US20140274915A1 - Methods of Use for Recombinant Human Secretoglobins - Google Patents

Methods of Use for Recombinant Human Secretoglobins Download PDF

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US20140274915A1
US20140274915A1 US13/843,289 US201313843289A US2014274915A1 US 20140274915 A1 US20140274915 A1 US 20140274915A1 US 201313843289 A US201313843289 A US 201313843289A US 2014274915 A1 US2014274915 A1 US 2014274915A1
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respiratory
patient
exacerbation
protein
rhscgb3a2
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Aprile L. Pilon
Melissa E. Winn
John K. Zehmer
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THERABRON THERAPEUTICS Inc
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Clarassance Inc
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Assigned to CLARASSANCE, INC. reassignment CLARASSANCE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZELMER, JOHN K, PILON, APRILE L, WINN, MELISSA E
Priority to RU2015144359A priority patent/RU2662665C2/ru
Priority to BR112015021265A priority patent/BR112015021265A2/pt
Priority to PCT/US2014/030101 priority patent/WO2014145357A2/en
Priority to UAA201509797A priority patent/UA118756C2/uk
Priority to SG11201507276UA priority patent/SG11201507276UA/en
Priority to CN201480015596.0A priority patent/CN105722524B/zh
Priority to JP2016503332A priority patent/JP6513632B2/ja
Priority to CA2907395A priority patent/CA2907395A1/en
Priority to EP14763146.9A priority patent/EP2968464B1/en
Priority to KR1020157029167A priority patent/KR20160011621A/ko
Priority to AU2014233116A priority patent/AU2014233116A1/en
Priority to MX2015013091A priority patent/MX2015013091A/es
Priority to ES14763146T priority patent/ES2883933T3/es
Publication of US20140274915A1 publication Critical patent/US20140274915A1/en
Assigned to THERABRON THERAPEUTICS INC. reassignment THERABRON THERAPEUTICS INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: CLARASSANCE INC.
Priority to IL241418A priority patent/IL241418A0/en
Priority to US14/880,908 priority patent/US9765127B2/en
Priority to US15/707,934 priority patent/US10556938B2/en
Priority to US16/785,221 priority patent/US11512121B2/en
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates

Definitions

  • the invention relates to pharmaceutical compositions, methods of production, analytical methods, and methods of use for secretoglobin proteins, including SCGB1A1 (CC10), SCGB3A1, and SCGB3A2. Novel physiologic roles and therapeutic uses for these secretoglobins have been identified.
  • the present invention relates to novel methods of use for rhCC10, rhSCGB3A2, and rhSCGB3A1 in preventing or delaying hospitalizations due to severe respiratory exacerbations up to 10 months after a course of treatment.
  • the present also relates to novel methods of production and pharmaceutical compositions of rhSCGB3A2 that is stable and possesses anti-inflammatory properties.
  • the invention further provides a method to prevent severe respiratory exacerbations by administering rhCC10.
  • the invention further provides a method for treating bronchiectasis and preventing exacerbations of bronchiectasis by administering rhSCGB3A2.
  • the invention provides a method for reversing airway remodeling in chronic lung diseases and preventing airway remodeling in acute lung injuries by administering rhCC10, rhSCGB3A2, or rhSCGB3A1.
  • these secretoglobins modify airway remodeling indirectly by restoring normal numbers of Clara cells and their associated structures, termed neuro-epithelial bodies (aka NEBs) or neuroendocrine cell clusters (aka NECs) that are identified by their immunoreactivity to anti-CGRP1 antibodies, in the airway epithelium.
  • NEBs neuro-epithelial bodies
  • NECs neuroendocrine cell clusters
  • the Clara cells and other CGRP1+ cells secrete these secretoglobins and other components of the normal mucosal milieu, contributing to homeostasis and normal functioning of the respiratory mucosa and epithelium that is then more resistant to inhaled challenges without experiencing severe exacerbations.
  • Natural human Clara Cell 10 kDa protein (CC10), also known as uteroglobin, Clara cell 16 kDa protein (CC16), Clara cell secretory protein (CCSP), blastokinin, urine protein-1, and secretoglobin 1A1 (SCGB1A1), is one of a family of related proteins called secretoglobins believed to exist in all vertebrate animals. There are two additional secretoglobins that are also expressed at very high levels in the respiratory tract, called SCGB3A1 and SCGB3A2 (Porter, 2002). These three proteins; SCGB1A1, SCGB3A1, and SCGB3A2, are herein referred to as “respiratory secretoglobins.” Table 1 shows Genebank loci and amino acid sequences for each respiratory secretoglobin.
  • respiratory secretoglobins are highly expressed in both the upper and lower respiratory tracts; the upper respiratory tract includes the nasal passages and sinuses and the lower respiratory tract includes the trachea, bronchi, and alveoli of the lungs. A significant amount of respiratory secretoglobins are also present in serum and urine, which is largely derived from pulmonary sources.
  • SCGB3A1 is also expressed in the stomach, heart, small intestine, uterine and mammary glands, and SCGB3A2 is expressed at a low level in the thyroid (Porter, 2002).
  • CC10 is also produced by reproductive tissues (uterus, seminal vesicles), exocrine glands (prostate, mammary gland, pancreas), endocrine glands (thyroid, pituitary, adrenal, and ovary) and by the thymus and spleen (Mukherjee, 1999; Mukherjee, 2007).
  • the major recoverable form of human CC10 in vivo is a homodimer, comprised of two identical 70 amino acid monomers, with an isoelectric point of 4.8.
  • All three respiratory secretoglobins may be made by synthetic (Nicolas, 2005) or recombinant methods (Mantile, 1993), although there have been no reports to date describing the successful synthesis of human SCGB3A1 and SCGB3A2 and the biochemical characterization of these proteins in vitro.
  • CC10 is an anti-inflammatory and immunomodulatory protein that has been characterized with respect to various interactions with other proteins, receptors and cell types (reviewed in Mukherjee, 2007, Mukherjee, 1999, and Pilon, 2000).
  • Lower levels of CC10 protein or mRNA have been found in various tissue and fluid samples for a number of clinical conditions characterized by some degree of inflammation including asthma (Lensmar, 2000; Shijubo, 1999; Van Vyve, 1995), pneumonia (Nomori, 1995), bronchiolitis obliterans (Nord, 2002), sarcoidosis (Shijubo, 2000), and in patients suffering from chronic rhinitis with recurrent sinusitis and nasal polyposis (Liu, 2004).
  • Pulmonary epithelial cells, the body's primary source for endogenous CC10 are often adversely affected in these conditions, depleted or even ablated (Shijubo, 1999).
  • CC10 knockout (KO) mice have been important in characterizing the role of CC10 in pulmonary homeostasis, reproduction, and certain types of renal disease.
  • One knockout strain exhibits several extreme phenotypes, including systemic inflammation, poor reproductive capability (small litter sizes), and a lethal renal phenotype resembling human IgA nephropathy (Zhang, 1997; Zheng, 1999).
  • the other knockout strain does not possess these extreme phenotypes and is more viable, enabling a greater number of experiments to be performed (Stripp, 1997).
  • both strains share an airway epithelial phenotype characterized by significantly decreased numbers of Clara cells and associated structures called neuro-epithelial bodies (NEBs; Lau, 2000) or neuro-endocrine cell clusters (NECs; Hong, 2001; Reynolds, 2000), as identified by positive staining with calcitonin-gene related protein 1 (CGRP1).
  • NEBs neuro-epithelial bodies
  • NECs neuro-endocrine cell clusters
  • CGRP1 calcitonin-gene related protein 1
  • rhCC10 premature infants who experience respiratory distress syndrome (RDS) are deficient in native CC10.
  • RDS respiratory distress syndrome
  • a single dose of rhCC10 was administered on the day of birth and mediated potent short-term anti-inflammatory effects for 3-7 days in the lungs.
  • Pharmacokinetic analyses showed that surplus CC10 was cleared within 48 hours of the single dose administered.
  • rhCC10 did not prevent development of neonatal bronchopulmonary dysplasia (BPD) (Levine, 2005), as defined by clinical parameters, including 1) opacity of chest X-ray at 28 days after birth or 2) use of supplemental oxygen at 36 weeks of postmenstrual age (PMA).
  • BPD neonatal bronchopulmonary dysplasia
  • PMA postmenstrual age
  • Severe respiratory exacerbations are characterized by shortness of breath, labored breathing, nasal and chest congestion, overproduction of mucus, and sometimes respiratory distress. Severe respiratory exacerbations occur when patients encounter environmental exposures and infections through inhalation of dust, smoke, allergens, pollutants, chemicals, bacteria, fungi, and viruses.
  • Environmental irritants that trigger exacerbations include, but are not limited to, dust, particulates, smoke, allergens, pollutants, chemicals, contaminants, bacteria, fungi, and viruses may be inhaled, ingested, swallowed, absorbed through the skin, or otherwise come in contact topically with a wet mucosal surface of the patient's body.
  • ALF respiratory airway lining fluids
  • MAS meconium aspiration syndrome
  • MAS meconium aspiration syndrome
  • MAS meconium aspiration syndrome
  • MAS meconium aspiration syndrome
  • Secretoglobin proteins that are expressed in the respiratory tract facilitate development of Clara cells and other respiratory epithelial cells and resident immune structures in the functional respiratory epithelium.
  • secretoglobins that are highly expressed in the human respiratory tract, including SCGB1A1 (aka CC10, uteroglobin, CCSP, CC16, etc.), SCGB3A2 (aka UGRP1, HIN-2) and SCGB3A1 (aka UGRP2, HIN-1).
  • the invention generally pertains to the use of respiratory secretoglobins to delay and prevent severe exacerbations of chronic diseases caused by environmental exposures, particularly respiratory diseases.
  • the respiratory secretoglobins mediate an increase in the numbers of secretoglobin-secreting cells and associated structures in respiratory tissues, which may be measured indirectly through increases in the amounts of their secretoglobin secretion products in body fluids.
  • rhCC10 administration mediates an increase in the number of Clara cells, NEBs, and NECs, restoring the respiratory airway epithelia.
  • rhCC10 did not avert the development of neonatal BPD, it did confer long-term protection from severe respiratory exacerbations requiring re-hospitalization was observed at 6 months PMA, which is the time at which the infant would have been 6 months old after 40 weeks gestation. Since the trial enrolled infants between 24-28 weeks PMA, this endpoint is up to 10 months after a single dose of rhCC10.
  • FIG. 1 Human SCGB3A2 amino acid sequences, alignment of human SCGB3A2 amino acid sequences with comparison of predicted and actual N-termini
  • FIG. 2 SDS-PAGE of purified rhSCGB3A2, SDS-PAGE of purified rhSCGB3A2.
  • Samples containing 5 micrograms each with and without 1 mM DTT were mixed with SDS Sample Buffer, boiled 5 minutes and loaded on a 10-20% tricine gel. The gel was run and stained with Coomassie R250. The gel was de-stained and imaged with a digital camera.
  • FIG. 3 Isoelectric focusing of purified rhSCGB3A2, Isoelectric focusing of purified rhSCGB3A2, compared to rhCC10 and UBL and Den-1. Samples containing 5 micrograms each were loaded on a Novex IEF gel. The gel was run and stained with Coomassie R250. The gel was de-stained and imaged with a digital camera. Arrows represent major and minor isoforms of rhSCGB3A2 with ATA N-terminus.
  • FIG. 4 In vitro inhibition of sPLA 2 -1B with rhSCGB3A2
  • Panel A UNIBIPY substrate; no PLA 2 ; no rhSCGB3A2.
  • Panel B UNIBIPY substrate plus PLA 2 ; no rhSCGB3A2.
  • Panel C UNIBIPY substrate plus PLA2 plus rhSCGB3A2. Peak #1 is the UNIBIPY phospholipid substrate, peak #2 is the product after cleavage by sPLA 2 .
  • FIG. 5 Western blot of SCGB3A2 in human TAF.
  • Western blot of tracheal aspirate fluids from human infants compared to purified rhSCGB3A2 using anti-rhSCGB3A2 rabbit polyclonal antibody.
  • Samples containing 20 microliters of each TAF were loaded on a Novex 10-20% tricine gel; rhSCGB3A2 is in lane 1 (5 nanograms) and lane 8 (1 nanogram). The gel was de-stained and imaged with a digital camera.
  • CC10 and SCGB3A2 are structurally similar, and, therefore, believed to share some functions, there are no reports pertaining to the stimulation of growth or development of airway epithelial cells by CC10, and rhCC10 is, in fact, well-known to suppress the growth of tumor cells of epithelial origin (Kundu, 1996; Leyton, 1994), including an airway epithelial cell line, A549 (Szabo, 1998).
  • rhCC10 administered to premature infants on the day of birth stimulated the development of CC10-secreting cells, which, in turn, produced native CC10, which stimulated development of more CC10-secreting cells, and so on.
  • the end result was a more normal and resilient respiratory epithelium in the rhCC10-treated infants who were more resistant to all environmental challenges (dust, smoke, allergens, RSV infection, influenza infection, etc.) compared to the placebo-treated infants.
  • a single dose of rhCC10 on the day of birth conferred 100% protection from re-hospitalization due to severe respiratory exacerbation, contrasting the 50% re-hospitalization rate observed in the placebo-treated infants.
  • CC10 to stimulate development of CC10-secreting cells in the respiratory epithelium will also work in adults with chronic respiratory diseases in which airway remodeling has resulted in loss of Clara cells.
  • a course of treatment with rhCC10 may not cure the disease, but, we believe, would restore, to some extent, Clara cells and associated structures, resulting in a more normal epithelium that is then more resistant to subsequent environmental challenges.
  • the clinical outcome of a course of rhCC10 treatment would then be an increase in the time interval to the next severe exacerbation.
  • CC10 is an autocrine and paracrine factor required for the development of Clara cells, associated structures, and other normal cell populations of the airway epithelium.
  • CC10 is an autocrine and paracrine factor required for the development and maintenance of CC10-secreting cells outside of the respiratory tract, including the gastrointestinal tract and urogenital tract.
  • secretoglobins share structural similarities that they will also share similar function, however, no biological activity has ever been previously shown to be shared between any two secretoglobins either in vitro or in vivo.
  • rhSCGB3A2 shares with CC10, the ability to inhibit porcine pancreatic phospholipase A 2 in vitro. This is the first report that any other secretoglobin, besides CC10, actually inhibits any phospholipase A 2 enzyme or possesses any type of anti-inflammatory activity. Based on these results, we infer that other secretoglobins, including respiratory secretoglobins, which share structural similarities with rhCC10, can stimulate the development and maintenance of the cells that secrete them to effect long-term clinical benefits such as increased time to next exacerbation, decreased severity of next exacerbation, and prevention of severe exacerbations following acute injury.
  • RDS respiratory distress syndrome
  • rhCC10-treated infants showed significant reductions in TAF total cell counts (P ⁇ 0.001), neutrophil counts (P ⁇ 0.001), and total protein concentrations (P ⁇ 0.01) and decreased IL-6 (P ⁇ 0.07) over the first 3 days of life.
  • the rhCC10 was safe and well tolerated.
  • FIG. 2 shows the amino acid sequence of rhSCGB3A2 that was made for these studies.
  • the sequence was taken from Genebank locus AAQ89338.
  • UBL ubiquitin-like
  • a synthetic DNA coding sequence for rhSCGB3A2 was designed using jcat (www.jcat.de), with codon usage optimized for expression in E. coli bacteria K12 strain. Once the DNA sequence was generated, restriction sites were added to the ends to facilitate directional cloning of the gene into the bacterial expression vector, pTXB1, already containing the UBL. SCGB3A2 was cloned as a C-terminal extension of the UBL. An AfIII site was placed at the 5′ end and a BamHI site was placed at the 3′ end for directional cloning.
  • the new gene for rhSCGB3A2 was synthesized from overlapping oligonucleotides using PCR.
  • the DNA sequence for the rhSCGB3A2 gene is:
  • the pTXB1 plasmid containing the UBL-rhSCGB3A2 fusion was transformed into E. coli strain HMS174/DE3 which contains a DE3 prophage encoding the T7 RNA polymerase that enables inducible expression of the fusion protein. Colonies were screened for expression of the fusion protein and the rhSCGB3A2 gene was reconfirmed by DNA sequencing in high expressers.
  • a four liter fermentation culture containing SuperBroth media with ampicillin was inoculated from a 120 ml overnight culture of the highest-expressing clone and grown at 37° C.
  • the culture was induced to overexpress the UBL-rhSCGB3A2 fusion protein at an OD 600 of 8.75 using 0.3 mM IPTG, then allowed to grow for another 2 hours.
  • Cell paste was harvested by centrifugation and the wet cell paste yield was 67 grams. The cell paste was then used for purification of rhSCGb3A2.
  • the cell paste was resuspended in 20 mM NaH 2 PO 4 , 0.5 M NaCl, pH 7.2, then the cells were ruptured by freeze-thaw to generate a crude lysate.
  • the crude lysate was clarified by centrifugation at 19,800 ⁇ g for 20′ at 4° C. DNA, endotoxin, and other bacterial contaminants were precipitated out of the clarified lysate supernatant using polyethylimine (PEI) at a concentration of 0.025% and a second centrifugation at 19,800 ⁇ g for 10′ at 4° C.
  • PEI polyethylimine
  • the PEI supernatant was then filtered through a 0.22 micron filter and 10 mM imidazole was added to the filtrate.
  • Both the UBL and the UBL protease contain a histidine tag so that they bind to an immobilized metal affinity chromatography column.
  • the filtrate containing the UBL-rhSCGB3A2 fusion protein was then passed over an IMAC column (nickel chelating sepharose fast flow) previously equilibrated in 20 mM NaH 2 PO 4 , 0.5 M NaCl, 10 mM imidazole, pH 7.2, the column was washed with the same buffer, then the UBL-rhSCGB3A2 fusion protein was eluted with 20 mM NaH 2 PO 4 , 100 mM NaCl, 300 mM imidazole, pH 7.2.
  • IMAC column nickel chelating sepharose fast flow
  • the IMAC eluate was then concentrated and buffer exchanged using tangential flow filtration with a 5 kDa NMWCO filter in 15 mM Tris, 15 mM BisTris, 40 mM NaCl, pH 7.0.
  • the UBL-rhSCGB3A2 was further purified over a Macro Prep High Q column (BioRad) in which contaminants were bound and the UBL-rhSCGB3A2 flowed through.
  • the rhSCGB3A2 was then separated from the UBL by digestion with UBL protease Den-1 (1:100 molar ratio) in 5 mM DTT, with pH adjusted to 6.5 with HCl, at 37° C. for 2 hours.
  • the rhSCGB3A2 was then purified from the digestion mixture using cation exchange chromatography (GE Sepharose SP High Performance).
  • the SP column was equilibrated with 15 mM Tris, 15 mM BisTris, 40 mM NaCl, pH 6.5, the digestion mixture loaded, and contaminants bound to the column while rhSCGB3A2 flowed through.
  • the SP flow through was then extensively dialyzed against 0.9% NaCl using a 3.5 kDa MWCO regenerated cellulose membrane.
  • the sample was concentrated using centrifugal concentrators (3.5 kDa MWCO), then filtered through a 0.22 micron filter.
  • the filtrate was purified rhSCGB3A2.
  • the isoelectric point (pi) of a protein is a measure of the total surface charge of that protein. pl is measured using standard isoelectric focusing (IEF) methods. Approximately 5 micrograms of rhSCGB3A2, rhCC10, UBL, and Den-1 were loaded onto an IEF gel (Novex) in order to determine the pl of rhSCGB3A2 as shown in FIG. 3 . When a protein migrates as a single band on SDS-PAGE and multiple bands are observed in the IEF gel, alternate isoforms of the protein are likely present. In contrast to rhCC10, which shows a single band at pl 4.8, rhSCGB3A2 shows two bands at pl 6.7 and 6.3.
  • the predicted pl of our rhSCGB3A2 sequence is 6.1 (www.expasy.edu; Protein tool “Compute MW/pl”), yet the vast majority of the protein migrates at a position corresponding to a pl of 6.7. Not even the minor band at 6.3 corresponds to the predicted pl of 6.1. That there are two rhSCGB3A2 IEF bands means that either alternatively folded isoforms are present or that they represent monomers and dimers, as visualized in non-reducing SDS-PAGE.
  • this preparation is an unknown and unpredicted isoform of rhSCGB3A2 that is unique.
  • the unique folding pattern of a recombinant protein is often determined by the synthetic process, in this case, the selection of N-terminus, expression of the protein as a C-terminal fusion with an ubiquitin-like protein, IMAC purification of the fusion protein, cleavage of the SCGB3A2 from the UBL, and separation of the SCGB3A2 from the UBL and UBL-protease.
  • the uniqueness of this preparation may be due to the synthetic process, the non-native N-terminus, or a combination of these or other unknown factors.
  • the biological activity of rhSCGB3A2 was evaluated in a fluorescent and quantitative HPLC assay that evaluates inhibition of porcine pancreatic secretory PLA 2 enzyme (sPLA 2 ) that was developed to evaluate the potency of different batches of rhCC10. Inhibition of PLA 2 enzymes is thought to be a major anti-inflammatory mechanism of action for CC10. Many have speculated that other secretoglobins may also inhibit PLA 2 enzymes, due to their structural similarities with CC10.
  • the rhSCGB3A2 (5.5 micrograms) was mixed with of 100 nanograms porcine sPLA 2 1B (0.1 microgram) and incubated at 37° C.
  • the reaction was started through the addition of the fluorescent phospholipid analogue 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecylysn-glycero-3-phosphocholine (aka UNIBIPY; 47.6 nanograms).
  • the reaction was terminated by the addition of 2-propanol/n-hexane.
  • the cleavage product was separated from the substrate on a Waters Spherisorb silica HPLC column. The separation was followed with a G1321A fluorescence detector.
  • Results of the assay are shown in FIG. 4 .
  • Panel A shows the UNIBIPY substrate without sPLA 2 or rhSCGB3A2;
  • panel B shows the UNIBIPY substrate plus sPLA 2 , and
  • panel C shows the UNIBIPY substrate plus sPLA 2 plus rhSCGB3A2.
  • the sPLA 2 cleaves the substrate (peak #1), giving rise to a product (peak #2).
  • peak #2 peak #2
  • the product peak is significantly reduced.
  • Each reaction set was run in duplicate.
  • the rhSCGB3A2 showed 83% inhibition of sPLA 2 -1B activity in the assay, which is comparable to rhCC10 protein (data not shown).
  • Purified rhSCGB3A2 was used to immunize two New Zealand white rabbits, using a standard immunization protocol.
  • the protein was conjugated to KLH, mixed with Freund's adjuvant, and injected into the animals. Both animals produced excellent antibody responses with very high titers.
  • IgG was purified from each set of animal sera using a Pierce Protein A IgG Purification Kit and the purified IgGs were dialyzed into PBS, pH 7.2, aliquoted and stored at ⁇ 80° C.
  • the antibodies were qualified by Western blot using tracheal aspirate fluids (TAF) obtained from premature human infants.
  • TAF tracheal aspirate fluids
  • Samples containing 20 microliters of TAF from 6 infants were run on non-reducing SDS-PAGE and compared to rhSCGB3A2 (5 nanograms).
  • the gel was electro-blotted to PVDF membrane, blocked with 4% non-fat milk, then the highest titer rabbit anti-rhSCGB3A2 IgG (1:5000 dilution) was incubated with the blot, followed by a goat anti-rabbit-HRP conjugate (1:20,000 dilution).
  • the blot was developed using enhanced chemiluminescence (4IPBA-ECL—100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 5.3 mM hydrogen peroxide and 2 mM 4IPBA).
  • Immunoreactive bands appeared in 5/6 of the TAF samples.
  • Two of the samples, (lane 3 and lane 6) contained bands that migrated at the same size as the rhSCGB3A2 homodimer, indicating that the rhSCGB3A2 preparation resembled native human SCGB3A2 in some patients.
  • Heterologous expression of recombinant proteins, especially hydrophobic proteins, for use in animal or human studies often yields misfolded, inactive, immunogenic, or otherwise unusable preparations.
  • a competitive ELISA was developed using standard methods.
  • the antibody that captures the target is coated onto the wells of the microtiter plate, then an enzyme-conjugated target molecule (labeled target) is used to compete with unconjugated target in the sample for binding to available sites in the well.
  • labeled target an enzyme-conjugated target molecule
  • the rabbit anti-rhSCGB3A2 antibody was coated onto 96 well Maxisorb plates (200 ng/well) then the wells were blocked with 5% sucrose, 2.5% BSA in PBS, then plates are dried and stored at 4° C.
  • HRP horse radish peroxidase
  • rhSCGB3A2 was made (Pierce kit—EZ-Link Maleimide Activated HRP kit, Cat#31494) and was used in the assay diluted 1:130,000. Calibrators (1-500 ng) were made using rhSCGB3A2 and the standard curve was generated as shown in FIG. 6 . Native SCGB3A2 was then quantitated in human TAF samples as shown in Table 3.

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UAA201509797A UA118756C2 (uk) 2013-03-15 2014-03-16 Композиція для лікування захворювань дихальних шляхів
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WO2018178051A1 (en) * 2017-03-28 2018-10-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Scgb1a1 polymorphism for the prediction and therapy or prevention of primary graft dysfunction
WO2019143934A1 (en) 2018-01-19 2019-07-25 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-cancer activity of scbg3a2 and lps

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KR20200138684A (ko) * 2020-11-17 2020-12-10 김유신 토양 파쇄기를 장착한 친환경 고온스팀 토양소독기
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