Long-lasting LIF proteins and formulations

Abstract

Provided herein are chimeric polypeptides and compositions comprising LIF peptides having an extended half-life, and methods of use thereof.

Description

LONG-LASTING LIF PROTEINS AND FORMULATIONSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 665,077, filed June 27, 2024, which application is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 60196-707_601_SL.xml, created June 25, 2025, which is 30,433 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Angiogenesis is a physiological process required for embryonic development, adult vascular homeostasis, and tissue repair. Yet, angiogenesis also contributes to a variety of pathological conditions such as tumors and several intraocular disorders including wet (neovascular) age-related macular degeneration (AMD). During tumor progression, the new vessels provide neoplastic tissues with nutrients and oxygen and thus play an essential role. In intraocular disorders, growth of abnormal, leaky blood vessels may damage the retina and lead to visual loss. Extensive efforts to dissect the molecular basis of angiogenesis and to identify therapeutic targets for neoplasms and other diseases resulted in the discovery of key signaling pathways involved in vascular development and differentiation. In particular, numerous studies have established the pivotal role of the VEGF pathway in physiological and pathological angiogenesis and therapies targeting this pathway, e.g., anti-VEGF therapy, have achieved success in treatments of cancer and ocular disorders such as wet AMD. Conversely, stimulating angiogenesis holds the promise of improving outcomes of patients with a variety of ischemic disorders through improved perfusion. This hypothesis led to a series of clinical trials in the past decades, testing angiogenic factors such as VEGF or bFGF, delivered by gene therapy or as recombinant proteins in coronary or limb ischemia patients. Unfortunately, none of these studies were successful, in spite of promising preclinical studies.SUMMARY

[0004] In some aspects disclosed herein, is a composition comprising a chimeric polypeptide comprising a leukemia inhibitor factor (LIF) peptide, or a functional fragment thereof, and an Fc region of an IgG molecule. In some cases, the LIF peptide described herein can have unexpected pro-angiogenic properties (e.g., in the eyes). Contrary to a LIF that rapidly clears from a subject’s system (e.g., from the vitreous), the LIF peptide described herein may have an extended lifetime (e.g, halflife).

[0005] In some aspects, a chimeric polypeptide comprising an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide. In some embodiments, the Fc region comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide. In some embodiments, the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the Fc region comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide. In some embodiments, the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

[0006] Also provided herein is a composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the chimeric polypeptide described herein. In some embodiments, the first polypeptide and the second polypeptide form a dimer through a disulfidelinkage. In some embodiments, the second polypeptide comprises an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide. In some embodiments, the Fc region comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, the second LIF peptide. In some embodiments, the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the Fc region comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide comprises from N- terminus to C-terminus: the first LIF peptide, the Fc region, the second LIF peptide. In some embodiments, the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the first polypeptide comprises the chimeric polypeptide of any one of claims 1-23. In some embodiments, the second polypeptide comprises the chimeric polypeptide described herein. In some embodiments, the first polypeptide and the second polypeptide are identical in same amino acid sequence. In some embodiments, the composition has a half-life of about 5.4 days. In some embodiments, the composition inhibits growth of bovine aortic endothelial cells. In some embodiments, the composition promotes growth of bovine choroidal endothelial cells. In some embodiments, the composition preserves choriocapillaris, retinalpigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model. In some embodiments, the composition promotes growth of retinal capillary endothelial cells. In some embodiments, administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject promotes retinal angiogenesis. In some embodiments, administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject increases proliferation of choroidal endothelial cells. In some embodiments, administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject does not induce vascular leakage. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the LIF peptide comprises the amino acid sequence SEQ ID NO: 1. In some embodiments, the LIF peptide consists of the amino acid sequence SEQ ID NO: 1.INCORPORATION BY REFERENCE

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present specification, including definitions, will control.BRIEF DESCRIPTION OF DRAWINGS

[0008] Various features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0009] FIGs. 1A-1C show the EC50 of commercial Leukemia Inhibitory Factor (LIF) (FIG. 1A), LIF-Fc (a dimer of SEQ ID NO: 9; FIG. IB) and LIF-KiH (knob in hole; a dimer of SEQ ID NO: 10 and SEQ ID NO: 11; FIG. 1C) in a pStat3 assay performed on bovine aortic endothelial (BAE) cells.

[0010] FIGs. 2A-2C show the IC50 of commercial LIF (FIG. 2A), LIF-Fc (FIG. 2B), and LIF-KiH (FIG. 2C) in a BAE proliferation assay.

[0011] FIG. 3 shows the melting temperature of LIF-Fc and LIF-KiH as determined by dynamic scanning fluorimetry.

[0012] FIGs. 4A-4D show the polyspecificity due to hydrophobicity of LIF-Fc (FIG. 4C) and LIF- KiH (FIG. 4D) as determined by hydrophobic interaction chromatography and as compared to efalizumab (FIG. 4A) and bavituxumab (FIG. 4B).

[0013] FIG. 5 shows self-interaction scores of LIF-Fc and LIF-KiH as compared to efalizumab and bavituxumab.

[0014] FIG. 6 shows the Baculovirus Particle (BVP) assay results of LIF-Fc and LIF-KiH.

[0015] FIGs. 7A-7U show results from a stability study conducted at 4°C and 40°C over two weeks. Samples were collected at day 0, day 7, and day 14. FIGs. 7A-7E show purity of LIF-Fc at the indicated timepoints, as determined by SEC-HPLC measured at Day 0 (FIG. 7A), at 4 °C measured at Day 7 (FIG. 7B), at 4 °C measured at Day 14 (FIG. 7C), at 40 °C measured at Day 7 (FIG. 7D), and at 40 °C measured at Day 14 (FIG. 7E). FIGs. 7F-7J show purity of LIF-KiH at the indicated timepoints, as determined by SEC-HPLC at Day 0 (FIG. 7F), at 4 °C measured at Day 7 (FIG. 7G), at 4 °C measured at Day 14 (FIG. 7H), at 40 °C measured at Day 7 (FIG. 71), and at 40 °C measured at Day 14 (FIG. 7J). FIGs. 7K-7P show binding kinetics for LIF-Fc to LIFR at the indicated time points, as determined by SPR measured at Day 0 (FIG. 7K), at 4 °C measured at Day 7 (FIG. 7L), at 4 °C measured at Day 14 (FIG. 7M), at 40 °C measured at Day 7 (FIG. 7N), and at 40 °C measured at Day 14 (FIG. 70). FIG. 7P shows a table summarizing KD (nM). FIG. 7Q and FIG. 7R show the EC50 of LIF-Fc (FIG. 7Q) and LIF-KiH (FIG. 7R) in pStat3 assay performed on BAE cells at day 0, day 7, and day 14 at 4°C. FIG. 7S and FIG. 7T show the EC50 of LIF-Fc (FIG. 7S) and LIF-KiH (FIG. 7T) in pStat3 assay performed on BAE cells at day 0, day 7, and day 14 at 4°C. 40°C. FIG. 7U summarizes EC50 of LIF-Fc and LIF-KiH.

[0016] FIGs. 8A-8E compare the EC50 of the commercial LIF (FIG. 8A), LIF-KiH (FIG. 8B), LIF- XTEN (a dimer of SEQ ID NO: 13; FIG. 8C), 4xLIF-Fc (a dimer of SEQ ID NO: 12; FIG. 8D), and adjusted 4xLIF-Fc (FIG. 8E) in a pStat3 assay performed on BAE cells.

[0017] FIGs. 9A-9D compare the IC50 of the commercial LIF (FIG. 9A), LIF-KIH (FIG. 9B), LIF- XTEN (FIG. 9C), and 4xLIF-Fc (FIG. 9D) in a BAE cell proliferation assay. FIG. 9E and FIG. 9F shows the fold change in BAE (FIG. 9E) and BCE (FIG. 9F) cell proliferation after administration of 4x LIF or LIF+XTEN at the concentrations (nM) indicated on the x-axis.

[0018] FIGs. 10A-10L show H&E stainings of retinas in animals administered sodium iodinate (NalOs) followed by injection of PBS, LIF, LIF-KiH, 4xLIF-Fc, or LIF-XTEN. FIG. 10A showsretinas after injection of 50ng of LIF. FIGs. 10B-10D show retinas after injection of 0.5 pM (35.81 ng / pL; FIG. 10B), 2.89 pM (207.95 ng / pL; FIG. IOC), or 5.52 pM (395.29 ng / pL; FIG. 10D) LIF- KiH. FIGs. 10E-10I show retinas after injection of 0.24 pM (32.30 ng / pL; FIG. 10E), 0.48 pM (64.59 ng / pL; FIG. 10F), 0.96 pM (129.19 ng / pL; FIG. 10G), 2.47 pM (332.39 ng / pL; FIG. 10H), or 4.94 pM (664.79 ng / pL; FIG. 101) 4xLIF-Fc. FIGs. 10J-10L show retinas after injection of 0.99 pM (146.49 ng / pL; FIG. 10J), 2.48 pM (367.98 ng / pL; FIG. 10K), or 4.90 pM (725 ng / pL; FIG. 10L) LIF-XTEN.

[0019] FIGs. 11A-11D show pharmacokinetic data for LIF (FIG. 11A), LIF-KiH (FIG. 11B), 4xLIF-Fc (FIG. 11C), and LIF-XTEN (FIG. 11D).

[0020] FIGs. 12 shows size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) analysis of 4xLIF-Fc.

[0021] FIG. 13 shows size -exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of 4xLIF-Fc formulated to at 10 mg / mL, 20 mg / mL, and 50 mg / mL in 50 mM phosphate, 5.8% sucrose, 0.03% P20, pH 7.2.

[0022] FIG. 14 shows pSTAT activation by 4xLIF-Fc.

[0023] FIGs. 15A-15F show pSTAT activation at day 0 of 4xLIF-Fc formulated at 10 mg / mL (FIG. 15A), 20 mg / mL (FIG. 15B), and 50 mg / mL (FIG. 15C). FIGs. 15D-15F show pSTAT activation at day 14 of 4xLIF-Fc formulated at 10 mg / mL (FIG. 15D), 20 mg / mL (FIG. 15E), and 50 mg / mL (FIG. 15F)

[0024] FIGs. 16A-16D show melting curve of 4xLIF-Fc (FIG. 16A), 4xLIF-Fc Variant 1 (FIG. 16B), 4xLIF-Fc Variant 2 (FIG. 16C), or 4xLIF-Fc Variant 3 (FIG. 16D).

[0025] FIGs. 17A-17D show pSTAT3 activation curve and EC50 (nM) of commercial LIF (FIG. 17A), 4xLIF-Fc (FIG. 17B), 4xLIF-Fc Variant 1 (FIG. 17C), or 4xLIF-Fc Variant 2 (FIG. 17D).

[0026] FIGs. 18A-18H show vitreous humor PK analysis. FIG. 18A shows a dose-response curve, and FIG. 18B shows a semi -logarithmic pharmacokinetic plot of the commercial product. FIG. 18C shows a dose-response curve, and FIG. 18D shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc. FIG. 18E shows a dose-response curve, and FIG. 18F shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 1. FIG. 18G shows a dose-response curve, and FIG. 18H shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 2.

[0027] FIGs. 19A-19H show aqueous humor PK analysis. FIG. 19A shows a dose-response curve, and FIG. 19B shows a semi -logarithmic pharmacokinetic plot of the commercial product. FIG. 19C shows a dose-response curve, and FIG. 19D shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc. FIG. 19E shows a dose-response curve, and FIG. 19F shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 1. FIG. 19G shows a dose-response curve, and FIG. 19H shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 2.

[0028] FIGs. 20A-20H show choroidal PK analysis. FIG. 20A shows a dose-response curve, and FIG. 20B shows a semi-logarithmic pharmacokinetic plot of the commercial product. FIG. 20Cshows a dose-response curve, and FIG. 20D shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc. FIG. 20E shows a dose-response curve, and FIG. 20F shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 1. FIG. 20G shows a dose-response curve, and FIG. 20H shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 2.

[0029] FIGs. 21A-21H show retinal PK analysis. FIG. 21A shows a dose-response curve, and FIG. 21B shows a semi-logarithmic pharmacokinetic plot of the commercial product. FIG. 21C shows a dose-response curve, and FIG. 21D shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc. FIG. 21E shows a dose-response curve, and FIG. 21F shows a semi-logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 1. FIG. 21G shows a dose-response curve, and FIG. 21H shows a semi- logarithmic pharmacokinetic plot of 4xLIF-Fc Variant 2.

[0030] FIGs. 22A-22C show plasma PK analysis. FIG. 22A shows a dose-response curve of 4xLIF- Fc. FIG. 22B shows a dose-response curve of 4xLIF-Fc Variant 1. FIG. 22C shows a dose-response curve of 4xLIF-Fc Variant 2.

[0031] FIG. 23A shows a quantification of vascular density as measured by CD31 immunostaining. FIG. 23B shows immuno staining of retinal layers (intermediate vascular plexus, inner nuclear layer, and deep vascular plexus).

[0032] FIG. 24A and FIG. 24B show retinal layer H&E staining of PBS-treated (FIG. 24A) and 4xLIF-Fc Variant 2 (FIG. 24B)-treated mice.

[0033] FIG. 25A-25E show quantifications of outer nuclear layer (ONL) thickness of LIF 50 ng / uL (FIG. 25A), 4xLIF-Fc Variant 2 0.25 uM (FIG. 25B), 4xLIF-Fc Variant 2 0.5 uM (FIG. 25C), 4xLIF-Fc Variant 2 2.5 uM (FIG. 25D), or 4xLIF-Fc Variant 2 5.0 uM (FIG. 25E)-treated mice.DETAILED DESCRIPTION

[0034] In some aspects, disclosed herein is a composition comprising a chimeric polypeptide comprising a LIF peptide, or a functional fragment thereof, and Fc region of an IgG molecule.Leukemia Inhibitory Factor (LIF)

[0035] In one aspect, provided herein are Leukemia Inhibitory Factor (LIF) peptides or functional fragments thereof. LIF is an interleukin 6 class cytokine. LIF may also be known as Differentiationstimulating factor (D factor), Melanoma-derived LPL inhibitor (MLPLI), Human interleukin for DA cells (HILDA) or Emfilermin. The LIF peptide may bind to a LIF receptor. In some embodiments, the LIF peptides are human LIF. In some embodiments, the LIF peptides are the full human LIF peptide. Non-limiting examples of a LIF peptide sequence are provided in Table 1.

[0036] In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 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%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the LIF peptide comprised in any one of the polypeptides or compositions described herein can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 1

[0037] In some embodiments, a polypeptide, fusion protein, composition, system, or kit described herein may comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least99% identical to SEQ ID NO: 1. In some embodiments, a polypeptide, fusion protein, composition, system, or kit described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 1.

[0038] In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein may comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 19. In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 19.

[0039] In some embodiments, a polypeptide, fusion protein, composition, system, or kit described herein may comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least99% identical to SEQ ID NO: 19. In some embodiments, a polypeptide, fusion protein, composition, system, or kit described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 19.

[0040] In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein may comprise a sequence that is at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 20. In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 20.

[0041] In some embodiments, a polypeptide, fusion protein, composition, system, or kit described herein may comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 20. In some embodiments, a polypeptide, fusion protein, composition, system, or kit described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 20.

[0042] In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more amino acid additions, deletions, or substitutions as compared to any one of SEQ ID NOs: 1, 19, and 20. In some embodiments, a LIF peptide comprised in any one of the polypeptides or compositions described herein may comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or less amino acid additions, deletions, or substitutions as compared to any one of SEQ ID NOs: 1, 19, and 20.Table 1. Non-limiting example of LIF peptidesAntibodies fragments

[0043] In one aspect, provided herein are antibody fragment sequences. In some embodiments, an antibody fragment sequence can allow the polypeptide comprising the sequence to modify the halflife and distribution, valency, affinity and avidity, tissue penetration and bioactivities of a polypeptide.

[0044] In some embodiments, the antibody fragment may be a fragment crystallizable (Fc) region or a functional fragment thereof. The antibody fragment sequence may be naturally occurring, partially modified, or fully synthetic. In some embodiments, the antibody fragment can comprise a Fc region of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM. In some embodiments, the antibody fragment can comprise a Fc region of IgGl. In some embodiments, the Fc region can have one or more amino acid modifications (e.g., addition, deletion, or substitution). In some embodiments, the Fc region can comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten amino acid modifications (e.g., addition, deletion, or substitution) when compared to a corresponding naturally occurring Fc (e.g., relative to SEQ ID NO: 22). In some embodiments, the Fc region can comprise at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid modifications (e.g., addition, deletion, or substitution) when compared to a corresponding naturally occurring Fc (e.g., relative to SEQ ID NO: 22). For example, in some embodiments, the Fc region can comprise a Fc-LALA mutation. In some embodiments, the Fc region can comprise a H to A substitution at position 193 when aligned to SEQ ID NO: 22 (or H to A substitution at position 310 of the antibody constant heavy chain region of human IgGl using the numbering according to EU Index of Kabat). In some embodiments, the Fc region can have one or more glycosylated amino acids. In some embodiments, the Fc region can comprise one or more mutations such that it interferes with glycosylation at that position (e.g., by replacing asparagine residue with another amino acid). In some embodiments, there may be one or more antibody fragments. In some embodiments, the antibody fragments may be heterodimers. In some embodiments, the heterodimeric antibody fragments may be modified to preferentially generate a heterodimer pairing.

[0045] In some embodiments, the antibody fragment sequence comprising one or more amino acid modifications at the Fc region as described herein (e.g., the Fc region comprising a Fc-LALA mutation) can alter or reduce FcR binding affinity when compared to that of a corresponding antibody fragment sequence lacking one or more amino acid modifications in the Fc region. In some embodiments, the antibody fragment sequence comprising one or more amino acid modifications at the Fc region as described herein (e.g., the Fc region comprising a Fc-LALA mutation) can reduce FcR binding affinity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, or at least 5fold when compared to that of a corresponding antibody fragment sequence lacking one or more amino acid modifications at the Fc region (e.g., lacking a Fc-LALA mutation).

[0046] In some embodiments, the antibody fragment sequence comprising one or more amino acid modifications at the Fc region as described herein (e.g. , a H to A substitution at position 193 when aligned to SEQ ID NO: 22) can alter or decrease neonatal Fc receptor (FcRN) recycling when compared to that of a corresponding antibody fragment sequence lacking one or more amino acid modifications at the Fc region. In some embodiments, the antibody fragment sequence comprising one or more amino acid modifications at the Fc region as described herein (e.g., a H to A substitution at position 193 when aligned to SEQ ID NO: 22) can decrease FcRN recycling by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, or at least 5 fold when compared to that of a corresponding antibody fragment sequence lacking one or more amino acid modifications at the Fc region (e.g., lacking a H to A substitution at position 193 when aligned to SEQ ID NO: 22).

[0047] In some embodiments the antibody fragment sequences may be a short peptide. The antibody fragment sequences can be coupled directly or indirectly to a polypeptide, such as LIF. The antibody fragment sequences can be coupled to any site of a polypeptide, e.g. , to or near the end or terminus of a polypeptide, to one or more internal sites, attached to a side chain, or to the amino-terminal amino acid (N -terminal) or to the carboxy-terminal amino acid (C-terminal). In some embodiments, the antibody fragment sequence is coupled to the C-terminal or N-terminal end of a polypeptide by placing a DNA sequence encoding the antibody or antibody fragment sequences within the same open reading frame of the recombinant protein by means of genetic engineering. In some embodiments, the antibody fragment sequence may be manufactured separately and introduced to a polypeptide after translation, secretion, isolation, or concentration of the polypeptide. Sequences for non-limiting examples of antibody fragments are provided in Table 2.

[0048] Various embodiments include an antibody fragment. The antibody fragment may be present on a chimeric polypeptide construct. The antibody fragment sequences may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to any one of the sequences in Table 2. In some embodiment, the antibody fragment sequence(s) may be at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to any one of the sequences in Table 2.

[0049] In some embodiments, an antibody fragment sequences can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 2. Insome embodiments, an antibody fragment can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 3. In some embodiments, an antibody fragment can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, an antibody fragment can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, an antibody fragment can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 22.

[0050] In some embodiments, an antibody fragment sequence can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 2. In some embodiments, an antibody fragment can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 3. In some embodiments, an antibody fragment can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 10. In some embodiments, an antibody fragment can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 21. In some embodiments, an antibody fragment can comprise a sequence that is at least 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 22.

[0051] In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 3. In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 22.

[0052] In some embodiments, any one of the polypeptides or compositions described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 2. In some embodiments, any one of the polypeptides or compositions described herein may comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 3. In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 10. In some embodiments, any one of the polypeptides or compositions described herein can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 21. In some embodiments, any one of the polypeptides or compositions described herein cancomprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most99% identical to SEQ ID NO: 22.Table 2. Non-limiting example of antibody fragments.Unstructured polypeptides

[0053] In one aspect, provided herein are unstructured recombinant polypeptide (URP) sequences (e.g., an extended recombinant polypeptide, or XTEN). In some embodiments, a URP sequence can enhance the pharmacokinetic properties, and / or increases the solubility and stability of the polypeptide comprising the sequence, while retaining or enhancing overall biologic and / or therapeutic activity of a biologically active portion of the polypeptide. Sequences for non-limiting examples of antibody fragments are provided in Table 3.

[0054] In some embodiments, a URP (e.g., XTEN) can comprise a sequence characterized in that a) it comprises at least 40 contiguous amino acids; b) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP constitutesmore than 80% of the total amino acids of the URP, and the remainder, when present, consists of arginine or lysine, and the remainder does not contain methionine, cysteine, asparagine, and glutamine; c) the URP comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein one of said at least three different types is glycine (G) or glutamine (E); and at least 50% of the at least 40 contiguous amino acids in the URP sequence are devoid of secondary structure as determined by Chou-Fasman algorithm. In some embodiments, the URP can comprise multiple units of two or more non-overlapping sequence motifs. In some embodiments, the sequence motif is 12 amino acids in length. In some embodiments, the motif is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0055] In some embodiments, the URP (e.g., XTEN) can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the URP (e.g., XTEN) can comprise a sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 4. In some embodiments, the URP can comprise an XTEN sequence that is identical to SEQ ID NO: 4.Table 3. Non-limiting example of URP sequences.Chimeric Polypeptides and Compositions

[0056] Non-limiting example peptides and fusion proteins are provided in Table 4. Each of the sequences in Table 4 may include an N-terminal methionine for recombinant protein expression.LIF-Fc

[0057] In one aspect, provided herein are chimeric polypeptides comprising a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG.

[0058] In some embodiments, the chimeric polypeptide comprising a LIF peptide, or functional fragment thereof, is coupled to an Fc region of an IgG via a linker. In some embodiments, the linker can comprise an amino acid sequence of any one of SEQ ID NOs: 5-8 or 26-28. In some embodiments, the linker can comprise the amino acid sequence of SEQ ID NO: 5. In some embodiments, the Fc region can comprise an amino acid sequence of any one of SEQ ID NOs: 2, 3, 10, or 21. In some embodiments, the Fc region can comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Fc region can comprise the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Fc region can comprise the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Fc region can comprise the amino acid sequence of SEQ ID NO: 21 .

[0059] In some embodiments, the chimeric polypeptide comprising a LIF peptide or functional fragment thereof coupled to an Fc region can comprise, from N-terminus to C-terminus: the LIF peptide, or functional fragment thereof, the linker, and the Fc region. In some embodiments, the chimeric polypeptide comprising a LIF peptide or functional fragment thereof coupled to an Fc region can comprise, from N-terminus to C-terminus: the Fc region, the linker, and the LIF peptide or functional fragment thereof. In some embodiments, the chimeric polypeptide can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 9. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 9.

[0060] In some aspects, provided herein are LIF-Fc compositions comprising a first polypeptide and a second polypeptide, wherein the first polypeptide can comprise a chimeric polypeptides comprising a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG. In some embodiments, the first polypeptide can comprise a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG via a linker, wherein the linker can comprise the amino acid sequence SEQ ID NO: 5. In some embodiments, the first polypeptide can comprise a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG via a linker, wherein the Fc region can comprise the amino acid sequence SEQ ID NO: 2. In some embodiments, the first polypeptide can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 9. In some embodiments, the first polypeptide is identical to SEQ ID NO: 9.

[0061] In some embodiments, the first polypeptide and the second polypeptide form a dimer. In some embodiments, the first polypeptide and the second polypeptide can form a dimer through a disulfide linkage.

[0062] In some embodiments, the second polypeptide can comprise a chimeric polypeptides comprising a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG. In some embodiments, the second polypeptide can comprise a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG via a linker, wherein the linker can comprise an amino acid sequence of any one of SEQ ID NOs: 5-8 and 26-28. In some embodiments, the second polypeptide can comprise a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG via a linker, wherein the linker can comprise the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second polypeptide can comprise a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG via a linker, wherein the Fc region can comprise an amino acid sequence of any one of SEQ ID NOs: 2, 3, 10, or 21. In some embodiments, the second polypeptide can comprise a LIF peptide or functional fragment thereof coupled to an Fc region of an IgG via a linker, wherein the Fc region can comprise the amino acid sequence SEQ ID NO: 2. In some embodiments, the second polypeptide can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 9. In some embodiments, the second polypeptide is identical to SEQ ID NO: 9.

[0063] In some embodiments, the first polypeptide and the second polypeptide are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical in amino acid sequence. In some embodiments, the first polypeptide and the second polypeptide are identical in amino acid sequence.

[0064] In some embodiments, the LIF-Fc composition has a melting temperature of about 65 to about 75°C, e.g., about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C°C. In some embodiments, the LIF-Fc composition has a melting temperature of about 69°C to about 71 °C.

[0065] In some embodiments, the LIF-Fc composition can have a hydrophobic interaction chromatography retention time of about 15 minutes.

[0066] In some embodiments, the LIF-Fc composition can have a self-interaction score of about 0.0.

[0067] In some embodiments, the LIF-Fc composition can be stable between 4 and 40°C for at least 14 days.

[0068] In some embodiments, the LIF-Fc composition can have a baculovirus particle assay score of about 5.

[0069] In some embodiments, the LIF-Fc composition can inhibit growth of bovine aortic endothelial cells. In some embodiments, the LIF-Fc composition can promote growth of bovine choroidal endothelial cells. In some embodiments, the LIF-Fc composition can preserve choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mousemodel. In some embodiments, the LIF-Fc composition can promote growth of retinal capillary endothelial cells.LIF-KiH (Knob in Hole)

[0070] In one aspect, provided herein are LIF-KiH compositions comprising a first polypeptide and a second polypeptide, wherein the first polypeptide can comprise a LIF peptide, or functional fragment thereof, coupled to an Fc region of an IgG, wherein the Fc region can comprise a knob into hole amino acid substitution. In some embodiments, the second polypeptide can comprise an Fc region of an IgG molecule, wherein the Fc region can comprise a knob into hole amino acid substitution. In some embodiments, the second polypeptide can comprise a LIF peptide coupled to the Fc region. In some embodiments, the Fc region of the first polypeptide can comprise a knob substitution and the Fc region of the second polypeptide can comprise a hole substitution. In some embodiments, the Fc region of the first polypeptide can comprise a hole substitution and the Fc region of the second polypeptide can comprise a knob substitution.

[0071] In some embodiments, the hole substitution is selected from any one or more ofY407T, Y407V, T366S, L368A, T394W, F405A (based on Kabat numbering of full length IgG heavy chain). In some embodiments, the knob substitution is selected from T366W and T366Y (based on Kabat numbering of full length IgG heavy chain). In some embodiments, the hole substitution is T366S and / or L368A. In some embodiments, the knob substitution is T366W.

[0072] In some embodiments, the Fc region of the first polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the Fc region of the first polypeptide is identical to SEQ ID NO: 21.

[0073] In some embodiments, the first polypeptide of the LIF-KiH composition can comprise, from N-terminus to C-terminus: LIF, optionally a linker, the first Fc region. In some embodiments, the first polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the first polypeptide of the LIF-KiH composition can be identical to SEQ ID NO: 11.

[0074] In some embodiments, the second polypeptide of the LIF-KiH composition can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identicalto SEQ ID NO: 10. In some embodiments, the second polypeptide of the LIF-KiH composition can be identical to SEQ ID NO: 10.

[0075] In some embodiments, the LIF-KiH composition can have a melting temperature of about 60 to about 70°C, e.g., about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C. In some embodiments, the LIF-KiH composition has a melting temperature of about 65°C.

[0076] In some embodiments, the LIF-KiH composition can have a hydrophobic interaction chromatography retention time of about 15 minutes.

[0077] In some embodiments, the LIF-KiH composition can have a self-interaction score of about 0.4.

[0078] In some embodiments, the LIF-KiH composition can be stable between 4 and 40°C for at least 14 days.

[0079] In some embodiments, the LIF-KiH composition can have a baculovirus particle assay score of about 4.

[0080] In some embodiments, the LIF-KiH composition can have a half-life of 2.6 days.

[0081] In some embodiments, the LIF-KiH composition can inhibit growth of bovine aortic endothelial cells. In some embodiments, the LIF-KiH composition can promote growth of bovine choroidal endothelial cells. In some embodiments, the LIF-KiH composition can preserve choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model. In some embodiments, the LIF-KiH composition can promote growth of retinal capillary endothelial cells.LIF-XTEN

[0082] In one aspect, provided herein are chimeric polypeptides comprising an Fc region (e.g., of an IgG) coupled to a LIF peptide, and an XTEN moiety.

[0083] In some embodiments, the Fc region can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 3.

[0084] In some embodiments, the XTEN moiety is characterized in that that a) it comprises at least 40 contiguous amino acids; b) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the XTEN moiety constitutes more than 80% of the total amino acids of the URP, and the remainder, when present, consists of arginine or lysine, and the remainder does not contain methionine, cysteine, asparagine, and glutamine; c) the XTEN moiety comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein one of said at least three different types is glycine (G) or glutamine (E); and at least 50% ofthe at least 40 contiguous amino acids in the XTEN moiety are devoid of secondary structure as determined by Chou-Fasman algorithm. In some embodiments, the XTEN moiety comprises a motif selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0085] In some embodiments, the XTEN moiety can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the XTEN moiety is identical to SEQ ID NO: 4.

[0086] In some embodiments, the chimeric polypeptide can comprise from N-terminus to C- terminus: LIF, the Fc region, the XTEN moiety. In some embodiments, the chimeric polypeptide can comprise from N-terminus to C-terminus: the XTEN moiety, the Fc region, and LIF. In some embodiments, the chimeric polypeptide further can comprise a linker between the Fc region and the XTEN moiety. In some embodiments, the chimeric polypeptide further can comprise a linker between the LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide can comprise from N-terminus to C-terminus: LIF, a first linker, the Fc region, a second linker, and the XTEN moiety. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the first linker can comprise an amino acid sequence according to SEQ ID NO: 5 and the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 13.

[0087] In some embodiments, provided herein are LIF-XTEN compositions comprising a first polypeptide and a second polypeptide, wherein the first polypeptide can comprise any one of the chimeric polypeptides comprising LIF and XTEN described herein (e.g., a chimeric polypeptides comprising an Fc region of an IgG coupled to a LIF peptide, and an XTEN moiety).

[0088] In some embodiments, the second polypeptide can comprise, from N-terminus to C-terminus: LIF, the Fc region, and the XTEN moiety. In some embodiments, the XTEN moiety can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the XTEN moiety is identical to SEQ ID NO: 4. In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: the XTEN moiety, the Fc region, LIF. In some embodiments, the second polypeptide further can comprise alinker between the Fc region and the XTEN moiety. In some embodiments, the second polypeptide further can comprise a linker between the LIF peptide and the Fc region. In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: LIF, a first linker, the Fc region, a second linker, and the XTEN moiety. In some embodiments, the first linker or the second linker can comprise an amino acid sequence of any one of the sequences of SEQ ID NO: 5-8 and 26-28. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the first linker can comprise an amino acid sequence according to SEQ ID NO: 5 and the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the second polypeptide can comprise an amino acid that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 13.

[0089] In some embodiments, the first polypeptide and the second polypeptides of the LIF-XTEN composition can form a dimer. In some embodiments, the first polypeptide and the second polypeptides of the LIF-XTEN composition can form a dimer through a disulfide linkage.

[0090] In some embodiments, the first polypeptide and the second polypeptide are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical in amino acid sequence. In some embodiments, the first polypeptide and the second polypeptide can be identical in amino acid sequence.

[0091] In some embodiments, the LIF-XTEN composition can have a half-life of 4.2 days.

[0092] In some embodiments, the LIF-XTEN composition can inhibit growth of bovine aortic endothelial cells. In some embodiments, the LIF-XTEN composition can promote growth of bovine choroidal endothelial cells. In some embodiments, the LIF-XTEN composition can preserve choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model. In some embodiments, the LIF-KiH composition can promote growth of retinal capillary endothelial cells.4xLIF-Fc

[0093] In one aspect, provided herein are chimeric polypeptides comprising an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide. In some embodiments, the Fc region can comprise an amino acid sequence of any one of the sequences of SEQ ID NO: 2, 3, 10, or 21. In some embodiments, the Fc region can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, 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%, or at least 99% identical to SEQ ID NO: 3. In some embodiments, the Fc region may comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 3. In some embodiments, the Fc region can comprise an amino acid sequence identical to SEQ ID NO: 3.

[0094] In some embodiments, the Fc region may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the Fc region may comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 2. In some embodiments, the Fc region can comprise an amino acid sequence identical to SEQ ID NO: 2.

[0095] In some embodiments, the chimeric polypeptide can comprise, from N-terminus to C- terminus: the first LIF peptide, the Fc region (e.g., SEQ ID NO: 3), and the second LIF peptide. In some embodiments, the chimeric polypeptide can further comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide can further comprise a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first polypeptide and the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the FC region may comprise an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 12. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 12.

[0096] In some embodiments, the chimeric polypeptide can comprise, from N-terminus to C- terminus: the first LIF peptide, the Fc region (e.g., SEQ ID NO: 2), and the second LIF peptide. In some embodiments, the chimeric polypeptide can further comprise a linker between the first LIFpeptide and the Fc region. In some embodiments, the chimeric polypeptide can further comprise a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the FC region may comprise an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 23. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 23. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 23.

[0097] In some embodiments, the chimeric polypeptide can comprise, from N-terminus to C- terminus: the first LIF peptide, the Fc region (e.g., SEQ ID NO: 2), and the second LIF peptide. In some embodiments, the chimeric polypeptide can further comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide can further comprise a linker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the FC region may comprise an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 24. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 24.

[0098] In some embodiments, the chimeric polypeptide can comprise, from N-terminus to C- terminus: the first LIF peptide, the Fc region (e.g., SEQ ID NO: 2), and the second LIF peptide. In some embodiments, the chimeric polypeptide can further comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the chimeric polypeptide can further comprise alinker between the Fc region and the second LIF peptide. In some embodiments, the chimeric polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the FC region may comprise an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 25. In some embodiments, the chimeric polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 25. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 25.

[0099] In some embodiments, provided herein are 4xLIF-Fc compositions comprising a first polypeptide and a second polypeptide, wherein the first polypeptide can comprise any one of the chimeric polypeptides described herein (e.g., a chimeric polypeptides comprising an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide). In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, the Fc region (e.g., SEQ ID NO: 2 or 3), and the second LIF peptide. In some embodiments, the second polypeptide further can comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the second polypeptide further can comprise a linker between the Fc region and the second LIF peptide.

[0100] In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the Fc region can comprise an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 12. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 12.

[0101] In some other cases, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide. In some embodiments, the second polypeptide further can comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the second polypeptide further can comprise a linker between the Fc region and the second LIF peptide. In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the Fc region can comprise an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 23. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 23. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 23.

[0102] In some other cases, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide. In some embodiments, the second polypeptide further can comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the second polypeptide further can comprise a linker between the Fc region and the second LIF peptide. In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the Fc region can comprise an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 24. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 24.

[0103] In some other cases, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide. In some embodiments, the second polypeptide further can comprise a linker between the first LIF peptide and the Fc region. In some embodiments, the second polypeptide further can comprise a linker between the Fc region and the second LIF peptide. In some embodiments, the second polypeptide can comprise from N-terminus to C-terminus: the first LIF peptide, a first linker, the Fc region, a second linker, and the second LIF peptide. In some embodiments, the first linker or the second linker can comprise an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the Fc region can comprise an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 25. In some embodiments, the second polypeptide can comprise an amino acid sequence that is at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% identical to SEQ ID NO: 25. In some embodiments, the chimeric polypeptide is identical to SEQ ID NO: 25.

[0104] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) can be a dimer of the first polypeptide (e.g. , any one of the chimeric polypeptides described herein) and the second polypeptide. In some embodiments, the first polypeptide (e.g., any one of the chimeric polypeptides described herein) and the second polypeptide of the 4xLIF-Fc or 4xLIF-Fc variant (e.g, 4xLIF-Fc Variants 1-3) composition form a dimer through a disulfide linkage.

[0105] In some embodiments, the first polypeptide and the second polypeptide are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical in amino acid sequence. In some embodiments, the first polypeptide and the second polypeptide are identical in amino acid sequence.

[0106] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition may have a half-life of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 14 days, or more. In some cases, the 4xLIF-Fc composition may have a half-life of 5.4 days. In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g, 4xLIF-Fc Variants 1-3) composition described herein may have at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or longer half-life than that of a control molecule. In some embodiments, a control molecule may have single or multimeric LIF molecules (e.g., one LIF peptide, two LIF peptides, threeLIF peptides, four LIF peptides or more) but lacking one or more domains (e.g., one or more linkers, one or more Fc regions) and / or lacking one or more mutations (e.g., one or more mutations in the Fc regions).

[0107] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition described herein can have a melting temperature of from about 60 to about 75°C, e.g., about 60°C, about 61 °C, about 62°C, about 63 °C, about 64°C, about 65 °C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, or about 75°C. In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant composition described herein can have a melting temperature of at least 60°C, at least 61 °C, at least 62°C, at least 63 °C, at least 64°C, at least 65°C, at least 66°C, at least 67°C, at least 68°C, at least 69°C, at least 70°C, at least 71°C, at least 72°C, at least 73°C, at least 74°C, at least about 75°C or more. In some embodiments, the 4xLIF- Fc or 4xLIF-Fc variant composition described herein can have a melting temperature of at most 60°C, at most 61 °C, at most 62°C, at most 63 °C, at most 64°C, at most 65 °C, at most 66°C, at most 67°C, at most 68°C, at most 69°C, at most 70°C, at most 71°C, at most 72°C, at most 73°C, at most 74°C, at most about 75 °C or less.

[0108] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition can inhibit growth of bovine aortic endothelial cells. For example, in some cases, the 4xLIF-Fc composition may inhibit growth of bovine aortic endothelial cells by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more compared to that of no treatment (without 4xLIF-Fc composition treatment) or as compared to that of a control molecule treatment. In some embodiments, a control molecule may have single or multimeric LIF molecules (e.g., one LIF peptide, two LIF peptides, three LIF peptides, four LIF peptides or more) but lacking one or more domains (e.g. , one or more linkers, one or more Fc regions) and / or lacking one or more mutations (e.g., one or more mutations in the Fc regions).

[0109] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition can promote growth of bovine choroidal endothelial cells. In some embodiments, the 4xLIF-Fc composition can promote growth of bovine choroidal endothelial cells by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to that of no treatment (without 4xLIF-Fc composition treatment) or as compared to that of a control molecule treatment. In some embodiments, a control molecule may have single or multimeric LIF molecules (e.g., one LIF peptide, two LIF peptides, three LIF peptides, four LIF peptides or more) but lacking one or more domains (e.g., one or more linkers, one or more Fc regions) and / or lacking one or more mutations (e.g, one or more mutations in the Fc regions).

[0110] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition can preserve choriocapillaris, retinal pigment epithelium, and / or photoreceptors aftersystemic administration of sodium iodate in a mouse model. In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition preserves choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to that of no treatment (without 4xLIF-Fc or variant composition treatment) or as compared to that of a control molecule treatment. In some embodiments, a control molecule may have single or multimeric LIF molecules (e.g., one LIF peptide, two LIF peptides, three LIF peptides, four LIF peptides or more) but lacking one or more domains (e.g. , one or more linkers, one or more Fc regions) and / or lacking one or more mutations (e.g. , one or more mutations in the Fc regions).[oni] In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition can promote growth of retinal capillary endothelial cells. In some embodiments, the 4xLIF-Fc or 4xLIF-Fc variant (e.g., 4xLIF-Fc Variants 1-3) composition can promote growth of retinal capillary endothelial cells by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to that of no treatment (without 4xLIF-Fc or variant composition treatment) or as compared to that of a control molecule treatment. In some embodiments, a control molecule may have single or multimeric LIF molecules (e.g., one LIF peptide, two LIF peptides, three LIF peptides, four LIF peptides or more) but lacking one or more domains (e.g., one or more linkers, one or more Fc regions) and / or lacking one or more mutations (e.g., one or more mutations in the Fc regions).

[0112] Non-limiting example peptides and fusion proteins are provided in Table 4. Each of the sequences in Table 4 may include an N-terminal methionine for recombinant protein expression. In some embodiments, the chimeric polypeptide described herein (e.g., a chimeric polypeptide as shown in Table 4 or a variant thereof) can be glycosylated at one or more positions. In some embodiments, the chimeric polypeptide described herein (e.g., a chimeric polypeptide as shown in Table 4 or a variant thereof) can have reduced glycosylation. For example, in some embodiments, the antibody can be produced in a non-mammalian cell production system such that there is no glycosylation. In some embodiments, glycosylation is reduced by removing a carbohydrate group already present in the chimeric polypeptide. In some embodiments, the Fc region of the chimeric polypeptide can comprise one or more mutation such that interferes with glycosylation at that position (e.g., by replacing asparagine residue with another amino acid).

[0113] In some embodiments, the chimeric polypeptide described herein (e.g., a chimeric polypeptide as shown in Table 4 or a variant thereof) results in a reduction in systemic exposure of the composition upon an intravitreal injection.

[0114] In some embodiments, the chimeric polypeptide described herein can be retained within the injection site or surrounding area of the injection site. For example, in some cases, the chimericpolypeptide described herein can be retained within the injection site or surrounding area of the injection site for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1.5 months, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, or more. In some cases, the chimeric polypeptide described herein can be retained within the injection site or surrounding area of the injection site for at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 2 weeks, at most about 3 weeks, at most about 4 weeks, at most about 1.5 months, at most about 2 months, at most about 3 months, at most about 4 months, at most about 5 months, at most about 6 months, at most about 7 months, at most about 8 months, at most about 9 months, at most about 10 months, at most about 11 months, at most about 12 months, or less.

[0115] In some embodiments, the chimeric polypeptide described herein can lead to a decrease in immune cells (e.g., Natural Killer cells) recruitment in the injection site (e.g., an eye). In some embodiments, the chimeric polypeptide described can lead to a decrease in immune cells (e.g., Natural Killer cells) recruitment in the injection site (e.g., an eye) by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more as compared to that of no treatment area (e.g. , an eye that was not treated with the chimeric polypeptide described herein)

[0116] In some embodiments, the chimeric polypeptide described herein (e.g., a chimeric polypeptide as shown in Table 4 or a variant thereof) can elicit Stat3 signaling in a cell. In some embodiments, the chimeric polypeptide described herein (e.g., a chimeric polypeptide as shown in Table 4 or a variant thereof) can enhance Stat3 signaling in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2- fold, at least 3 fold, at least 4 fold, or at least 5 fold compared to that of a control polypeptide lacking one or more components described herein or compared to that of a LIF polypeptide.Table 4. Non-limiting examples of chimeric polypeptides.Linkers

[0117] In some aspects, polypeptides, fusion proteins, and compositions herein comprise a linker. The linker may be positioned between a first peptide and a second agent (e.g, a Fc region). There may be one or more linkers. In some embodiments, if there are more than one linker, each linker may be the same linker or a different linker.

[0118] In some embodiments, the linker can comprise a flexible linker, where at least about four of the amino acids have no regular secondary structure. In some embodiments, regular secondary structure can comprise any helical structure (e.g, an alpha helix, 310 helix, n helix), a beta turn, omega loop, and / or a beta sheet. In some embodiments, the flexible linker is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% glycine, serine, or glycine and serine residues.

[0119] In some embodiments, the linker can comprise a rigid linker, where at least seven of the amino acids form a helical structure. In some embodiments, the linker can comprise a flexible and a rigid linker.

[0120] In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, 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%, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 7. In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 8. In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 26. In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 27. In some embodiments, the linker can comprise a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to SEQ ID NO: 28.

[0121] In some embodiments, the linker can comprise a sequence of about 5 to about 50 amino acids. For example, a sequence of about 4 to about 45, about 4 to about 40, about 5 to about 35, about 4 to about 30, about 4 to about 20, about 4 to about 15, about 10 to about 50, about 10 to about 45, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 15, about 15 to about 50, about 15 to about 45, about 15 to about 40, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 15 to about 20, about 20 to about 50, about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 25 to about 45, about 25 to about 40, about 25 to about 35, about 25 to about 30, about 30 to about 50, about 30 to about 45, about 30 to about 40, about 30 to about 35, about 35 to about 50, about 35 to about 45, about 35 to about 40, about 40 to about 50, about 40 to about 45, about 45 to about 50, about 4, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.Table 5. Non-limiting examples of linkersMethod of making chimeric polypeptides

[0122] Also provided herein are methods of producing polypeptide (e.g, chimeric polypeptides) described there that include: (a) culturing a cell (e.g, any of the cells described herein) including any of the nucleic acids encoding any of the polypeptides or any components thereof (e.g, LIF-Fc, L1F- KiH, LIF-XTEN, 4xLlF-Fc, or 4xLlF-Fc variants) described herein, or any of the expression vectors described herein that include nucleic acid encoding any of the polypeptides described herein, in a culture medium under conditions sufficient to allow for the production of the polypeptide; and (b) harvesting the polypeptide from the host cell or the culture medium. In some embodiments of any of the methods described herein, the method further includes isolating the polypeptide (e.g, through performance of one or more column chromatography steps, ultrafiltration / diafiltration, and / or viral inactivation). In some embodiments of any of the methods described herein, the method further includes formulating the isolated polypeptide into a composition (e.g, a pharmaceutical composition).

[0123] Any of the polypeptides described herein can be produced by any cell, (e.g, a mammalian cell). Non-limiting examples of a mammalian cell include: a human cell, a rodent cell (e.g, a rat cell or a mouse cell), a rabbit cell, a dog cell, a cat cell, a porcine cell, or a non-human primate cell. For example, a host cell can be a CHO cell, Expi293, or a HEK cell.

[0124] Methods of culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor cell proliferation, cell growth, and / or cell differentiation. For example, cells can be cultured by contacting a cell (e.g, any of the cells described herein) with a cell culture medium that includes supplemental growth factors to support cell viability and cell growth.

[0125] Methods of introducing nucleic acids (e.g. , any of the exemplary nucleic acids described herein) and / or expression vectors (e.g. , any of the exemplary expression vectors described herein (e.g, an AAV vector)) into cells (e.g, mammalian cells) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid (e.g. , DNA, RNA, ssRNA, siRNA, microRNA, or mRNA) and / or an expression vector (e.g, any of the exemplary expression vectors described herein (e.g, an AAV vector) include: electroporation, lipofection, transfection, microinjection, calcium phosphate transfection, dendrimer-based transfection, anionic polymer transfection, cationic polymer transfection, transfection using highly branched organic compounds, cell-squeezing, sonoporation, optical transfection, magnetofection, particle-based transfection (e.g, nanoparticle transfection), transfection using liposomes (e.g, cationic liposomes), and viraltransduction (e.g., lentiviral transduction, adenoviral transduction). In some embodiments, a nucleic acid encoding one or more components of the polypeptide described herein can be integrated into the genome of the host cell. In some embodiments, a nucleic acid encoding one or more components of the polypeptide described herein can be transiently expressed in the host cells.

[0126] Some methods described herein further include isolating or purifying the polypeptide from cell culture medium or from a cell (e.g., a mammalian cell) using techniques well-known in the art (e.g. , ion exchange chromatography (anionic or cation), metal-affinity chromatography, ligand- affmity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and precipitation (e.g. , ammonium sulfate precipitation, polyethylene glycol precipitation).Methods of Treatment

[0127] Provided herein are methods of treatment for a condition related to inadequate choriocapillaris and / or retinal capillary perfusion in the eye of a subject, the method comprising delivering an effective amount of any one of the compositions described herein to promote angiogenesis and / or preserve capillaries that supply the retinal with its oxygen and nutrients.

[0128] In some embodiments, delivering an effective amount of any one of the compositions described herein can increase retinal microvessel density. In embodiments, delivering an effective amount of any one of the compositions described herein can increase proliferation of choroidal endothelial cells. In embodiments, the delivering an effective amount of any one of the compositions described herein can stimulate angiogenesis. In some embodiments, delivering an effective amount of any one of the compositions described herein can preserve capillaries. In some embodiments, delivering an effective amount of any one of the compositions described herein can reduce the rate of capillary attrition, e.g., by aging or a disease process.

[0129] In some embodiments, the condition can be a retinovascular disease. In some embodiments, the condition can be a disease of the choriocapillaris. In some embodiments, the condition can be a disease of the retinal capillaries.

[0130] In some embodiments, the condition can be a disease of the choriocapillaris. In some embodiments, the condition can be dry macular degeneration with or without geographic atrophy (e.g. , geographic atrophy, complete or incomplete retinal pigment epithelium and outer retinal atrophy (cRORA and iRORA) and hypertransmission defects (hyperTD)); wet macular degeneration with or without exudation; age related macular degeneration; myopia, high myopia, pathologic myopia; hereditary choroidal dystrophies; polypoidal choroidal vasculopathy; pachychoroid spectrum disorder; or other ischemic diseases of the choroid.

[0131] In some embodiments, the condition can be a disease of the retinal capillaries. In some embodiments, the condition can be diabetic retinopathy, retinal vein occlusion, retinal artery occlusive disease, retinopathy of prematurity (ROP), sickle cell retinopathy, radiation retinopathy, familialexudative vitreoretinopathy, Coats’ disease, Eales’ disease, sarcoid, ocular ischemic syndrome, or other retinal capillary ischemic diseases. In some cases, the condition can be radiation retinopathy.

[0132] In some embodiments, the condition can be glaucoma.

[0133] In some embodiments, intraocular delivery can be via intravitreal injection. In some embodiments, delivery can be via suprachoroidal injection. In embodiments, delivering an effective amount of any one of the compositions described herein does not induce significant vascular leakage. In some embodiments, delivering an effective amount of any one of the compositions described herein reduces retinal ischemia. In embodiments, delivering an effective amount of any one of the compositions described herein does not induce edema.

[0134] In some embodiments, the provided herein is a method of inducing blood vessel formation and / or preserving existing capillaries in the eye of a subject comprising delivering to a subject in need thereof an effective amount of any one of the compositions described herein. In embodiments, the invention can provide that the administration increases retinal angiogenesis and / or preserves existing capillaries in the retina. In embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein can increase proliferation of choroidal endothelial cells.

[0135] In some embodiments, the subject can have retino vascular disease. In some embodiments, the subject can have a disease of the choriocapillaris. In some embodiments, the subject can have a disease of the retinal capillaries. In some embodiments, the subject can have age-related macular degeneration. In some embodiments, the subject can have glaucoma.

[0136] In some embodiments, the subject can have a disease of the choriocapillaris. In some embodiments, the subject can have dry macular degeneration with or without geographic atrophy (e.g. , geographic atrophy, complete or incomplete retinal pigment epithelium and outer retinal atropy (cRORA and iRORA) and hypertransmission defects (hyperTD)); wet macular degeneration with or without exudation; myopia, high myopia, pathologic myopia; hereditary choroidal dystrophies; polypoidal choroidal vasculopathy; pachychoroid spectrum disorder; radiation retinopahy; or other ischemic diseases of the choroid.

[0137] In some embodiments, the subject can have a disease of the retinal capillaries. In some embodiments, the subject has diabetic retinopathy, retinal vein occlusion, retinal artery occlusive disease, retinopathy of prematurity (ROP), sickle cell retinopathy, radiation retinopathy, familial exudative vitreoretinopathy, Coats’ disease, Eales’ disease, sarcoid, ocular ischemic syndrome, or other retinal capillary ischemic diseases.

[0138] In some embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein can be via intravitreal injection. In some embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein can be via suprachoroidal injection. In embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein does not induce exudation. In some embodiments, delivering to a subject in need thereof an effective amount of any one of thecompositions described herein does not induce vascular leakage. In embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein does not induce edema. In some embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein does not induce subretinal hemorrhage. In some embodiments, delivering to a subject in need thereof an effective amount of any one of the compositions described herein reduces vascular atrophy and / or scarring.Certain Definitions

[0139] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0140] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0141] The “percent sequence identity” between a reference amino acid sequence and a query amino sequence (i.e., the amino sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference amino acid sequence) is determined by optimally aligning the sequences using the Needleman- Wunsch alignment algorithm with a gap existence penalty of 11 and a gap extension penalty of 1 and comparing the sequences. The number of exact matches, divided by the total number of positions in the alignment (which corresponds with the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query amino acid sequence and the reference amino acid sequence (i.e., percent sequence identity = (# of exact matches / (total # of positions in alignment)* 100). An alignment using the Needleman-Wunsch alignment algorithm (with a gap existence penalty of 11 and a gap extension penalty of 1) can be generated using the “Global Align” BLAST program available at htt s: / / blast.ncbi.nlm.nih. ov / Blast.c i. The “percent sequence identity” between a reference nucleic acid sequence and a query nucleic acid sequence (i.e., the nucleic acid sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference nucleic acidsequence) is determined by optimally aligning the sequences using the Needleman-Wunsch alignment algorithm (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap extension penalty of 2) and comparing the aligned nucleic acids. The number of exact match-es divided by the total number of nucleotides in the alignment (which corresponds with the number of nucleotides in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query nucleic acid sequence and the reference nucleic acid sequence (i.e., percent sequence identity = (# of exact matches) / (total # of nucleotides in the alignment)* 100). An alignment using the Needleman- Wunsch alignment algorithm (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap ex-tension penalty of 2) can be generated using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0142] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0143] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0144] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0145] The term “in vivo” is used to describe an event that takes place in a subject’s body.

[0146] The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0147] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0148] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial ordesired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0149] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Exemplary Embodiments

[0150] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[0151] Embodiment 1. A chimeric polypeptide comprising a leukemia inhibitor factor (L1F) peptide coupled to an Fc region of an IgG molecule, wherein the L1F is coupled to the Fc region via a linker, wherein the linker comprises the amino acid sequence of SEQ ID NO: 5.

[0152] Embodiment 2. A chimeric polypeptide comprising a leukemia inhibitor factor (L1F) peptide coupled to an Fc region of an IgG molecule, wherein the L1F is coupled to the Fc region via a linker, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 2.

[0153] Embodiment 3. The chimeric polypeptide of embodiment 1, wherein the Fc region comprises the amino acid sequence SEQ ID NO: 2.

[0154] Embodiment 4. The chimeric polypeptide of any one of embodiments 1-3, wherein the chimeric polypeptide comprises, from N-terminus to C-terminus: L1F, the linker, and the Fc region.

[0155] Embodiment 5. The chimeric polypeptide of any one of embodiments 1-4, wherein the chimeric polypeptide comprises SEQ ID NO: 9.

[0156] Embodiment 6. A composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the chimeric polypeptide of any one of embodiments 1-5.

[0157] Embodiment 7. The composition of embodiment 6, wherein the first polypeptide and the second polypeptide form a dimer through a disulfide linkage.

[0158] Embodiment 8. The composition of embodiment 7, wherein the second polypeptide is a chimeric polypeptide comprising a L1F peptide coupled to an Fc region of an IgG molecule.

[0159] Embodiment 9. The composition of embodiment 8, wherein the L1F peptide is coupled to the Fc region via a linker.

[0160] Embodiment 10. The composition of embodiment 8 or 9, wherein the linker comprises the amino acid sequence of SEQ ID NO: 5.

[0161] Embodiment 11. The composition of any one of embodiments 8-10, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 2.

[0162] Embodiment 12. The composition of any one of embodiments 8-11, wherein the second polypeptide comprises, from N -terminus to C-terminus: L1F, the linker, and the Fc region.

[0163] Embodiment 13. The composition of any one of embodiments 8-12, wherein the second polypeptide comprises SEQ ID NO: 9.

[0164] Embodiment 14. The composition of any one of embodiments 6-13, wherein the first polypeptide and the second polypeptide are identical in amino acid sequence.

[0165] Embodiment 15. The composition of any one of embodiments 6-14, wherein the composition has a melting temperature of 65 to 75°C.

[0166] Embodiment 16. The composition of any one of embodiments 6-15, wherein the composition has a melting temperature of 69 to 71 °C.

[0167] Embodiment 17. The composition of any one of embodiments 6-16, wherein the composition has a hydrophobic interaction chromatography retention time of about 15 minutes.

[0168] Embodiment 18. The composition of any one of embodiments 6-17, wherein the composition has a self-interaction score of about 0.0.

[0169] Embodiment 19. The composition of any one of embodiments 6-18, wherein the composition is stable between 4 and 40°C for at least 14 days.

[0170] Embodiment 20. The composition of any one of embodiments 6-19, wherein the composition has a baculo virus particle assay score of about 5.

[0171] Embodiment 21. The composition of any one of embodiments 6-20, wherein the composition inhibits growth of bovine aortic endothelial cells.

[0172] Embodiment 22. The composition of any one of embodiments 6-21, wherein the composition promotes growth of bovine choroidal endothelial cells.

[0173] Embodiment 23. The composition of any one of embodiments 6-22, wherein the composition preserves choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model.

[0174] Embodiment 24. The composition of any one of embodiments 6-23, wherein the composition promotes growth of retinal capillary endothelial cells.

[0175] Embodiment 25. A composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a chimeric polypeptide comprising a L1F peptide coupled to an Fc region of an IgG molecule, wherein the Fc region comprises a knob into hole amino acid substitution.

[0176] Embodiment 26. The composition of embodiment 25, wherein the second polypeptide comprises an Fc region of an IgG molecule, wherein the Fc region comprises a knob into hole amino acid substitution.

[0177] Embodiment 27. The composition of embodiment 26, wherein the Fc region is coupled to a L1F peptide.

[0178] Embodiment 28. The composition of any one of embodiments 25-27, wherein the Fc region of the first polypeptide comprises a knob substitution and the Fc region of the second polypeptide comprises a hole substitution.

[0179] Embodiment 29. The composition of any one of embodiments 25-28, wherein the Fc region of the first polypeptide comprises a hole substitution and the Fc region of the second polypeptide comprises a knob substitution.

[0180] Embodiment 30. The composition of any one of embodiments 25-29, wherein the hole substitution is selected from one of more or Y407T, Y407V, T366S, L368A, T394W, and F405A.

[0181] Embodiment 31. The composition of any one of embodiments 25-30, wherein the knob substitution is selected from T366W and T366Y.

[0182] Embodiment 32. The composition of any one of embodiments 25-31, wherein the Fc region of the first polypeptide comprises the amino acid sequence of SEQ ID NO: 21.

[0183] Embodiment 33. The composition of any one of embodiments 25-32, wherein the first polypeptide comprises, from N -terminus to C-terminus: L1F, optionally a linker, and the first Fc region.

[0184] Embodiment 34. The composition of any one of embodiments 25-33, wherein the first polypeptide comprises SEQ ID NO: 11.

[0185] Embodiment 35. The composition of any one of embodiments 25-34, wherein the second polypeptide comprises the amino acid sequence SEQ ID NO 10.

[0186] Embodiment 36. The composition of any one of embodiments 25-35, wherein the composition has a melting temperature of about 60 to about 70°C.

[0187] Embodiment 37. The composition of any one of embodiments 25-36, wherein the composition has a melting temperature of about 65°C.

[0188] Embodiment 38. The composition of any one of embodiments 25-37, wherein the hydrophobic interaction chromatography retention time of the composition is about 15 minutes.

[0189] Embodiment 39. The composition of any one of embodiments 25-38, wherein the composition has a self-interaction score of about 0.4.

[0190] Embodiment 40. The composition of any one of embodiments 25-39, wherein the composition is stable between 4 and 40°C for at least 14 days.

[0191] Embodiment 41. The composition of any one of embodiments 25-40, wherein the composition has a baculovirus particle assay score of about 4.

[0192] Embodiment 42. The composition of any one of embodiments 25-40, wherein the composition has a half-life of about 2.6 days.

[0193] Embodiment 43. The composition of any one of embodiments 25-41, wherein the composition inhibits growth of bovine aortic endothelial cells.

[0194] Embodiment 44. The composition of any one of embodiments 25-43, wherein the composition promotes growth of bovine choroidal endothelial cells.

[0195] Embodiment 45. The composition of any one of embodiments 25-44, wherein the composition preserves choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model.

[0196] Embodiment 46. The composition of any one of embodiments 25-45, wherein the composition promotes growth of retinal capillary endothelial cells.

[0197] Embodiment 47. A chimeric polypeptide comprising an Fc region of an IgG, coupled to a LIF peptide, and an XTEN moiety.

[0198] Embodiment 48. The chimeric polypeptide of embodiment 47, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 3.

[0199] Embodiment 49. The chimeric polypeptide of embodiment 47 or 48, wherein the XTEN moiety is characterized in that:(a) it comprises at least 40 contiguous amino acids;(b) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the XTEN moiety constitutes more than 80% of the total amino acids of the XTEN moiety, and the remainder, when present, consists of arginine or lysine, and the remainder does not contain methionine, cysteine, asparagine, and glutamine;(c) the XTEN moiety comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein one of said at least three different types is glycine (G) or glutamine (E); and(d) at least 50% of the at least 40 contiguous amino acids in the XTEN moiety sequence are devoid of secondary structure as determined by Chou -Fasman algorithm.

[0200] Embodiment 50. The chimeric polypeptide of any one of embodiments 47-49, wherein the XTEN moiety comprises a motif selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0201] Embodiment 51. The chimeric polypeptide of any one of embodiments 47-49, wherein the XTEN moiety comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4.

[0202] Embodiment 52. The chimeric polypeptide of any one of embodiments 47-50, wherein the XTEN moiety comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4.

[0203] Embodiment 53. The chimeric polypeptide of any one of embodiments 47-52, wherein the XTEN moiety comprises an amino acid sequence that is identical to SEQ ID NO: 4.

[0204] Embodiment 54. The chimeric polypeptide of any one of embodiments 47-53, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: LIF, the Fc region, and the XTEN moiety.

[0205] Embodiment 55. The chimeric polypeptide of embodiment 54, wherein the chimeric polypeptide further comprises a linker between the LIF and the Fc region.

[0206] Embodiment 56. The chimeric polypeptide of embodiment 54 or 55, wherein the chimeric polypeptide further comprises a linker between the Fc region and the XTEN moiety.

[0207] Embodiment 57. The chimeric polypeptide of any one of embodiments 54-56, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: LIF, a first linker, the Fc region, a second linker, and the XTEN moiety.

[0208] Embodiment 58. The chimeric polypeptide of any one of embodiments 54-57, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 5.

[0209] Embodiment 59. The chimeric polypeptide of any one of embodiments 54-57, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0210] Embodiment 60. The chimeric polypeptide of any one of embodiments 54-59, wherein the first linker comprises an amino acid sequence according to SEQ ID NO: 5 and the second linker comprises an amino acid sequence according to SEQ ID NO: 7

[0211] Embodiment 61. The chimeric polypeptide of any one of embodiments 47-60, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 13.

[0212] Embodiment 62. A composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the chimeric polypeptide of any one of embodiments 47-61.

[0213] Embodiment 63. The composition of embodiment 62, wherein the first polypeptide and the second polypeptide form a dimer through a disulfide linkage.

[0214] Embodiment 64. The composition of embodiment 62 or 63, wherein the second polypeptide is a chimeric polypeptide comprising an Fc region of an IgG molecule coupled to a LIF peptide and an XTEN moiety.

[0215] Embodiment 65. The composition of embodiment 64, wherein the XTEN moiety of the second polypeptide is characterized in that:(a) it comprises at least 40 contiguous amino acids;(b) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the XTEN moiety constitutes more than 80% of the total amino acids of the XTEN moiety, and the remainder, when present, consists of arginine or lysine, and the remainder does not contain methionine, cysteine, asparagine, and glutamine;(c) the XTEN moiety comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P), wherein one of said at least three different types is glycine (G) or glutamine (E); and(d) at least 50% of the at least 40 contiguous amino acids in the XTEN moiety sequence are devoid of secondary structure as determined by Chou-Fasman algorithm.

[0216] Embodiment 66. The composition of embodiment 64 or 65, wherein the XTEN moiety of the second polypeptide comprises a motif selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0217] Embodiment 67. The composition of any one of embodiments 64-66, wherein the Fc region of the second polypeptide comprises the amino acid sequence SEQ ID NO: 4.

[0218] Embodiment 68. The composition of any one of embodiments 64-67, wherein the XTEN moiety of the second polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4.

[0219] Embodiment 69. The composition of any one of embodiments 64-68, wherein the XTEN moiety of the second polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4.

[0220] Embodiment 70. The composition of any one of embodiments 64-69, wherein the XTEN moiety of the second polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 4.

[0221] Embodiment 71. The composition of any one of embodiments 64-70, wherein the chimeric polypeptide of the second polypeptide comprises from N-terminus to C-terminus: LIF, the Fc region, and the XTEN moiety.

[0222] Embodiment 72. The composition of any one of embodiments 64-71, wherein the chimeric polypeptide of the second polypeptide comprises from N-terminus to C-terminus: LIF, a first linker, the Fc region, a second linker, and the XTEN moiety.

[0223] Embodiment 73. The composition of any one of embodiments 64-72, wherein the first linker or the second linker of the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 5.

[0224] Embodiment 74. The composition of any one of embodiments 64-72, wherein the first linker or the second linker of the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 7.

[0225] Embodiment 75. The composition of any one of embodiments 64-74, wherein the first linker of the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 5 and the second linker of the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 7.

[0226] Embodiment 76. The composition of any one of embodiments 64-75, wherein the chimeric polypeptide of the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 13.

[0227] Embodiment 77. The composition of any one of embodiments 62-76, wherein the first polypeptide and the second polypeptide are identical in amino acid sequence.

[0228] Embodiment 78. The composition of any one of embodiments 62-77, wherein the composition has a half-life of about 4.2 days.

[0229] Embodiment 79. The composition of any one of embodiments 62-78, wherein the composition inhibits growth of bovine aortic endothelial cells.

[0230] Embodiment 80. The composition of any one of embodiments 62-79, wherein the composition promotes growth of bovine choroidal endothelial cells.

[0231] Embodiment 81. The composition of any one of embodiments 62-80, wherein the composition preserves choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model.

[0232] Embodiment 82. The composition of any one of embodiments 62-81, wherein the composition promotes growth of retinal capillary endothelial cells.

[0233] Embodiment 83. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject promotes retinal angiogenesis.

[0234] Embodiment 84. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject increases proliferation of choroidal endothelial cells.

[0235] Embodiment 85. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject does not induce vascular leakage.

[0236] Embodiment 86. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.

[0237] Embodiment 87. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

[0238] Embodiment 88. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.

[0239] Embodiment 89. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

[0240] Embodiment 90. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

[0241] Embodiment 91. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 1.

[0242] Embodiment 92. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1.

[0243] Embodiment 93. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1.

[0244] Embodiment 94. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0245] Embodiment 95. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide comprises the amino acid sequence SEQ ID NO: 1.

[0246] Embodiment 96. The chimeric polypeptide or composition of any one of the preceding embodiments, wherein the LIF peptide consists of the amino acid sequence SEQ ID NO: 1.

[0247] Embodiment 97. A method of treatment for a condition related to inadequate vascularization in the eye of a subject, the method comprising delivering an effective amount of the composition of any one of embodiments 6-46 or 62-96 to the eye of the subject.

[0248] Embodiment 98. The method of embodiment 97, wherein the effective amount promotes angiogenesis.

[0249] Embodiment 99. The method of embodiment 97, wherein the effective amount increases retinal angiogenesis.

[0250] Embodiment 100. The method of any one of embodiments 97-99, wherein the effective amount increases retinal microvessel density.

[0251] Embodiment 101. The method of any one of embodiments 97-100, wherein the effective amount increases proliferation of choroidal endothelial cells.

[0252] Embodiment 102. The method of any one of embodiments 97-101, wherein the effective amount does not induce vascular leakage.

[0253] Embodiment 103. The method of any one of embodiments 97-102, wherein the effective amount does not induce edema.

[0254] Embodiment 104. The method of any one of embodiments 97-103, wherein the delivery is via intravitreal injection.

[0255] Embodiment 105. The method of any one of embodiments 97-103, wherein the delivery is via suprachoroidal injection.

[0256] Embodiment 106. The method of any one of embodiments 97-105, wherein the condition is a disease of the choriocapillaris.

[0257] Embodiment 107. The method of embodiment 106, wherein the disease is dry macular degeneration with or without geographic atrophy (e.g., geographic atrophy, complete or incomplete retinal pigment epithelium and outer retinal atropy (cRORA and iRORA) and hypertransmission defects (hyperTD)); wet macular degeneration with or without exudation; myopia, high myopia, pathologic myopia; hereditary choroidal dystrophies; polypoidal choroidal vasculopathy; or pachychoroid spectrum disorder.

[0258] Embodiment 108. The method of any one of embodiments 97-105, wherein the condition is a disease of the retinal capillaries.

[0259] Embodiment 109. The method of embodiment 108, wherein the disease is diabetic retinopathy, retinal vein occlusion, retinal artery occlusive disease, retinopathy of prematurity (ROP), sickle cell retinopathy, radiation retinopathy, familial exudative vitreoretinopathy, Coats’ disease, Eales’ disease, sarcoid, or ocular ischemic syndrome.

[0260] Embodiment 110. The method of any one of embodiments 97-105, wherein the condition is age-related macular degeneration.

[0261] Embodiment 111. The method of any one of embodiments 97-105, the condition is glaucoma.

[0262] Embodiment 112. A chimeric polypeptide comprising an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide.

[0263] Embodiment 113. The chimeric polypeptide of embodiment 112, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 3.

[0264] Embodiment 114. The chimeric polypeptide of embodiment 112 or 113, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide.

[0265] Embodiment 115. The chimeric polypeptide of any one of embodiment 112-114, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

[0266] Embodiment 116. The chimeric polypeptide of any one of embodiments 112-115, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

[0267] Embodiment 117. The chimeric polypeptide of any one of embodiments 112-116, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

[0268] Embodiment 118. The chimeric polypeptide of any one of embodiments 115-117, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0269] Embodiment 119. The chimeric polypeptide of any one of embodiments 115-118wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0270] Embodiment 120. The chimeric polypeptide of any one of embodiments 112-119, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 12.

[0271] Embodiment 121. The chimeric polypeptide of claim 112, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 2.

[0272] Embodiment 122. The chimeric polypeptide of embodiment 121, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide.

[0273] Embodiment 123. The chimeric polypeptide of embodiment 121 or 122, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

[0274] Embodiment 124. The chimeric polypeptide of any one of embodiments 121-123, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

[0275] Embodiment 125. The chimeric polypeptide of any one of embodiments 121-124, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

[0276] Embodiment 126. The chimeric polypeptide of embodiment 125, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0277] Embodiment 127. The chimeric polypeptide of embodiment 125, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0278] Embodiment 128. The chimeric polypeptide of embodiment 126 or 127 wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

[0279] Embodiment 129. The chimeric polypeptide of embodiment 125, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

[0280] Embodiment 130. The chimeric polypeptide of embodiment 125, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

[0281] Embodiment 131. The chimeric polypeptide of embodiment 129 or 130, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 24.

[0282] Embodiment 132. The chimeric polypeptide of embodiment 125, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

[0283] Embodiment 133. The chimeric polypeptide of embodiment 125, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

[0284] Embodiment 134. The chimeric polypeptide of embodiment 132 or 133, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

[0285] Embodiment 135. A composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the chimeric polypeptide of any one of embodiments 112- 134.

[0286] Embodiment 136. The composition of embodiment 135, wherein the first polypeptide and the second polypeptide form a dimer through a disulfide linkage.

[0287] Embodiment 137. The composition of embodiment 135 or 136, wherein the second polypeptide comprises an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide.

[0288] Embodiment 138. The composition of embodiment 137, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 3.

[0289] Embodiment 139. The composition of embodiment 137 or 138, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, the second LIF peptide.

[0290] Embodiment 140. The composition of any one of embodiments 137-139, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

[0291] Embodiment 141. The composition of any one of embodiments 137-140, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

[0292] Embodiment 142. The composition of any one of embodiments 137-141, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

[0293] Embodiment 143. The composition of any one of embodiments 137-142, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0294] Embodiment 144. The composition of any one of embodiments 137-143, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0295] Embodiment 145. The composition of any one of embodiments 135-144, wherein the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 12.

[0296] Embodiment 146. The composition of embodiment 137, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 2.

[0297] Embodiment 147. The composition of embodiment 146, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, the second LIF peptide.

[0298] Embodiment 148. The composition of embodiment 146 or 147, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

[0299] Embodiment 149. The composition of any one of embodiments 146-148, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

[0300] Embodiment 150. The composition of any one of embodiments 146-149, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

[0301] Embodiment 151. The composition of embodiment 150, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0302] Embodiment 152. The composition of any one of embodiments 150 or 151, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

[0303] Embodiment 153. The composition of embodiment 152, wherein the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

[0304] Embodiment 154. The composition of embodiment 150, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

[0305] Embodiment 155. The composition of embodiment 150, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

[0306] Embodiment 156. The composition of embodiment 155, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 24.

[0307] Embodiment 157. The composition of embodiment 150, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

[0308] Embodiment 158. The composition of embodiment 150, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

[0309] Embodiment 159. The composition of embodiment 158, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

[0310] Embodiment 160. The composition of any one of embodiments 135-159, wherein the first polypeptide comprises the chimeric polypeptide of any one of embodiments 112-134.

[0311] Embodiment 161. The composition of any one of embodiments 135-160, wherein the first polypeptide and the second polypeptide are identical in same amino acid sequence.

[0312] Embodiment 162. The composition of any one of embodiments 135-161, wherein the composition has a half-life of about 5.4 days.

[0313] Embodiment 163. The composition of any one of embodiments 135-162, wherein the composition inhibits growth of bovine aortic endothelial cells.

[0314] Embodiment 164. The composition of any one of embodiments 135-163, wherein the composition promotes growth of bovine choroidal endothelial cells.

[0315] Embodiment 165. The composition of any one of embodiments 135-164, wherein the composition preserves choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model.

[0316] Embodiment 166. The composition of any one of embodiments 135-165, wherein the composition promotes growth of retinal capillary endothelial cells.

[0317] Embodiment 167. The chimeric polypeptide or composition of any one of embodiments 112-166, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject promotes retinal angiogenesis.

[0318] Embodiment 168. The chimeric polypeptide or composition of any one of embodiments 112-167, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject increases proliferation of choroidal endothelial cells.

[0319] Embodiment 169. The chimeric polypeptide or composition of any one of embodiments 112-168, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject does not induce vascular leakage.

[0320] Embodiment 170. The chimeric polypeptide or composition of any one of embodiments 112-169, wherein the LIF peptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1

[0321] Embodiment 171. The chimeric polypeptide or composition of any one of embodiments 112-170, wherein the LIF peptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

[0322] Embodiment 172. The chimeric polypeptide or composition of any one of embodiments 112-171, wherein the LIF peptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.

[0323] Embodiment 173. The chimeric polypeptide or composition of any one of embodiments 112-172, wherein the LIF peptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

[0324] Embodiment 174. The chimeric polypeptide or composition of any one of embodiments 112-173, wherein the LIF peptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

[0325] Embodiment 175. The chimeric polypeptide or composition of any one of embodiments 112-174, wherein the LIF peptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 1.

[0326] Embodiment 176. The chimeric polypeptide or composition of any one of embodiments 112-175, wherein the LIF peptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1.

[0327] Embodiment 177. The chimeric polypeptide or composition of any one of embodiments 112-176, wherein the LIF peptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:

[0328] Embodiment 178. The chimeric polypeptide or composition of any one of embodiments 112-177, wherein the LIF peptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0329] Embodiment 179. The chimeric polypeptide or composition of any one of embodiments 112-178, wherein the LIF peptide comprises the amino acid sequence SEQ ID NO: 1.

[0330] Embodiment 180. The chimeric polypeptide or composition of any one of embodiments 112-179, wherein the LIF peptide consists of the amino acid sequence SEQ ID NO: 1.

[0331] Embodiment 181. A method of treatment for a condition related to inadequate vascularization in the eye of a subject, the method comprising delivering an effective amount of the composition of any one of embodiments 135-180 to the eye of the subject.

[0332] Embodiment 182. The method of embodiment 181, wherein the effective amount promotes angiogenesis.

[0333] Embodiment 183. The method of embodiment 181, wherein the effective amount increases retinal angiogenesis.

[0334] Embodiment 184. The method of any one of embodiments 181-183, wherein the effective amount increases retinal microvessel density.

[0335] Embodiment 185. The method of any one of embodiments 181-184, wherein the effective amount increases proliferation of choroidal endothelial cells.

[0336] Embodiment 186. The method of any one of embodiments 181-185, wherein the effective amount does not induce vascular leakage.

[0337] Embodiment 187. The method of any one of embodiments 181-186, wherein the effective amount does not induce edema.

[0338] Embodiment 188. The method of any one of embodiments 181-187, wherein the delivery is via intravitreal injection.

[0339] Embodiment 189. The method of any one of embodiments 181-187, wherein the delivery is via suprachoroidal injection.

[0340] Embodiment 190. The method of any one of embodiments 181-189, wherein the condition is a disease of the choriocapillaris.

[0341] Embodiment 191. The method of embodiment 190, wherein the disease is dry macular degeneration with or without geographic atrophy (e.g., geographic atrophy, complete or incomplete retinal pigment epithelium and outer retinal atropy (cRORA and iRORA) and hypertransmission defects (hyperTD)); wet macular degeneration with or without exudation; myopia, high myopia, pathologic myopia; hereditary choroidal dystrophies; polypoidal choroidal vasculopathy; or pachychoroid spectrum disorder.

[0342] Embodiment 192. The method of any one of embodiments 181-189, wherein the condition is a disease of the retinal capillaries.

[0343] Embodiment 193. The method of embodiment 192, wherein the disease is diabetic retinopathy, retinal vein occlusion, retinal artery occlusive disease, retinopathy of prematurity (ROP), sickle cell retinopathy, radiation retinopathy, familial exudative vitreoretinopathy, Coats’ disease, Eales’ disease, sarcoid, or ocular ischemic syndrome.

[0344] Embodiment 194. The method of any one of embodiments 181-189, wherein the condition is age-related macular degeneration.

[0345] Embodiment 195. The method of any one of embodiments 181-189, the condition is glaucoma.EXAMPLES

[0346] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.Example 1: Activation of BAE Cells by LIF-Fc and LIF-KiH constructs

[0347] LIF-Fc (a dimer of SEQ ID NO: 9) and LIF-KiH (knob in hole; a dimer of SEQ ID NO: 10 and SEQ ID NO: 11) were tested for their ability to elicit Stat3 signaling in BAE cells. LIF-Fc had an EC50 of 64 pM and LIF-KiH of 29 pM in that assay (FIGs. 1A-1C). These constructs were —5-10- fold more potent in eliciting Stat3 signaling in BAE cells compared to HSA-LIF fusions. LIF-Fc and LIF-KiH were also able to inhibit BAE proliferation. LIF-Fc had an IC50 of 4.4 pM, and LIF-KiH of 32 pM (FIGs. 2A-2C). Combined, these data demonstrate LIF-Fc and LIF-KiH bind to LIFR and elicit LIFR signaling.Example 2: Developmentability of LIF-Fc and LIF-KiH constructs

[0348] LIF constructs were subjected to a series of developability assessments to identify any potential risks. LIF-Fc and LIF-KiH constructs were formulated to a high concentration (10 mg / mL) in PBS and assessed for purity by SDS-PAGE and SEC-HPLC. All candidates had >90% purity by SDS-PAGE. By SEC-HPLC, LIF-Fc had >90% purity, while LIF-KiH had 85% purity. Neither construct changed in purity upon formulation to 10 mg / mL. Combined, these data suggest LIF-Fc and LIF-KiH can be formulated to a high concentration.

[0349] Next, the melting temperature (TM) of all candidates was assessed by dynamic scanning fluorimetry. Generally, a TM of >57°C is considered acceptable from a developability standpoint. LIF-Fc had a TM of 67.0°C and LIF-KiH of 65.0°C. As a control, lysozyme was found to have a TM of 68.4°C (FIG. 3).

[0350] The propensity of LIF-Fc and LIF-KiH to interact nonspecifically with a hydrophobic stationary phase via hydrophobic interaction chromatography (HIC)-HPLC was assessed, where a longer retention time indicates more interaction with the stationary phase and thus nonspecificity. Efalizumab, an antibody control with good performance in this assay, had a retention time of 16.3 minutes, while bavituxumab, an antibody control with poor performance in this assay, had a retention time of 25.2 minutes. LIF-Fc and LIF-KiH had retention times of 15.6 and 15 minutes, respectively, better than the good antibody control (FIGs. 4A-4D). These data demonstrate LIF-Fc and LIF-KiH have low poly specificity due to hydrophobicity.

[0351] To determine the propensity for LIF-Fc and LIF-KiH to self-interact and thus aggregate, selfassociation was monitored by BLI. Briefly, a candidate is immobilized on the surface of a BLI and dipped into a solution of itself. Any binding is detected by the sensor and converted into a selfinteraction score. Blosozumab was used as a poor antibody control and had a self-interaction score of 0.85, while eculizumab was used as a good antibody control and had a self-interaction score of 0.35. LIF-Fc and LIF-KiH had self-interactions scores of 0. 17 and 0.4, respectively, suggesting a low likelihood of self-interaction (FIG. 5).

[0352] As another measure of polyspecificity, LIF-Fc and LIF-KiH were tested for binding to baculovirus particles (BVP) by ELISA. For the Fc-containing candidates, an anti-IgG HRP was used for detection. Alternatively, for non-Fc containing candidates, candidates and control antibodies werebiotinylated and detected with streptavidin-HPR. For controls, pantilumumab and efalizumab are known antibodies with good performance while dupilumab and blosozumab are antibodies that are known to perform poorly. In the anti-IgG BVP ELISA, pantilumumab and efalizumab had BVP scores of 1.42 and 3.91, respectively, while dupilumab and blosozumab had scores of 10.44 and 17.46, respectively. LIF-Fc and LIF-KiH had BVP sores of 2.92, and 3.95, respectively (FIG. 6). These values are similar to the performance of the good antibody controls, suggesting that the Fc- fusion candidates have low polyspecificity. In the streptavidin-HRP BVP ELISA, pantilumumab and efalizumab had BVP scores of 3.80 and 4.40, respectively, while duplimumab and blosozumab had BVP scores of 18.00 and 16.80, respectively.

[0353] Finally, LIF-Fc and LIF-KiH were subjected to a two-week 4°C and 40°C stability study to determine if the compositions lost activity or purity over time (FIGs. 7A-7U). LIF-Fc and LIF-KiH were formulated at 10 mg / mL in PBS and samples were collected and analyzed at day 0, day 7, and day 14. By SEC-HPLC, LIF-Fc and LIF-KiH had similar levels of purity after 14 days at both 4°C and 40°C (FIGs. 7A-7J) and did not lose affinity after 14 days at 4°C and 40°C (FIGs. 7K-7P). Similarly, there was no change in EC50 in the Stat3 BAE signaling assay for either LIF-Fc and LIF- KiH after 14 days and 4°C and 40°C (FIG. 7Q and FIG. 7R). Combined, these data demonstrate no issues with thermal stability under formulation at 10 mg / mL in PBS.Example 3: Assessment of 4 x LIF-Fc and XTEN-domain fused LIF-Fc constructs

[0354] As a strategy to increase the half-life of LIF in the vitreous, 4 x LIF-Fc (4xLIF-Fc; a dimer of SEQ ID NO: 12) and XTEN-domain fused LIF-Fc (LIF-XTEN; a dimer of SEQ ID NO: 13) constructs were designed. With 4 x LIF-Fc, the large size and heavy glycosylation of LIF is expected to lead to slow diffusion out of the vitreous. Alternatively, XTEN is a large, globular domain that has similar properties to PEG. 4xLIF-Fc and LIF-XTEN were expressed via transient transfection in Expi293. Constructs had good purity (>90% purity) by SDS-PAGE and SEC-HPLC. In the Stat3 phosphorylation assay, 4xLIF-Fc had an EC50 of 0.00048 nM compared to LIF-KiH (EC50 = 0.33 nM), suggesting a significant increase in potency (FIGs. 8A-8E). Even upon normalization to moles of LIF, 4xLIF-Fc had an EC50 of 0.039 nM, a 10-fold increase compared to LIF-KiH on a per-mole basis. LIF-XTEN had an EC50 of 3.58 nM — a 10-fold loss in activity compared to LIF-KiH which could be due to steric inhibition of the XTEN fusion. In the inhibition of proliferation of BAE cell assay, 4xLIF-Fc had an IC50 of 0.04 nM and LIF-XTEN had an IC50 of 0.04 nM, similar to LIF-KiH with an IC50 of 0.03 nM (FIGs. 9A-9D). This data demonstrates that 4xLIF-Fc and LIF-XTEN express well and have good stability and activity profiles.

[0355] LIF stimulates growth of bovine choroidal endothelial cells (BCECs) and inhibits growth of bovine aortic endothelial cells (BAECs). 4xLIF-Fc and LIF-XTEN were assessed for their abilities to stimulate BCEC growth and inhibit BAEC growth. As shown in FIG. 9E (top), both constructsinhibited BAEC growth, with 4xLIF-Fc inhibiting growth even at the lowest concentration tested.Similarly, both constructs effectively stimulated growth of BCEC (FIG. 9F, bottom).Example 4: Sodium Iodate Model

[0356] The NalOs mouse model has been widely used as a pre-clinical model of atrophic AMD. In this model, the RPE layer, photoreceptors, and choriocapillaris are damaged. LIF-KiH, 4xLIF-Fc, and LIF-XTEN were tested fortheir ability to promote choroid capillary recovery in this model.

[0357] After intravenous injection of NalOs, LIF, LIF-KiH, 4xLIF-Fc, or LIF-XTEN, or PBS were injected intravitreally. As shown in FIGs. 10B-10D, retinal thickness is significantly thicker in animals injected with LIF-KIH than those injected with PBS, and comparable to animals injected with LIF (FIG. 10A). Similarly, retinas were thicker in animals administered 4xLIF-Fc after NalOs than those receiving PBS (FIGs 10E-10I). Retinas in animals receiving the lowest dose of LIF-XTEN also had thicker retinas than those receiving PBS (FIGs. 10J-10L).Example 5: Pharmacokinetic Assessment

[0358] A pharmacokinetic study was performed in Zealand white rabbits for LIF-KiH, 4xLIF-Fc, and LIF-XTEN to determine the half-life of the constructs. LIF fused to a His tag was used as a control. An overview of the study is shown in Table 6.Table 6: PK study overview

[0359] Test article concentration in the vitreous was quantified using an anti-human LIF ELISA.Validated standard curves (12 point, 1:2 serial dilutions) were run alongside each corresponding test article in duplicate. High and low QC samples prepared in vitreous were run alongside each corresponding test article in duplicate. Data is shown in FIG. 11A-11D. Half-life was then determined by log transforming the data and performing a simple linear regression.

[0360] As shown in FIGs. 11A-11D, LIF-KiH had a half life of 2.6 days, 4xLIF-Fc had a half-life of 5.4 days, and LIF-XTEN had a half-life of 4.2 days. By comparison, His-tagged LIF had a half-life of 2.6 days.Example 6: 4xLIF-Fc (a dimer of SEQ ID NO: 12) Profile

[0361] The purity of the 4xLIF-Fc was assessed by size -exclusion chromatography-high performance liquid chromatography (SEC-HPLC). SEC-HPLC was performed on elution fractions from the ProA purification. As shown in FIG. 12, size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) analysis showed that the early fractions were enriched for main peak (95%), while the later fractions were predominantly high molecular weight (HMW) species (5%). The main peak had a molecular weight of 147.9 kDa, which was expected. On the other hand, the high molecular weight species was at 307.4 kDa. This suggested that the 4xLIF-Fc existed as a mixture of monomer (main peak) and dimer (HMW), which further confirms that the main species in solution was likely a monomer.

[0362] Next, the melting temperature (Tm) and the aggregation temperature (Tagg) of 4xLIF-Fc was observed. A Tm for 4xLIF-Fc was about 59 °C, but it did not have a measurable Tagg. In hydrophobic interaction chromatography (HIC), the 4xLIF-Fc had a retention time of 11.9 minutes. This confirmed that the 4xLIF-Fc was less hydrophobic than the other benchmark controls. 4xLIF-Fc also showed no self-interaction.

[0363] A Baculovirus Particle (BVP) ELISA assay was conducted to assess propensity for nonspecific binding (e.g., polyspecificity). The 4xLIF-Fc had an elevated BVP score of 30.24 (similar to that of other commercial molecules), but this BVP score appears to be inherent to LIF itself.Example 7: Assessment of 4xLIF-Fc stability

[0364] 4xLIF-Fc molecule was also subjected to a stability study. Briefly, 4xLIF-Fc was formulated to 10, 20 and 50 mg / mL in 50 mM phosphate, 5.8% sucrose, 0.03% P20, pH 7.2 and subjected to 40 °C for 2 weeks. Samples were collected at days 0, 1, 3, 7, and 14 days.

[0365] Stability was evaluated by taking aliquots and analysing for concentration by UV absorbance at 280 nm (A280), volume recovery, and total mass balance. The A280 readings remained consistent across all time points, indicating no significant change in protein concentration over time. Volume measurements showed a slight decrease in the Day 14 sample, attributed to minor evaporation during storage. Mass balance calculations confirmed that the overall protein content remained stable throughout the study period. This indicates that the 4xLIF-Fc formulation maintained its concentration and integrity over the 14-day period.

[0366] SDS-PAGE and SEC-HPLC showed no significant increase in high molecular weight (HMW) species over time, suggesting dimer formulation may occur during the protein production stage, but not during the storage condition (e.g., 40 °C for 2 weeks). As shown in FIG. 13, low molecularweight (LMW) was increased over time: +1.5% for 10 mg / mL, +1.5% for 20 mg / mL, and +1.4% for 50 mg / mL. This showed that starting concentration did not affect degree degradation, only driven by time at 40 °C storage conditions. HMW was increased about -0.1% for 10 mg / mL, +0.5% for 20 mg / mL, and+1.8% for 50 mg / mL. This suggested that the higher starting concentration results in higher degree of aggregation. The main peak purities modestly decreased for all sample sets over time at 40 °C: -1.4% for 10 mg / mL, -2.0 % for 20 mg / mL, and -3.2% for 50 mg / mL. Overall, these results indicate minimal degradation and / or aggregation of the 4xLIF-Fc formulation, and that the formulation remains chemically and physically stable under the conditions for at least 14 days.

[0367] Liquid chromatography-mass spectrometry (LS-MS) analysis was conducted to assess purity and heterogeneity of the 4xLIF-Fc formulation. A cleavage product was observed to form over time, albeit at low amounts (< 2% after 14 days). Specifically, the main peak was observed at about 6.4 min, which corresponds to the expected mass for the 4xLIF-Fc, and the peak at about 6.2 min was observed, which corresponds to a truncation impurity. The impurity seemed to become more abundant in the samples kept at 40 °C for 14 days regardless of concentration as estimated using the UV-HPLC chromatograms. MS analysis further revealed that the cleavage occurs within the CH2 domain of the Fc via an acid labile D-P linkage. The cleavage could be due to contaminating proteases, which could be removed via additional purification, or this process could be controlled via formulating at a lower pH.

[0368] Despite the observed cleavage, 4xLIF-Fc showed no loss of activity as measured by the BAE pStat3 inhibition assay. As shown in FIG. 14, ELISA assay for pStat3 measurement showed similar trend shape across all concentrations (10 mg / mL, 20 mg / mL and 50 mg / mL) at Day 0 and Day 14. As shown in FIGs. 15A-15F, EC50 of the Stat3 BAE signaling assay showed consistency among different time points & concentrations (Table 7).Table 7. EC50 of 4xLIF-Fc

[0369] To further confirm the activity, 4xLIF-Fc binding to LIFR was assessed using bio-layer interferometry (BLI) assay. The study was conducted at 3 different concentrations of 4xLIF-Fc (10, 20 and 50 mg / mL) and at 2 different time points (0 and 14 day).Table 8. Summary of 4xLIF-Fc Binding to LIFR by BLI Assay| Storage | Binding | koff(l / s) | kon(l / Ms) Rmax |

[0370] As shown in Table 8, the dissociation was too slow to measure koff and thus KD, but Rmax and kon were consistent across different samples, suggesting no change in affinity.

[0371] Overall, the data suggests that 4xLIF-Fc shows an acceptable developability profile.Example 8: 4XLIF-Fc Variants & Improved Characteristics

[0372] 4xLIF-Fc variants (e.g., 4xLIF-Fc Variants 1-3) were designed to increase Tm. Since 4xLIF- Fc was previously designed to lack Fc glycosylation as a strategy to minimize effector function, this may have led to the construct having a lower TM. To that end, 4xLIF-Fc Variant 1 (SEQ ID NO: 23), which is the same as 4xLIF-Fc except it has native Fc glycosylation intact, 4xLIF-Fc Variant 2 (SEQ ID NO: 24), which has a shorter (G4S)2 linker (SEQ ID NO: 26) between LIF peptides and the Fc compared to 4xLIF-Fc Variant 1 (G4S)4 linker (SEQ ID NO: 7), and 4xLIF-Fc Variant 3 (SEQ ID NO: 25), which contains an IgGl hinge as a linker between the Fc and LIF, were designed (Table 9). All constructs were expressed transiently in Expi293 with similar expression levels to 4xLIF-Fc. As measured using SEC-HPLC, a dimer was present in all constructs at similar levels to 4xLIF-Fc, except 4xLIF-Fc Variant 3 which was unable to be purified at >90% purity.Table 9. 4xLIF-Fc and 4xLIF-Fc Variants Description & Melting Temperature

[0373] As shown in FIGs. 16A-16D, the 4xLIF-Fc variants were subjected to developability assessment. 4xLIF-Fc Variant 1 had a Tm of 69.5 °C. 4xLIF-Fc Variant 2 (had a Tm of 70.3 °C.4xLIF-Fc Variant 3 had a Tm of 68.5 °C. All constructs had about 10 °C increase in Tm compared to 4xLIF-Fc. No aggregation was observed for any constructs.

[0374] To measure polyspecificity, 4xLIF-Fc Variants were tested for binding to baculovirus particles (BVP) by ELISA. 4xLIF-Fc Variants had slightly elevated BVP scores (5 < 4xLIF-Fc Variants BVP score < 15). Specifically, 4xLIF-Fc Variant 1 and 4xLIF-Fc Variant 2 were able to bind to BVP particles to a similar level as 4xLIF-Fc, while 4xLIF-Fc Variant 3 had a higher BVP score. All constructs had similar retention times by HIC as 4xLIF-Fc (about 12.5 min). No construct had measurable self-interaction by BLI. Combined, these data suggest that the new variants (e.g., 4xLIF- Fc Variants 1-3) have an improved developability profile compared to 4xLIF-Fc. However, given that 4xLIF-Fc Variant 3 had higher dimer formation (as observed using SEC-HPLC analysis), only 4xLIF- Fc Variant 1 and 4xLIF-Fc Variant 2 were used for subsequent analysis.

[0375] 4xLIF-Fc Variants’ pStat3 activation was assessed. As shown in FIGs. 17A-17D, similar to 4xLIF-Fc, 4xLIF-Fc Variant 1 and 4xLIF-Fc Variant 2 had improved activity relative to native LIF in the BAE pStat3 activity assay. 4xLIF-Fc Variant 1 had an EC50 of 0.0086 nM. 4xLIF-Fc Variant 2 had an EC50 of 0.0080 nM. Given that the native LIF had an EC50 of 0.026 nM, the 4xLIF-Fc Variants demonstrated enhanced potency relative to the control (corresponds to approximately 3.1- fold and 3.3 -fold improvement in potency, respectively).Example 9: In vivo half-life assessment of 4XLIF-Fc & Variants

[0376] The intravitreal half-lives of 4xLIF-Fc and variants were assessed by carrying out a 7-day intravitreal PK study in New Zealand White rabbits. All test articles were dosed at 400 ug per eye. Tissues were collected at days 1, 3, and 7, and analyzed for test article concentration via ELISA.

[0377] In the vitreous, the commercial product had an average half-life of 5.37 days, 4xLIF-Fc of 4.92 days, 4xLIF-Fc Variant 1 (SEQ ID NO: 23) of 5.60 days, and 4xLIF-Fc Variant 2 (SEQ ID NO: 24) of 5.00 days (FIGs. 18A-18H). Thus, all test articles had a half-life of roughly 5 days in the vitreous. Aqueous humor (FIGs. 19A-19H), retina (FIGs. 21A-21H), choroid (FIGs. 20A-20H), and plasma (FIGs. 22A-22C) samples were also analyzed. In general, the commercial product reached maximal concentration in the aqueous humor, retina, and choroid at day 1, while 4xLIF-Fc, 4xLIF-Fc Variant 1, and 4xLIF-Fc Variant 2 reached maximal concentration at day 3. Very low levels of test articles were observed in the plasma, as expected.MethodsSEC-HPLC

[0378] SEC-HPLC analysis was performed on a TOSOH TSKgel Super SW mAb column (TOSOH Cat# 0022854). The column was equilibrated in 100 mM sodium phosphate, 150 mM NaCl, 0.5mM EDTA pH 7.2 at 0.5mL / min flow rate. Isocratic elution over 45 minutes at 0.5mL / min was performed on an Agilent series 1200 system. Detection was monitored at 280 run, 16 run bandwidth andreference at 800 nm with a 100 nm bandwidth. Sample (10 - 20 pg) were injected with up to 100 pL volume. Quantitation was achieved using a BSA standard curve with injection amounts from 2 pg - 32 pg. BSA peaks were integrated to obtain a standard curve. A molecular weight standard curve was also generated using gel fdtration chromatography standards. Target protein samples were integrated, and molecular weights were calculated using the BSA standard curve with a correction for extinction coefficient.

[0379] SEC-MALS was performed on Wyatt OptiLab RID with miniDawn MALS using similar methods and columns as outlined above.Surface plasmon resonance (SPR) measurement of affinity of LIF constructs to LIFR and gpl 30

[0380] The affinities of LIF variants towards LIFR and gpl30 were determined using Surface Plasmon Resonance (SPR) on a Biacore 3000 (Cytiva) equipped with a CM5 sensor chip (Cytiva cat.# BR100399) at 25°C and a flow rate of 30 pL min-1. The running buffer contained 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% p20, 3 mM EDTA, 1.0 mg / mL BSA IgG-Free and was adjusted to a final pH of 7.4, then sterile filtered and degassed by sonication under reduced pressure.Recombinant human LIFR alpha and gpl30 from R&D Systems (cat. # 7487-LR-050, and 228-GP- 050 / CF respectively) were immobilized on the SPR chip. The different LIF constructs expressed and purified in-house were tested for binding while in solution at different concentrations. During the experiments, a 6 min association was conducted with a dissociation phase of 30 min. No regeneration was required. The response was double-referenced by subtracting the responses from a reference flow cell (without protein immobilized), and a blank injection over the same flow cell. Equilibrium and kinetic parameters were determined by fitting the response to a 1 : 1 Langmuir binding model using Scrubber (BioLogic) software.SPR Measurements with Surface Immobilized LIFR and gpl 0

[0381] For immobilization, recombinant human LIFR alpha and gpl30 were immobilized onto carboxymethyldextran-containing (CM5) SPR chips by NHS / EDC activation following manufacturer’s protocol. After immobilization of each receptor (LIFR or gpl 30) in 10 mM sodium acetate pH 5.0 to approximately 1000 RU, deactivation was performed with a 7 min injection of 1 M ethanolamine, pH 9.5. LIF constructs were then screened at different concentrations for binding. Capture levels of 30 RU or greater were achieved.Leukemia inhibitory factor (LIF) activation of Signal transducer and activator of transcription 3 (STAT3)

[0382] Bovine Aortic Endothelial (BAE) cells were plated onto 12-well culture plates at a density 5 x 104cells / well and cultured for 48hrs in low glucose DMEM + 10% BCS at 37°C with 5% CO2. Following a 3-hour serum starve in low glucose DMEM supplemented with 1% BCS, cells were treated with titrated concentrations (0.05 - 50nM) of recombinant human LIF (R&D Systems, Cat# 7734-LF-025 / CF), mosaic-generated LIF constructs, or vehicle control (PBS) for 15 minutes. Cells were rinsed with ice-cold PBS 2X and lysed in Cell Lysis Buffer (Cell Signaling, Cat# 9803) plusprotease and phosphatase inhibitors (Halt™ Protease Inhibitor Cocktail, Cat # 87786). Following centrifugation, protein concentration in cell lysates were determined using BCA assay (Pierce™, Cat# 23225). Levels of STAT3 activation in BAE cells was determined using a Phospho-Stat3 (Tyr705) Sandwich ELISA kit (Cell Signaling, Cat# 7300) following the manufacturers’ instructions.Inhibition of proliferation in BAE cells

[0383] BAE cells were seeded in 96 well plates in low glucose DMEM supplemented with 1% BCS and 100 U / 100 mg Penicillin / Streptomycin and containing recombinant human LIF (R&D Systems, Cat# 7734-LF-025 / CF), LIF constructs, or vehicle control (PBS) and incubated at 37°C with 5% CO2. After 48 hours, the media was changed, and treatments were replenished. Following another 48-hour incubation, cells were incubated with alamar blue (Invitrogen, Cat#: DAL1025) for 4 hours and fluorescence was measured.BCEC cell proliferation

[0384] Bovine choroidal microvascular endothelial cells (BCECs, VEC Technologies) were seeded at the density of 1,000 cells per well in a 200 microliter volume in 96-well plates in low glucose DMEM supplemented with 10% bovine calf serum. Cells were incubated with 10 ng / ml of hVEGF165 (R&D, 293-VE), or different concentrations of LIF or LIF-related molecules, as indicated. After 6 days, media were removed and cell viability reagent was added (Advanced BioReagents, K020) for 4h. Fluorescence was then measured at 530 nm excitation wavelength and 590 nm emission wavelength. The experiments were performed in triplicate and repeated three times, with similar results. The fold changes induced by different concentrations of samples are shown in the figure. Statistical significance was assessed by Student’s t-test (significant change P<0.05).Differential Scanning Fluorimetry (DSF)

[0385] Samples (15 pL) were prepared containing 5 pM test antibody and 5 x SYPRO Orange (Invitrogen) in a 96-well plate. The plate was covered with optical seal film and placed in a StepOne Plus Real-Time PCR system (Applied Biosystems). The sample was heated from 25 °C to 95 °C in 1 °C increments. Melting temperatures are obtained from StepOne Plus software via the sample derivative curves.

[0386] DSF experiments were also conducted on the Unchained Labs Uncle instrument. Samples were mixed with 5X SYPRO dye and loaded into Uni sample wells, and then loaded into the Uncle instrument. The plate was exposed to a thermal ramp from 25 to 95 °C at a heating rate of 0.5 °C / min (continuous). Thermal melting curves of each sample were monitored by the change in area under the fluorescence spectra at -570 nm resulting from 4X LIF unfolding. Tm was determined automatically in the Uncle Analysis software from the maximum derivative of the melt curve. Thermal aggregation curves of each sample were monitored by the change in static light scattering signal at 473 nm. Taggwas determined automatically in the Uncle Analysis software as the temperature where the derivative equals 10% of the maximum derivative of the aggregation curve (i.e. aggregation onset).Hydrophobic Interaction Chromatography (HIC)

[0387] Samples were diluted to 2 mg / mL in buffer A (1.8 M ammonium sulfate, 0. 1 M sodium phosphate, pH 6.5) to also achieve a final ammonium sulfate concentration of ~ IM. Samples were injected over a HIC Butyl column (Sepax) on an Agilent 1100 series HPLC at 0.6 mL / mL flow over a 30-minute gradient from 100% buffer A to 100% buffer B (0. 1 M sodium phosphate, pH 6.5). Self-interaction by BLI Assay

[0388] To evaluate humanized monoclonal antibody constructs for propensity to self-associate, a biolayer interferometry assay using an Octet® QK384 system was developed to measure self-binding response. Eculizumab was used as a low self-association control, and Blosozumab was used as a high self-association control. Based on optimization conditions determined in Xu et. al. antibodies of interest and controls were loaded onto Sartorius AHC (Anti-Human Fc or biotin-labelled) sensors to a surface density of 0.8 nm. Post loading, sensors are blocked using inert human IgGl isotype control (MOPC) to quench remaining capture surface. Loaded antibodies were tested for self-binding through a 5-minute association test with 1 pM of the same antibody in solution (PBS, 0.1% BSA). For each antibody tested, a reference signal was subtracted from the test sensor. This reference was collected from complete blocking of a separate sensor with MOPC that was associated in 1 pM of the same antibody to capture any non-specific binding to the MOPC quench. Maximum response of test antibody with reference subtraction after 5 minutes of self-association provides a self-interaction output in nm that can be compared to Eculizumab and Blosozumab controls.Baculovirus Particle (BVP) Assay

[0389] 0.5% BVP (25 pL) in 50 mM sodium carbonate, pH 9.5 were added to a high binding 96-well plate (Greiner Bio-One, Microplate 96 well, PS, half area, transparent) and covered with a plate sealer for 18 hours at 4 °C. Following incubation, all steps are performed at room temperature. All unbound BVPs were then aspirated from the wells. Blocking buffer (37.5 pL, 2% BSA in PBS, pH 7.4) was added and incubated for 1 hour with shaking (400 rpm), then washed 3 x with 100 pL PBS, pH 7.4. Test antibodies (12.5 pL at 1 pM) were added to individual wells (n = 6) and allowed to incubate for 1 hour, followed by 3 x PBS washes. Goat-anti human IgG (Fey fragment specific) conjugated to HRP (12.5 pL of at 10 ng / mL in blocking buffer, Jackson ImmunoResearch) was incubated for 1 hour with shaking (400 rpm), followed by 6 x PBS washes. Finally, TMB substrate (25 pL, Abeam) was added to each well and incubated for 10 - 15 minutes. The reactions were stopped with ELISA stop solution (25 pL, Invitrogen). Absorbance values were read at 450 nm and the BVP score was determined by normalizing the absorbance values of the test antibodies with the control wells. A similar approach was used for the non-Fc constructs except they were first biotinylated and detected using streptavidin-HRP.Sodium Iodate Model

[0390] Before experiments, eight-week-old C57BL / 6J male mice were randomly and blindly allocated to different 5 -mouse groups. Mice were anesthetized with ketamine / xylazine cocktail. Sterilized NalOs was administered as a single intravenous injection (20 mg / kg body weight). Controlmice were injected with PBS. PBS, LIF (50 ng), or LIF-KiH was injected intravitreally in the left eye; the right eye was injected with PBS as a control. Five, seven and nine days after injection, choroid capillaries were monitored by OCT-A system. Ten days after injection, mice were sacrificed, and eyes were harvested for H&E staining. Retinal thickness was quantified using ImageJ.New Zealand White Rabbit Intravitreal PK Study

[0391] IVT PK study was carried out in New Zealand white rabbits by PharmOptima. Briefly, animals were dosed intravitreally with aflibercept, 4xLIF-Fc (a dimer of SEQ ID NO: 12) from stable CHO-K1, PRO3446 (a dimer of SEQ ID NO: 23), or (a dimer of SEQ ID NO: 24) from Expi293 with 400 pg test article. Animals (n = 2) were sacrificed at day 1, day 3, or day 7 and tissues were collected for analysis.PK Sample Testing

[0392] Lumitrac high binding white 96-well, flat bottom microplates were coated with 0.5 pg per mb goat anti-human IgG Fc polyclonal antibody (Thermo, Sa5-10273) in coating buffer (50 mM sodium carbonate pH 9.5) (100 pl per well). After overnight incubation at 4 °C, the coated wells were washed manually four times with 295 pL wash buffer (IX PBS + .05% Tween 20 ).The plate was blocked with 3% IgG Free BSA (Jackson ImmunoResearch) in wash buffer (245 pL per well). The plate was left to block while shaking at 37 °C for at least 1 hour. The calibrator, controls and samples were diluted in wash buffer. Standards and samples were added to the plate at 100 pL per well. Standards, QC, and experimental samples were run in duplicate. The plate was incubated at room temperature while shaking at 420 rpm for 1 hour. The plate was washed four times manually with 295 pl per well of wash buffer, blotting on absorbent paper towel wipes between each wash. After four washes, HRP- conjugated goat anti-Human Fc (Thermo A 18817) diluted 1 / 2,000 (to 0.5 pg / mL) in washing buffer containing IgG-free BSA (.08%) was added to the wells and incubated for one hour at room temperature while shaking at 420 rpm. The plate was washed five times manually with 295 pl per well of wash buffer, blotting on absorbent paper towel wipes between each wash. The plate was developed with chemiluminescent substate (ELISA BRIGHT Advansta) which had been diluted 1 part to six total parts with IX PBS. The plate was read with a Spectramax M5 plate reader under ELISA end point luminescent and using 25 ms integration. The data were processed using a four-parameter logistic fit on PRISM 10.10.

[0393] A three-fold serial dilution of each test construct was used to generate an eight-point calibrator curve with a range from 0.686 to 500 ng / ml. The standard curve was prepared each time by diluting a 50 pg per mb solution that was kept at -80 °C. QC samples were prepared daily from the 500 ng / mL standard. When assaying vitreous and aqueous humor samples, the standard and QC samples were prepared using wash buffer as diluent. When assaying retina or choroid samples, the standard curve was prepared in blank control tissue, which had been homogenized and diluted to 0.01 mg per mb in wash buffer. The protein concentration was determined using BCA assay (Thermo) according to the manufacturer’s instructions.

[0394] All test samples were kept at -80 °C. They were thawed and diluted before performing the ELISA Assay. Vitreous samples were diluted by a factor of 10000 in wash buffer. Aqueous humor samples were diluted by a factor of 500 in wash buffer. Protein concentrations in retina and choroid samples were first determined using the BCA assay as above. The samples were then diluted to 0.01 mg / mL before performing the ELISA Assay.Example 10: In vivo vascular density of 4XLIF-Fc Variants

[0395] The effects of 4xLIF-Fc Variant 2 (a dimer of SEQ ID NO: 24) on vascular density were assessed. Eight-week-old C57BL / 6J male mice were anesthetized with a ketamine / xylazine cocktail. Before experiments, mice were allocated to different groups randomly and blindly. PBS, LIF (50 ng / uL), and Testing Construct (4xLIF-Fc Variant 2 at 0.5 uM, 2.5 uM, and 5uM) were injected intravitreally into the eye. Five eyes were injected per group. Seven days after injection, mice were sacrificed, and eyes were harvested and whole-mounted for CD-31 staining. Retinas were imaged by using a confocal microscope. The retinal vasculature density (intermediate and deep plexus) was quantified by using ImageJ. The results were analyzed with ordinary one-way ANOVA for multiple comparisons and showed as fold changes, normalized to PBS (control).

[0396] As shown in FIG. 23A and FIG. 23B, LIF and 4xLIF-Fc Variant 2 treated mice showed an increase in vascular density as compared to that of a control group (PBS treatment), suggesting enhanced tissue perfusion and potential support for retinal repair mechanisms.Example 11 : Dry AMD model with Sodium Iodate (NalO d

[0397] Eight-week-old C57BL / 6J male mice were anesthetized with a ketamine / xylazine cocktail. Before experiments, mice were allocated to different groups randomly and blindly. Sterilized NalOs was administered as a single intravenous tail injection (20 mg / kg body weight). Control mice were injected with PBS. LIF (50 ng) and Testing Construct (4xLIF-Fc Variant 2 at 0.25 uM, 0.5 uM, 2.5 uM, and 5uM; SEQ ID NO: 24) were injected intravitreally into the left eye. The right eye was injected with PBS as a control. There were 6 mice per group. Ten days after injection, mice were sacrificed, and eyes were harvested for H&E staining. The retinal thickness was quantified by ImageJ, and the results were analyzed with a paired T-Test and showed as fold changes, normalized to PBS (control).

[0398] Hematoxylin and eosin (H&E) staining of retinal of the sodium iodate treated mice (FIG. 24A) typically shows a reduced outer nuclear layer (ONL), absences of photoreceptor inner and outer segments (IS / OS), extensive degeneration of the RPE monolayer, and damaged / thinning of choriocapillaris. An increase in OND thickness was observed with 4xLIF-FC Variant 2 treated mice (FIG. 24B) As shown in FIGs. 25A-25E, both LIF and 4xLIF-Fc Variant 2 treated mice showed an increase in ONL thickness as compared to PBS treated mice. Together, these results indicate that the4xLIF-Fc Variant 2 not only mitigate NaIO3-indcued retinal damage, but also promote structure recovery of key outer retinal layers, supporting its potential as a therapeutic candidate for dry AMD.

[0399] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A chimeric polypeptide comprising an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide.

2. The chimeric polypeptide of claim 1, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 3.

3. The chimeric polypeptide of claim 1 or 2, wherein the chimeric polypeptide comprises from N- terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide.

4. The chimeric polypeptide of any one of claims 1-3, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

5. The chimeric polypeptide of any one of claims 1-4, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

6. The chimeric polypeptide of any one of claims 1-5, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

7. The chimeric polypeptide of any one of claims 4-6, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

8. The chimeric polypeptide of any one of claims 4-6, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

9. The chimeric polypeptide of any one of claims 1-8, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 12.

10. The chimeric polypeptide of claim 1, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 2.

11. The chimeric polypeptide of claim 10, wherein the chimeric polypeptide comprises from N- terminus to C-terminus: the first LIF peptide, the Fc region, and the second LIF peptide.

12. The chimeric polypeptide of claim 10 or 11, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

13. The chimeric polypeptide of any one of claims 10-12, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

14. The chimeric polypeptide of any one of claims 10-13, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

15. The chimeric polypeptide of claim 14, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

16. The chimeric polypeptide of claim 14, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

17. The chimeric polypeptide of claim 15 or 16, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

18. The chimeric polypeptide of claim 14, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

19. The chimeric polypeptide of claim 14, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

20. The chimeric polypeptide of claim 18 or 19, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 24.

21. The chimeric polypeptide of claim 14, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

22. The chimeric polypeptide of claim 14, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

23. The chimeric polypeptide of claim 21 or 22, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

24. A composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the chimeric polypeptide of any one of claims 1-23.

25. The composition of claim 24, wherein the first polypeptide and the second polypeptide form a dimer through a disulfide linkage.

26. The composition of claim 24 or 25, wherein the second polypeptide comprises an Fc region of an IgG coupled to a first LIF peptide and a second LIF peptide.

1. The composition of claim 26, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 3.

28. The composition of claim 26 or 27, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, the Fc region, the second LIF peptide.

29. The composition of any one of claims 26-28, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

30. The composition of any one of claims 26-29, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

31. The composition of any one of claims 26-30, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

32. The composition of any one of claims 26-31, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

33. The composition of any one of claims 26-32, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

34. The composition of any one of claims 24-33, wherein the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 12.

35. The composition of claim 26, wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 2.

36. The composition of claim 35, wherein the chimeric polypeptide comprises from N-terminus to C- terminus: the first LIF peptide, the Fc region, the second LIF peptide.

37. The composition of claim 35 or 36, wherein the chimeric polypeptide further comprises a linker between the first LIF peptide and the Fc region.

38. The composition of any one of claims 35-37, wherein the chimeric polypeptide further comprises a linker between the Fc region and the second LIF peptide.

39. The composition of any one of claims 35-38, wherein the chimeric polypeptide comprises from N-terminus to C-terminus: the first LIF peptide, optionally a first linker, the Fc region, optionally a second linker, and the second LIF peptide.

40. The composition of claim 39, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

41. The composition of claim 39, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 7.

42. The composition of claim 40 or 41, wherein the second polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

43. The composition of claim 39, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

44. The composition of claim 39, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 26.

45. The composition of claim 43 or 44, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 24.

46. The composition of claim 39, wherein the first linker or the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

47. The composition of claim 39, wherein the first linker and the second linker comprises an amino acid sequence according to SEQ ID NO: 28.

48. The composition of claim 46 or 47, wherein the chimeric polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

49. The composition of any one of claims 24-48, wherein the first polypeptide comprises the chimeric polypeptide of any one of claims 1-23.

50. The composition of any one of claims 24-49, wherein the second polypeptide comprises the chimeric polypeptide of any one of claims 1-23.

51. The composition of any one of claims 24-50, wherein the first polypeptide and the second polypeptide are identical in same amino acid sequence.

52. The composition of any one of claims 24-51, wherein the composition has a half-life of about 5.4 days.

53. The composition of any one of claims 24-52, wherein the composition inhibits growth of bovine aortic endothelial cells.

54. The composition of any one of claims 24-53, wherein the composition promotes growth of bovine choroidal endothelial cells.

55. The composition of any one of claims 24-54, wherein the composition preserves choriocapillaris, retinal pigment epithelium, and / or photoreceptors after systemic administration of sodium iodate in a mouse model.

56. The composition of any one of claims 24-55, wherein the composition promotes growth of retinal capillary endothelial cells.

57. The chimeric polypeptide or composition of any one of the preceding claims, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject promotes retinal angiogenesis.

58. The chimeric polypeptide or composition of any one of the preceding claims, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject increases proliferation of choroidal endothelial cells.

59. The chimeric polypeptide or composition of any one of the preceding claims, wherein administration of an effective amount of the chimeric polypeptide or composition to an eye of a subject does not induce vascular leakage.

60. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.

61. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

62. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.

63. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

64. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

65. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 1.

66. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1.

67. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1.

68. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

69. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide comprises the amino acid sequence SEQ ID NO: 1.

70. The chimeric polypeptide or composition of any one of the preceding claims, wherein the LIF peptide consists of the amino acid sequence SEQ ID NO: 1.

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