https://www.dls.com/). Histology and Immunohistostaininq

[00265] Assessments of liver damage and hepatocyte death such as TUNEL and fibrosis were performed as described previously. Identification of inflammatory cells using macrophage and neutrophil markers was carried out using F4/80 and NIMP-R14 antibodies. Image J was used for quantification of signals. Antibodies against HGF were as follows: N-terminal HGF antibody called Ab1 and Ab2 were from Sigma Aldrich.

RNA-SEQ Analyses

[00266] RNA-Seq and bioinformatics analyses were carried out by ArrayStar Inc (arraystar.com). Differentially expressed genes and transcripts analyses were performed using Ballgown R package. Fold change (cutoff 1.5), P-value (< .05), and FPKM (>0.5 mean in one group) were used for filtering differentially expressed genes and transcripts. Reads were aligned against human genomic reference (and mouse genomic reference in the case of humanized livers, where indicated in the results). Human NASH and normal livers were 3 cases per group, and humanized NASH and normal livers consisted of 2 to 4 cases per group. In the case of human liver samples, as expected, greater than 95% (mean value n = 6) of the reads were mapped to the human reference. Only approximately 24% (mean value n = 6) of the reads from humanized livers (on HFD or on RD) mapped to the human genomic reference. Conversely, about 75% of the reads from humanized liver mapped to the mouse genomic reference, whereas greater than 95% of the reads from the nontransplanted livers mapped to the mouse genomic reference. These outcomes are anticipated because the humanized liver is composed of mouse parenchymal and nonparenchymal cells plus the transplanted human hepatocytes (see also Discussion).

Microarrav Studies

[00267] Expression profiling was carried out at the High Throughput Genome Center, UPMC Department of Pathology (http://path.upmc.edu/genome/lndex.htm) core using the Affymetrix platform. We used the human Affymetrix U133 Plus 2.0 Array. This array has more than 54,000 probes. We detected about 1 1 ,000 probe/genes being expressed in human liver and in humanized liver. All RNA samples were processed and subjected to array analyses side-by-side to minimize variation; livers from 2 different subjects/mice were used. To control for probe specificity, we also used FRGN mouse liver in these experiments. As expected, most probes are specific for human targets and are not conserved in mouse, and we detected about 3800 genes/probes expressed in the mouse liver. Microarray analysis was carried out as we described. Reverse Transcription Polymerase Chain Reaction Analysis and Sequence Verification for NK1/2

[00268] RNA was prepared from human liver tissues using TRIzol (Thermo Fisher, cat# 15596026) according to the manufacturer’s instructions. NK1 and NK2 expression were detected by reverse transcription PCR analysis using 5 pg of RNA in 20 pl of reactions comprised of components of Promega GoScript Reverse Transcription System (Fisher Scientific, cat# A5000) according to the instructions provided. Briefly, RNA mixture was denatured at 65 °C for 10 minutes and chilled on ice, then the mixture was incubated at 42 °C for 1 hour, and reverse transcriptase was inactivated at 70 °C for 15 minutes. For amplification, 1 pl of the synthesized cDNA was added to 25 pl of PCR mixture containing Taq DNA Polymerase System (Thermo Fisher, cat#: 10342020). PCR analysis was performed for 40 cycles; [3-actin was used as internal control. The forward PCR primer sequence for NK1 is: 5'- GCATCATTGGTAAAGGACGCAGC-3' (SEQ ID NO: 45), and the reverse primer sequence for NK1 is: 5'-GCATTAATCTGGTGATAATCCAACAG-3' (SEQ ID NO: 46). The amplified PCR product for NK1 is 508 bp. The forward PCR primer of NK2 is: 5'- CGCTACGAAGTCTGTGACATTCC-3' (SEQ ID NO: 47), and the reverse PCR primer for NK2 is: 5'-CTTCACTGCAGCCTCTGTCACTC-3' (SEQ ID NO: 48). The amplified PCR product for NK2 is 344 bp. The PCR products were analyzed on 2% of agarose gel. The specific DNA bands were cut off from gels and purified using QIAquick Gel Extraction Kit (QIAGEN, cat#: 28704); they were subcloned into PCR 2.1 vector using TA Cloning™ Kit (Thermo Fisher, cat#: K200001 ). Clones were grown; plasmid DNA was isolated and subjected to DNA sequencing by the University of Pittsburgh Genomic Core facility.

Production and Characterization of META4

[00269] Mouse monoclonal antibodies against the extracellular domain of human MET were produced according to standard methods. In brief, mice were immunized with the extracellular domain of purified recombinant human MET (R&D hMET-Fc). Enzyme-linked immunosorbent assay-positive hybridoma clone supernatant purified by protein-A was assayed in our laboratory for MET activation. Production of the antibody, its cDNA cloning from hybridomas (its heavy and light chains) and generation of META4 expression vectors were all carried out by the vendor Creative Biolabs (www.creative-biolabs.com). Recombinant META4 was also produced in our laboratory by transfecting HEK-293 cells with META4 expression vectors and purified by protein-A chromatography.

Statistics

[00270] The 2-tailed Student t test, 1 -way analysis of variance, and the Fisher Exact test were used to analyze data as indicated. A P value equal to .05 or less was considered significant in all statistical analyses.

[00271] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.

Claims

CLAIMS:

1. An antibody that binds to a hepatocyte growth factor (HGF) receptor, comprising: a heavy chain variable region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 20, SEQ ID NO: 32, and SEQ ID NO: 33; and a light chain variable region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 21 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , and SEQ ID NO: 42.

2. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 20; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 21 .

3. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

4. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

5. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 37.

6. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 37.

7. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 39.

8. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 40.

9. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 41 .

10. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 42.

11. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 37.

12. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 38.

13. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 39.

14. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 40.

15. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 41 .

16. The antibody of claim 1 , wherein: the heavy chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 33; and the light chain variable region has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 42.

17. The antibody of any one of claims 1 -16, wherein the amino acid sequence comprises a complementarity-determining region (CDR) having the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.

18. The antibody of claim 17, wherein the CDR comprises one or more mutation selected from S31 N, F33Y, S35G, Y59E, Y60E, P61 S, P61 Q, P61 L, P61T, S103I, K187I, S190G, and S226D.

19. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 2 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

20. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 6 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

21. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 9 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4.

22. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 11 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 13.

23. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 15 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 17.

24. An antibody that binds HGF receptor comprising a heavy chain variable region and a heavy chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 19 and a light chain variable region and a light chain constant region having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 21 .

25. An scFv having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 22.

26. A composition comprising an antibody of any one of claims 1 -24 and a pharmaceutically-acceptable excipient.

27. A method of treating a patient having a disease responsive to a MET agonist, comprising administering to the patient an amount of a MET agonist antibody compound of any one of claims 1 -24 effective to treat the disease in the patient.

28. The method of claim 27, wherein the disease is one or more of: organ failure such as liver and kidney failure/disease, a hepatitis (e.g., steatohepatitis, alcohol hepatitis, viral hepatitis, autoimmune hepatitis, or drug induced hepatitis, such as Tylenol poisoning), or type 2 diabetes and/or its associated pathologies such as retinopathy and macular degeneration.

29. A method of treating a cancer in a patient having a cancer in which MET/HGF are overexpressed, e.g., highly overexpressed, such as a breast, colon, glioma, or thyroid cancer, comprising administering to the patient an amount of a MET antagonist (scFv) antibody compound of any one of claims 1 -24 to the patient effective to treat the cancer.