# US 2019/0169231 A1 · Compositions containing hc-ha/ptx3 complexes and methods of use thereof

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- Number: US 2019/0169231 A1
- Document ID: US20190169231A1
- Status: Published application
- Kind: A1
- Application number: 16/274,123
- Filed: 2019-02-12
- Published: 2019-06-06
- Earliest claimed: 2012-07-11
- Assignees: Tissuetech, Inc.
- Applicants: TissueTech, Inc.
- Inventors: Scheffer TSENG; Hua HE; Sean TIGHE; Suzhen ZHANG; Ying-Tieng ZHU
- Web page: https://patentgrep.com/patent/US20190169231A1

## CPC

- C07K 1/36: by a combination of two or more processes of different types
- A61K 38/191: {Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta}
- A61K 47/55: the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
- A61K 47/69: the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K 47/6957: {the form being a device or a kit, e.g. stents or microdevices}
- A61L 27/34: Macromolecular materials
- A61L 27/54: Biologically active materials, e.g. therapeutic substances {(A61L27/227 takes precedence)}
- A61L 29/085: {Macromolecular materials}
- A61L 29/16: Biologically active materials, e.g. therapeutic substances {(A61L29/048 takes precedence)}
- A61L 31/10: Macromolecular materials
- A61L 31/16: Biologically active materials, e.g. therapeutic substances {(A61L31/047 takes precedence)}
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/04: for non-specific disorders of the connective tissue
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 19/10: for osteoporosis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/47: from mammals
- C07K 14/525: Tumour necrosis factor [TNF]
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61L 2300/236: Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
- A61L 2300/252: Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
- A61L 2300/41: Anti-inflammatory agents, e.g. NSAIDs
- A61L 2300/412: Tissue-regenerating or healing or proliferative agents
- A61L 2300/426: Immunomodulating agents, i.e. cytokines, interleukins, interferons
- C07K 14/81: Protease inhibitors
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Provided herein are methods for the production of native and reconstituted hyaluronan (HA) complexes containing pentraxin-3 (PTX3) and heavy chain 1 (HC1) of inter alpha inhibitor (IαI). Compositions containing the complexes and therapeutic methods using the complexes are provided. Combinations and kits for use in practicing the methods also are provided.

## Claims

### Claim 1

A method for producing a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprising contacting (i) high molecular weight hyaluronan (HMW HA) immobilized to a solid support, (ii) pentraxin 3 (PTX3) protein, (iii) inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and (iv) Tumor necrosis factor α-stimulated gene 6 (TSG-6) to form an immobilized rcHC-HA/PTX3 complex.

### Claim 2 (depends on 1)

The method of claim 1, comprising:
(a) contacting (i) high molecular weight hyaluronan (HMW HA) immobilized to a solid support, and (ii) pentraxin 3 (PTX3) protein, to form an immobilized complex of PTX3 and HMW HA (immobilized PTX3/HA); and
(b) contacting the immobilized PTX3/HA with an inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and Tumor necrosis factor α-stimulated gene 6 (TSG-6) to form an immobilized rcHC-HA/PTX3 complex.

### Claim 3 (depends on 1)

The method of claim 1, comprising:
(a) contacting (i) high molecular weight hyaluronan (HMW HA) immobilized to a solid support, (ii) an inter-α-inhibitor (Id) protein comprising heavy chain 1 (HC1) and (iii) TSG-6 to form an rcHC-HA complex pre-bound to TSG-6; and
(b) contacting rcHC-HA complex pre-bound to TSG-6 with a pentraxin 3 (PTX3) protein to form an rcHC-HA/PTX3 complex.

### Claim 4 (depends on 1, 2, 3)

The method of any of claims 1-3, wherein TSG-6 catalyzes the covalent linkage of IαI HC1 to HA.

### Claim 5 (depends on 2)

The method of claim 2, further comprising removing unbound PTX3 protein following step (a) and prior to performing step (b).

### Claim 6 (depends on 2, 3, 4, 5)

The method of any of claims 2-5, further comprising removing unbound TSG-6 following step (b).

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method of any of claims 1-6, wherein the PTX3 protein is native PTX3 protein or recombinant PTX3 protein, and the TSG-6 protein is native TSG-6 protein or recombinant TSG-6 protein.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The method of any of claims 1-7, wherein IαI protein further comprises HC2, bikunin, a chondroitin sulfate chain, or any combinations thereof.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of any of claims 1-8, wherein IαI protein is isolated from blood, serum, plasma, amniotic membrane, chorionic membrane, amniotic fluid, or a combination thereof.

### Claim 10 (depends on 7, 8, 9)

The method of claim 7-9, wherein IαI protein, native TSG-6 protein, and/or native PTX3 protein is produced by an amniotic membrane cell.

### Claim 11 (depends on 10)

The method of claim 10, wherein the amniotic membrane cell is an amniotic epithelial cell, amniotic stromal cell, umbilical cord epithelial cell, or umbilical cord stromal cell.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of any of claims 1-11, wherein PTX3 is a homomultimer.

### Claim 13 (depends on 12)

The method of claim 12, wherein the PTX3 homomultimer is an octamer.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The method of any of claims 1-13, wherein PTX3 comprises a modified multimerization domain or a heterogeneous multimerization domain.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The method of any of claims 1-14, wherein the PTX3, IαI HC1 or TSG-6 polypeptides comprise an affinity tag.

### Claim 16 (depends on 15)

The method of claim 15, wherein the affinity tag is selected from among a hemagglutinin tag, a poly-histidine tag, a myc tag, a FLAG tag, a glutathione-S-transferase (GST) tag.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any of claims 1-16, wherein the IαI and TSG-6 protein are contacted at a molar ratio of 3:1.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The method of any of claims 1-17, wherein the weight average molecular weight of the HMW HA is 3,000 kDa.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any of claims 1-18, wherein the HMW HA is immobilized by direct linkage to a solid support.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any of claims 1-18, wherein the HMW HA is immobilized by indirect linkage to a solid support.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any of claims 1-18, wherein the HMW HA is immobilized by linkage to a solid support via a cleavable linker.

### Claim 22 (depends on 21)

The method of claim 21, wherein the cleavable linker is a chemically or enzymatically cleavable linker.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any of claims 1-18, wherein the HMW HA is immobilized by covalent or non-covalent attachment to the solid support.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any of claims 1-18, wherein the HMW HA is immobilized by binding HMW HA to an intermediary polypeptide.

### Claim 25 (depends on 24)

The method of claim 24, wherein the intermediary polypeptide is covalently attached to the solid support.

### Claim 26 (depends on 24, 25)

The method of claim 24 or claim 25, wherein the HMW HA is bound to the intermediary polypeptide by a non-covalent bond.

### Claim 27 (depends on 24, 25, 26)

The method of any of claims 24-26, wherein the intermediary polypeptide is an HA binding protein (HABP).

### Claim 28 (depends on 27)

The method of claim 27, wherein the HABP is selected from among HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, or a portion thereof sufficient to bind HA.

### Claim 29 (depends on 27)

The method of claim 27, wherein the HABP is versican.

### Claim 30 (depends on 24, 25, 26, 27, 28, 29)

The method of any of claims 24-29, wherein the intermediary polypeptide comprises a link module.

### Claim 31 (depends on 30)

The method of claim 30, wherein the link module is a link module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2.

### Claim 32 (depends on 24, 25, 26, 27, 28, 29, 30, 31)

The method of any of claims 24-31, wherein the intermediary polypeptide comprises a polypeptide linker.

### Claim 33 (depends on 32)

The method of claim 32, wherein the polypeptide linker is attached to the solid support.

### Claim 34 (depends on 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)

The method of any of claims 24-33, wherein the intermediary polypeptide comprises a proteolytic cleavage sequence.

### Claim 35 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The method of any of claims 1-22, further comprising dissociation of the immobilized rcHC-HA/PTX3 complex to form an dissociated rcHC-HA/PTX3 complex.

### Claim 36 (depends on 35)

The method of claim 35, wherein dissociation of the immobilized rcHC-HA/PTX3 complex comprises cleavage of a cleavable linker.

### Claim 37 (depends on 35)

The method of claim 35, wherein dissociation of the immobilized rcHC-HA/PTX3 complex comprises contacting the immobilized rcHC-HA/PTX3 complex with a dissociation agent.

### Claim 38 (depends on 37)

The method of claim 37, wherein the dissociation agent is guanidine hydrochloride or urea.

### Claim 39 (depends on 35, 36, 37, 38)

The method of any of claims 35-38, wherein the method further comprises purification of the dissociated rcHC-HA/PTX3 complex.

### Claim 40 (depends on 39)

The method of claim 39, wherein purification comprises affinity purification, centrifugation, filtration, chromatography or a combination thereof.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The method of any of claims 1-40, further comprising contacting the immobilized PTX3/HA complex or rcHC-HA/PTX3 complex with a small leucine rich proteoglycan (SLRP).

### Claim 42 (depends on 41)

The method of claim 41, wherein the SLRP is selected from among decorin, biglycan, fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, osteoadherin, epipycan, and osteoglycin.

### Claim 43 (depends on 41)

The method of claim 41, wherein the SLRP is selected from among decorin, biglycan, and osteoglycin.

### Claim 44 (depends on 41, 42, 43)

The method of any of claims 41-43, wherein the SLRP is covalently attached to a glycosaminoglycan.

### Claim 45 (depends on 44)

The method of claim 44, wherein the glycosaminoglycan is keratan sulfate.

### Claim 46 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

A reconstituted HC-HA (rcHC-HA/PTX3) complex produced by the method of any of claims 1-45.

### Claim 47

A reconstituted HC-HA (rcHC-HA/PTX3) complex comprising high molecular weight hyaluronan (HMW HA), (ii) pentraxin 3 (PTX3) protein, and (iii) inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1).

### Claim 48 (depends on 47)

The rcHC-HA/PTX3 of claim 47 immobilized to a solid support.

### Claim 49

An isolated native HC-HA/PTX3 (nHC-HA/PTX3) complex purified from an amniotic membrane extract or an umbilical cord extract.

### Claim 50 (depends on 49)

The isolated nHC-HA/PTX3 complex of claim 49, wherein the umbilical cord extract or amniotic membrane extract is prepared from amniotic membrane, umbilical cord amniotic membrane, umbilical cord stroma or any combinations thereof.

### Claim 51 (depends on 49, 50)

The isolated nHC-HA/PTX3 complex of claim 49 or claim 50, wherein the nHC-HA/PTX3 complex is purified by ultracentrifugation of the amniotic membrane extract or umbilical cord extract.

### Claim 52 (depends on 51)

The isolated nHC-HA/PTX3 complex of claim 51, wherein the amniotic membrane extract or umbilical cord extract is a water insoluble extract.

### Claim 53 (depends on 52)

The isolated nHC-HA/PTX3 complex of claim 52, wherein the water insoluble extract is extracted with guanidine hydrochloride.

### Claim 54 (depends on 51, 52, 53)

The isolated nHC-HA/PTX3 complex of any of claims 51-53, further comprising a small leucine rich proteoglycan (SLRP).

### Claim 55 (depends on 54)

The isolated nHC-HA/PTX3 complex of claim 54, wherein the SLRP is selected from among decorin, biglycan, fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, osteoadherin, epipycan, and osteoglycin.

### Claim 56 (depends on 54)

The isolated nHC-HA/PTX3 complex of claim 54, wherein the SLRP is selected from among decorin, biglycan, and osteoglycin.

### Claim 57 (depends on 54, 55, 56)

The isolated nHC-HA/PTX3 complex of any of claims 54-56, wherein the SLRP is covalently attached to a glycosaminoglycan.

### Claim 58 (depends on 57)

The isolated nHC-HA/PTX3 complex of claim 57, wherein the glycosaminoglycan is keratan sulfate.

### Claim 59 (depends on 49, 50, 51)

The isolated nHC-HA/PTX3 complex of any of claims 49-51, wherein the amniotic membrane extract or umbilical cord extract is a water soluble extract.

### Claim 60 (depends on 59)

The isolated nHC-HA/PTX3 complex of claim 59, wherein the water soluble extract is extracted with an isotonic salt solution.

### Claim 61 (depends on 60)

The isolated nHC-HA/PTX3 complex of claim 60, wherein the isotonic salt solution is PBS.

### Claim 62 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

The rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61, wherein the complex promotes the M2 polarization of a macrophage.

### Claim 63 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

The rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61, wherein:
(i) the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex reduces the expression of IL-12p40 in LPS-stimulated macrophages, wherein the level of IL-12p40 expressed by LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted with the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex compared to the level of IL-12p40 expressed by LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex;
(ii) the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex reduces the expression of IL-12p70 protein in LPS-stimulated macrophages, wherein the amount of IL-12p70 protein expressed by LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex compared to the amount of IL-12p70 protein expressed by LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex;
(iii) the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex reduces the expression of IL-23 in LPS-stimulated macrophages, wherein the level of IL-23 expressed by LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are contacted the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex compared to the level of IL-23 expressed by LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex;
(iv) the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex increases the expression of IL-10 in LPS-stimulated macrophages, wherein the level of IL-10 expressed by LPS-stimulated macrophages is higher when the LPS-stimulated macrophages are contacted the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex compared to the level of IL-10 expressed by LPS-stimulated macrophages in the absence of rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex;
(v) the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex promotes apoptosis of LPS-stimulated neutrophils, wherein the number of LPS-stimulated neutrophils that are apoptotic in a sample of LPS-stimulated neutrophils is higher when the sample is contacted the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex compared to the number of LPS-stimulated neutrophils that are apoptotic in the sample in the absence of the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex;
(vi) the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein the number of apoptotic neutrophils that are phagocytosed by LPS-stimulated or resting macrophages in a sample of apoptotic neutrophils and LPS-stimulated or resting macrophages is higher when the sample is contacted the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex compared to the number of neutrophils that are phagocytosed by LPS-stimulated or resting macrophages in the sample in the absence of the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

### Claim 64 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

The rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61, wherein:
(i) the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex reduces the level of IL-12p40 mRNA or IL-12p40 protein;
(ii) the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex reduces the level of IL-12p70 protein;
(iii) the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex reduces the level of IL-23 mRNA or IL-23 protein;
(iv) the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex increases the level of IL-10 mRNA or IL-10 protein; or
any combination of (i)-(iv) thereof.

### Claim 65 (depends on 46, 47, 48)

The rcHC-HA/PTX3 complex of any of claims 46-48, wherein the complex promotes attachment of LPS-stimulated macrophages to at least the same level as a native HC-HA/PTX3 (nHC-HA/PTX3) complex isolated from human amniotic membrane, wherein attachment comprises the contacting LPS-stimulated macrophages to the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complexes immobilized to a solid support.

### Claim 66

A method for inducing the M2 polarization of macrophages comprising contacting an LPS-stimulated macrophage with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

### Claim 67 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 66)

The method of claim 66, wherein the rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex is rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61.

### Claim 68 (depends on 66, 67)

The method of claim 66 or claim 67, wherein contacting a macrophage with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex:
(i) reduces the level of IL-12p40 expressed by the macrophage:
(ii) reduces the level of IL-12p70 expressed by the macrophage;
(iii) reduces the level of IL-23 expressed by the macrophage; or
(iv) increases the level of IL-10 expressed by the macrophage.
(v) induces apoptosis of LPS-stimulated neutrophils;
(vi) promotes phagocytosis of apoptotic neutrophils; or
any combination of (i)-(vi) thereof.

### Claim 69 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

A pharmaceutical composition, comprising an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 and a pharmaceutically acceptable excipient.

### Claim 70 (depends on 69)

The pharmaceutical composition of claim 69, further comprising fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, or combinations thereof.

### Claim 71 (depends on 69, 70)

The pharmaceutical composition of claim 69 or claim 70, further comprising an anti-inflammatory agent, an anti-scarring agent, an anti-neoplastic agent, a chemotherapeutic agent, an immunosuppressive agent, a cytotoxic agent, an antimicrobial agent or a combination thereof.

### Claim 72 (depends on 69, 70, 71)

The pharmaceutical composition of any of claims 69-71, wherein the composition is a solution, suspension, gel, powder, ointment, tablet, capsule, or an aerosol.

### Claim 73 (depends on 72)

The pharmaceutical composition of claim 72, wherein the gel is a cross-linked gel, or a liposome.

### Claim 74 (depends on 72)

The pharmaceutical composition of claim 72, wherein the gel is a cross-linked hyaluronan hydrogel.

### Claim 75 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for preventing or reversing scar formation or fibrosis in a tissue, comprising contacting the tissue with an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61.

### Claim 76 (depends on 75)

The use of claim 75, wherein the scar is a dermatitis scar, a keloid scar, contracture scar, a hypertrophic scar, or a scar resulting from acne.

### Claim 77 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for preventing or reducing inflammation in a subject in need thereof.

### Claim 78 (depends on 77)

The use of claim 77, wherein the inflammation is acute inflammation or chronic inflammation.

### Claim 79 (depends on 77, 78)

The use of claim 77 or 78, wherein the inflammation is a macrophage-mediated inflammation, Th-17-mediated inflammation or T-cell mediated inflammation.

### Claim 80 (depends on 77, 78, 79)

The use of any of claims 77-79, wherein the inflammation is associated with an autoimmune disorder, an allergy, a leukocyte defect, an infection, graft versus host disease, tissue transplant rejection, or combinations thereof.

### Claim 81 (depends on 77, 78, 79)

The use of any of claims 77-79, wherein the inflammation is associated with rheumatoid arthritis.

### Claim 82 (depends on 77, 78, 79)

The use of any of claims 77-79, wherein the inflammation is associated with conjunctivitis, keratitis, blepharitis, blepharoconjunctivitis, scleritis, episcleritis, uveitis, retinitis, or choroiditis.

### Claim 83 (depends on 77, 78, 79)

The use of any of claims 77-79, wherein the inflammation is associated with myocardial infarction, stroke, endotoxin shock or sepsis.

### Claim 84 (depends on 77, 78, 79)

The use of any of claims 77-79, wherein the inflammation is associated with atherosclerosis.

### Claim 85 (depends on 77, 78, 79)

The use of any of claims 77-79, wherein the subject has cancer.

### Claim 86 (depends on 77, 78, 79, 80, 81, 82, 83, 84, 85)

The use of any of claims 77-85, for administration in combination with an additional anti-inflammatory agent to the subject.

### Claim 87 (depends on 86)

The use of claim 86, wherein the additional anti-inflammatory agent is selected from among an anti-TGF-β antibody, an anti-TGF-β receptor blocking antibody, an anti-TNF antibody, an anti-TNF receptor blocking antibody, an anti-IL1β antibody, an anti-IL1β receptor blocking antibody, an anti-IL-2 antibody, an anti-IL-2 receptor blocking antibody, an anti-IL-6 antibody, an anti-IL-6 receptor blocking antibody, an anti IL-12 antibody, an anti IL-12 receptor blocking antibody, an anti-IL-17 antibody, anti-IL-17 receptor blocking antibody, an anti-IL-23 antibody, or an anti-IL-23 receptor blocking antibody.

### Claim 88 (depends on 87)

The use of claim 87, wherein the Type 1 interferon is IFN-α or IFN-β.

### Claim 89 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for treating a skin wound or ulcer in a subject in need thereof.

### Claim 90 (depends on 89)

The use of claim 89, wherein the skin wound or ulcer is non-healing.

### Claim 91 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for promoting or inducing bone formation in a subject in need thereof.

### Claim 92 (depends on 91)

The use of claim 91, wherein the subject has arthritis, osteoporosis, alveolar bone degradation, Paget's disease, or a bone tumor.

### Claim 93 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for preventing or reducing unwanted angiogenesis in a subject in need thereof.

### Claim 94 (depends on 93)

The use of claim 93, wherein the unwanted angiogenesis is associated with wet age-related macular degeneration (wARMD) or diabetic proliferative retinopathy

### Claim 95 (depends on 93)

The use of claim 93, wherein the unwanted angiogenesis is associated with cancer.

### Claim 96 (depends on 93)

The use of claim 93, wherein the subject has a solid tumor.

### Claim 97 (depends on 95, 96)

The use of any of claims 95-96, for administration in combination with an anti-cancer therapy.

### Claim 98 (depends on 97)

The use of claim 97, wherein the anti-cancer therapy comprises administration of an anti-neoplastic agent, a cytotoxic agent, an antimicrobial agent, an anti-angiogenic agent, a chemotherapeutic agent, or radiation therapy.

### Claim 99 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for inhibiting or preventing transplant rejection in a transplant recipient.

### Claim 100 (depends on 99)

The use of claim 99, for administration before a transplantation procedure, after a transplantation procedure, or during a transplantation procedure.

### Claim 101 (depends on 99, 100)

The use of claim 99 or claim 100, for administration in combination with an immunosuppressive agent.

### Claim 102 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

A method of inducing stem cell expansion, comprising contacting a stem cell in vitro with an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61.

### Claim 103 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 in combination with a therapeutic cell for providing a cell therapy to a subject in need thereof.

### Claim 104 (depends on 103)

The use of claim 103, further comprising contacting a damaged tissue with the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex and the therapeutic cell.

### Claim 105 (depends on 103)

The use of claim 103, wherein the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex and therapeutic cell are formulated in a single composition or separate compositions.

### Claim 106 (depends on 103)

The use of claim 103, wherein the therapeutic cell is contained in a microcapsule.

### Claim 107 (depends on 104)

The use of claim 104, wherein the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex is attached to the microcapsule.

### Claim 108 (depends on 103, 104, 105, 106, 107)

The use of any of claims 103-107, wherein the therapeutic cell is a stem cell.

### Claim 109 (depends on 108)

The use of claim 108, wherein the therapeutic cell is an adult stem cell or an induced pluripotent stem cell.

### Claim 110 (depends on 108)

The use of claim 108, wherein the therapeutic cell is a mesenchymal stem cell, limbal epithelial progenitor cell, a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell

### Claim 111 (depends on 103, 104, 105, 106, 107, 108)

The use of any of claims 103-108, wherein therapeutic cell is a differentiated cell.

### Claim 112 (depends on 103, 104, 105, 106, 107, 108)

The use of any of claims 103-108, wherein therapeutic cell is an insulin producing cell.

### Claim 113 (depends on 112)

The use of claim 112, wherein the insulin producing therapeutic cell is an islet cell.

### Claim 114 (depends on 112, 113)

The use of claim 112 or claim 113, wherein the subject has diabetes mellitus type 1.

### Claim 115 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for inducing or promoting tissue regeneration in a subject in need thereof.

### Claim 116 (depends on 115)

The use of claim 115, wherein the tissue is bone or gum, corneal tissue, or conjunctival tissue.

### Claim 117 (depends on 115, 116)

The use of claim 115 or 116, for administration in combination with a therapeutic cell or a tissue transplant.

### Claim 118 (depends on 117)

The use of claim 117, wherein the tissue transplant is an allograft or an autograft.

### Claim 119 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for inhibiting or decreasing the amount of M1 adipose tissue macrophages in a subject.

### Claim 120 (depends on 119)

The use of claim 119, wherein the subject is diagnosed with obesity or insulin-resistance.

### Claim 121 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

Use of an effective amount of an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 for conjunctivochalasis in a subject in need thereof.

### Claim 122 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

A cell culture, comprising a substrate suitable for culturing a cell and an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61.

### Claim 123 (depends on 122)

The cell culture of claim 122, wherein the rcHA/PTX3 complex or the nHC-HA/PTX3 complex is immobilized to the substrate.

### Claim 124 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

A medical device, comprising a substrate coated with an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61.

### Claim 125 (depends on 124)

The medical device of claim 124, wherein the substrate comprises at least one of a stent, a joint, a screw, a rod, a pin, a plate, a staple, a shunt, a clamp, a clip, a suture, a suture anchor, an electrode, a catheter, a lead, a graft, a dressing, a pacemaker, a pacemaker housing, a cardioverter, a cardioverter housing, a defibrillator, a defibrillator housing, a prostheses, an ear drainage tube, an ophthalmic implant, an orthopedic device, a vertebral disk, a bone substitute, an anastomotic device, a perivascular wrap, a colostomy bag attachment device, a hemostatic barrier, a vascular implant, a vascular support, a tissue adhesive, a tissue sealant, a tissue scaffold, and an intraluminal device.

### Claim 126 (depends on 124)

The medical device of claim 124, wherein the substrate is a microcapsule, nanoparticle, or a bead.

### Claim 127 (depends on 126)

The medical device of claim 126, wherein microcapsule contains a stem cell.

### Claim 128 (depends on 124)

The medical device of claim 124, wherein the substrate is a contact lens.

### Claim 129 (depends on 124)

The medical device of claim 124, wherein the substrate is a surgical implant or prosthesis.

### Claim 130 (depends on 129)

The medical device of claim 129, wherein the implant or prosthesis is an artificial joint, a bone implant, a suture, or a stent.

### Claim 131 (depends on 130)

The medical device of claim 130, wherein the artificial joint is an artificial hip joint, an artificial knee, an artificial glenohumeral joint, or an artificial knee.

### Claim 132 (depends on 130)

The medical device of claim 130, wherein the stent is a coronary stent, a ureteral stent, a urethral stent, a prostatic stent, esophageal stent, or a bone stent.

### Claim 133 (depends on 124, 125, 126, 127, 128, 129, 130, 131, 132)

The medical device of any of claims 124-132, wherein the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex inhibits the formation of a bacterial biofilm.

### Claim 134 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

A method for modulating macrophage activity comprising contacting a macrophage with an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61 in an amount sufficient to (i) reduce or inhibit the expression of IL-12 or IL-23 or (ii) increase the expression of IL-10.

### Claim 135 (depends on 134)

The method of claim 134, wherein the macrophage has been stimulated with a pro-inflammatory mediator.

### Claim 136 (depends on 135)

The method of claim 135, wherein the pro-inflammatory mediator is lipopolysaccharide (LPS), tumor necrosis factor α (TNF-α), interferon-gamma (IFN γ) or a combination thereof.

### Claim 137 (depends on 134, 135, 136)

The method of any of claims 134-136, wherein the rcHC-HA/PTX3 complex or isolated nHC-HA/PTX3 complex contacts the macrophage in vitro.

### Claim 138 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

A kit, comprising a composition comprising an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61, a device for administration of the composition and, optionally, instructions for administration.

### Claim 139

A method for inducing or maintaining pluripotency in a cell, comprising culturing the cell with an nHC-HA/PTX3 complex or an rcHC-HA/PTX3 complex, thereby inducing or maintaining pluripotency in the cell.

### Claim 140 (depends on 139)

The method of claim 139, wherein the cell heterogeneously expresses a protein selected from among Sox2, myc, Oct4 and KLF4.

### Claim 141 (depends on 139)

The method of claim 139, wherein the cell heterogeneously expresses one, two, or three proteins selected from among Sox2, myc, Oct4 and KLF4.

### Claim 142 (depends on 139)

The method of claim 139, wherein the nHC-HA/PTX3 complex or rcHC-HA/PTX3 complex is immobilized onto a substrate.

### Claim 143 (depends on 139)

The method of claim 139, wherein the cell is an adult differentiated cell.

### Claim 144 (depends on 139)

The method of claim 139, wherein the cell is a fibroblast.

### Claim 145 (depends on 144)

The method of claim 144, wherein the cell is a human corneal fibroblast.

### Claim 146 (depends on 139)

The method of claim 139, wherein the cell is an adult stem cell, or an induced pluripotent stem cell.

### Claim 147 (depends on 139)

The method of claim 139, wherein the cell is a limbal epithelial progenitor cell, a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell.

### Claim 148 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 139, 140, 141, 142, 143, 144, 145, 146, 147)

The method of any of claims 139-147, wherein the nHC-HA/PTX3 complex or rcHC-HA/PTX3 complex is an rcHC-HA/PTX3 complex of any of claims 46-48 or isolated nHC-HA/PTX3 complex of any of claims 49-61.