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Pharmaceutical Research

, Volume 32, Issue 7, pp 2439–2449 | Cite as

Human Monoclonal Antibody Fragments Targeting Matrilin-3 in Growth Plate Cartilage

  • Crystal Sao-Fong Cheung
  • Zhongyu Zhu
  • Julian Chun-Kin Lui
  • Dimiter Dimitrov
  • Jeffrey Baron
Research Paper

Abstract

Purpose

Many genetic disorders, including chondrodysplasias, and acquired disorders impair growth plate function, resulting in short and sometimes malformed bones. There are multiple endocrine and paracrine factors that promote chondrogenesis at the growth plate, which could potentially be used to treat these disorders. Targeting these growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues.

Methods

Using yeast display technology, we selected single-chain variable antibody fragments that bound to human and mouse matrilin-3, an extracellular matrix protein specifically expressed in cartilage tissue. The ability of the selected antibody fragments to bind matrilin-3 and to bind cartilage tissue in vitro and in vivo was assessed by ELISA and immunohistochemistry.

Results

We identified antibody fragments that bound matrilin-3 with high affinity and also bound with high tissue specificity to cartilage homogenates and to cartilage structures in mouse embryo sections. When injected intravenously in mice, the antibody fragments specifically homed to cartilage.

Conclusions

Yeast display successfully selected antibody fragments that are able to target cartilage tissue in vivo. Coupling these antibodies to chondrogenic endocrine and paracrine signaling molecules has the potential to open up new pharmacological approaches to treat childhood skeletal growth disorders.

KEY WORDS

childhood growth drug targeting scFv skeletal diseases yeast display 

ABBREVIATION

ANOVA

Analysis of variance

BMP

Bone morphogenetic protein

BSA

Bovine serum albumin

CNP

C-type natriuretic peptide

DAB

3,3′-diaminobenzidine

EC50

Half maximal effective concentration

EDTA

Ethylenediaminetetraacetic acid

ELISA

Enzyme-linked immunosorbent assay

FCS

Fetal calf serum

GH

Growth hormone

HRP

Horse radish peroxidase

HZ

Hypertrophic zone

IGF

Insulin-like growth factor

IHH

Indian hedgehog

PBS

Phosphate buffered saline

PZ

Proliferative zone

RZ

Resting zone

scFv

Single-chain variable fragment

TMB

3,3′,5,5′-Tetramethylbenzidine

Notes

Acknowledgments

This work was supported by the Intramural Research Programs of the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Cancer Institute, National Institutes of Health. C.S.C., Z.Z., J.C.L., D.D. and J.B are co-inventors in a provisional patent application (U.S. Patent Application No. 61/927,904) submitted by the National Institutes of Health.

Supplementary material

11095_2015_1636_Fig6_ESM.gif (19 kb)
Figure S1

Coomassie-blue stained SDS-PAGE gel showing the purity of antibody fragments after protein A column chromatography. 5 μg of purified protein were loaded in each sample lane. (GIF 18 kb)

11095_2015_1636_MOESM1_ESM.tif (5.5 mb)
(TIFF 5605 kb)

References

  1. 1.
    Ranke MB, Lindberg A, Mullis PE, Geffner ME, Tanaka T, Cutfield WS, et al. Towards optimal treatment with growth hormone in short children and adolescents: evidence and theses. Horm Res Paediatr. 2013;79:51–67.CrossRefPubMedGoogle Scholar
  2. 2.
    Ranke MB. Human growth hormone therapy of non-growth hormone deficient children. Pediatrician. 1987;14:178–82.PubMedGoogle Scholar
  3. 3.
    Wilson TA, Rose SR, Cohen P, Rogol AD, Backeljauw P, Brown R, et al. Update of guidelines for the use of growth hormone in children: the Lawson Wilkins Pediatric Endocrinology Society Drug and Therapeutics Committee. J Pediatr. 2003;143:415–21.CrossRefPubMedGoogle Scholar
  4. 4.
    Wang SY, Tung YC, Tsai WY, Chien YH, Lee JS, Hwu WL. Slipped capital femoral epiphysis as a complication of growth hormone therapy. J Formos Med Assoc. 2007;106:S46–50.CrossRefPubMedGoogle Scholar
  5. 5.
    Canete R, Valle M, Martos R, Sanchez-Carrion A, Canete MD, van Donkelaar EL. Short-term effects of GH treatment on coagulation, fibrinolysis, inflammation biomarkers, and insulin resistance status in prepubertal children with GH deficiency. Eur J Endocrinol. 2012;167:255–60.PubMedGoogle Scholar
  6. 6.
    Darendeliler F, Karagiannis G, Wilton P. Headache, idiopathic intracranial hypertension and slipped capital femoral epiphysis during growth hormone treatment: a safety update from the KIGS database. Horm Res. 2007;68 Suppl 5:41–7.CrossRefPubMedGoogle Scholar
  7. 7.
    Cutfield WS, Wilton P, Bennmarker H, Albertsson-Wikland K, Chatelain P, Ranke MB, et al. Incidence of diabetes mellitus and impaired glucose tolerance in children and adolescents receiving growth-hormone treatment. Lancet. 2000;355:610–3.CrossRefPubMedGoogle Scholar
  8. 8.
    Yuen KC, Chong LE, Riddle MC. Influence of glucocorticoids and growth hormone on insulin sensitivity in humans. Diabet Med. 2013;30:651–63.CrossRefPubMedGoogle Scholar
  9. 9.
    Swerdlow AJ, Higgins CD, Adlard P, Preece MA. Risk of cancer in patients treated with human pituitary growth hormone in the UK, 1959-85: a cohort study. Lancet. 2002;360:273–7.CrossRefPubMedGoogle Scholar
  10. 10.
    Chau M, Forcinito P, Andrade AC, Hegde A, Ahn S, Lui JC, et al. Organization of the Indian hedgehog–parathyroid hormone-related protein system in the postnatal growth plate. J Mol Endocrinol. 2011;47:99–107.CrossRefPubMedGoogle Scholar
  11. 11.
    Kronenberg HM. PTHrP and skeletal development. Ann N Y Acad Sci. 2006;1068:1–13.CrossRefPubMedGoogle Scholar
  12. 12.
    Maeda Y, Nakamura E, Nguyen MT, Suva LJ, Swain FL, Razzaque MS, et al. Indian Hedgehog produced by postnatal chondrocytes is essential for maintaining a growth plate and trabecular bone. Proc Natl Acad Sci U S A. 2007;104:6382–7.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Long F, Joeng KS, Xuan S, Efstratiadis A, McMahon AP. Independent regulation of skeletal growth by Ihh and IGF signaling. Dev Biol. 2006;298:327–33.CrossRefPubMedGoogle Scholar
  14. 14.
    Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis AC. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol. 1994;126:1611–23.CrossRefPubMedGoogle Scholar
  15. 15.
    Nilsson O, Parker EA, Hegde A, Chau M, Barnes KM, Baron J. Gradients in bone morphogenetic protein-related gene expression across the growth plate. J Endocrinol. 2007;193:75–84.CrossRefPubMedGoogle Scholar
  16. 16.
    De LF, Barnes KM, Uyeda JA, De-Levi S, Abad V, Palese T, et al. Regulation of growth plate chondrogenesis by bone morphogenetic protein-2. Endocrinology. 2001;142:430–6.Google Scholar
  17. 17.
    Yoon BS, Pogue R, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, et al. BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways. Development. 2006;133:4667–78.CrossRefPubMedGoogle Scholar
  18. 18.
    Yoon BS, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, Lyons KM. Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Proc Natl Acad Sci U S A. 2005;102:5062–7.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kobayashi T, Lyons KM, McMahon AP, Kronenberg HM. BMP signaling stimulates cellular differentiation at multiple steps during cartilage development. Proc Natl Acad Sci U S A. 2005;102:18023–7.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Olney RC. C-type natriuretic peptide in growth: a new paradigm. Growth Horm IGF Res. 2006;16(Suppl A):S6–14.CrossRefPubMedGoogle Scholar
  21. 21.
    Olney RC, Bukulmez H, Bartels CF, Prickett TC, Espiner EA, Potter LR, et al. Heterozygous mutations in natriuretic peptide receptor-B (NPR2) are associated with short stature. J Clin Endocrinol Metab. 2006;91:1229–32.CrossRefPubMedGoogle Scholar
  22. 22.
    Agoston H, Khan S, James CG, Gillespie JR, Serra R, Stanton LA, et al. C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and -independent pathways. BMC Dev Biol. 2007;7:18.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mericq V, Uyeda JA, Barnes KM, De LF, Baron J. Regulation of fetal rat bone growth by C-type natriuretic peptide and cGMP. Pediatr Res. 2000;47:189–93.CrossRefPubMedGoogle Scholar
  24. 24.
    Teixeira CC, Agoston H, Beier F. Nitric oxide, C-type natriuretic peptide and cGMP as regulators of endochondral ossification. Dev Biol. 2008;319:171–8.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Yates KE, Shortkroff S, Reish RG. Wnt influence on chondrocyte differentiation and cartilage function. DNA Cell Biol. 2005;24:446–57.CrossRefPubMedGoogle Scholar
  26. 26.
    Andrade AC, Nilsson O, Barnes KM, Baron J. Wnt gene expression in the post-natal growth plate: regulation with chondrocyte differentiation. Bone. 2007;40:1361–9.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Yang Y, Topol L, Lee H, Wu J. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development. 2003;130:1003–15.CrossRefPubMedGoogle Scholar
  28. 28.
    Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, et al. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev. 2004;18:1072–87.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lui JC, Nilsson O, Chan Y, Palmer CD, Andrade AC, Hirschhorn JN, et al. Synthesizing genome-wide association studies and expression microarray reveals novel genes that act in the human growth plate to modulate height. Hum Mol Genet. 2012;21:5193–201.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Lui JC, Andrade AC, Forcinito P, Hegde A, Chen W, Baron J, et al. Spatial and temporal regulation of gene expression in the mammalian growth plate. Bone. 2010;46:1380–90.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Klatt AR, Nitsche DP, Kobbe B, Morgelin M, Paulsson M, Wagener R. Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development. J Biol Chem. 2000;275:3999–4006.CrossRefPubMedGoogle Scholar
  32. 32.
    Forcinito P, Andrade AC, Finkielstain GP, Baron J, Nilsson O, Lui JC. Growth-inhibiting conditions slow growth plate senescence. J Endocrinol. 2011;208:59–67.CrossRefPubMedGoogle Scholar
  33. 33.
    Zhang MY, Shu Y, Phogat S, Xiao X, Cham F, Bouma P, et al. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J Immunol Methods. 2003;283:17–25.CrossRefPubMedGoogle Scholar
  34. 34.
    Horton WA, Hall JG, Hecht JT. Achondroplasia. Lancet. 2007;370:162–72.CrossRefPubMedGoogle Scholar
  35. 35.
    Yasoda A, Komatsu Y, Chusho H, Miyazawa T, Ozasa A, Miura M, et al. Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nat Med. 2004;10:80–6.CrossRefPubMedGoogle Scholar
  36. 36.
    Igaki T, Itoh H, Suga SI, Hama N, Ogawa Y, Komatsu Y, et al. Effects of intravenously administered C-type natriuretic peptide in humans: comparison with atrial natriuretic peptide. Hypertens Res. 1998;21:7–13.CrossRefPubMedGoogle Scholar
  37. 37.
    Yokogawa K, Miya K, Sekido T, Higashi Y, Nomura M, Fujisawa R, et al. Selective delivery of estradiol to bone by aspartic acid oligopeptide and its effects on ovariectomized mice. Endocrinology. 2001;142:1228–33.CrossRefPubMedGoogle Scholar
  38. 38.
    Millan JL, Narisawa S, Lemire I, Loisel TP, Boileau G, Leonard P, et al. Enzyme replacement therapy for murine hypophosphatasia. J Bone Miner Res. 2008;23:777–87.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Yadav MC, Lemire I, Leonard P, Boileau G, Blond L, Beliveau M, et al. Dose response of bone-targeted enzyme replacement for murine hypophosphatasia. Bone. 2011;49:250–6.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Bhandari KH, Newa M, Uludag H, Doschak MR. Synthesis, characterization and in vitro evaluation of a bone targeting delivery system for salmon calcitonin. Int J Pharm. 2010;394:26–34.CrossRefPubMedGoogle Scholar
  41. 41.
    Bhandari KH, Newa M, Chapman J, Doschak MR. Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats. J Control Release. 2012;158:44–52.CrossRefPubMedGoogle Scholar
  42. 42.
    Yang Y, Bhandari KH, Panahifar A, Doschak MR. Synthesis, characterization and biodistribution studies of (125)I-radioiodinated di-PEGylated bone targeting salmon calcitonin analogue in healthy rats. Pharm Res. 2014;31:1146–57.CrossRefPubMedGoogle Scholar
  43. 43.
    Doschak MR, Kucharski CM, Wright JE, Zernicke RF, Uludag H. Improved bone delivery of osteoprotegerin by bisphosphonate conjugation in a rat model of osteoarthritis. Mol Pharm. 2009;6:634–40.CrossRefPubMedGoogle Scholar
  44. 44.
    Rothenfluh DA, Bermudez H, O’Neil CP, Hubbell JA. Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nat Mater. 2008;7:248–54.CrossRefPubMedGoogle Scholar
  45. 45.
    Hughes C, Faurholm B, Dell’Accio F, Manzo A, Seed M, Eltawil N, et al. Human single-chain variable fragment that specifically targets arthritic cartilage. Arthritis Rheum. 2010;62:1007–16.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Cheung CS, Lui JC, Baron J. Identification of chondrocyte-binding peptides by phage display. J Orthop Res. 2013;31:1053–8.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York (outside the USA) 2015

Authors and Affiliations

  • Crystal Sao-Fong Cheung
    • 1
  • Zhongyu Zhu
    • 2
  • Julian Chun-Kin Lui
    • 1
  • Dimiter Dimitrov
    • 2
  • Jeffrey Baron
    • 1
  1. 1.Section on Growth and DevelopmentNational Institute of Child Health and Development, National Institutes of HealthBethesdaUSA
  2. 2.Cancer and Inflammation ProgramNational Cancer InstituteFrederickUSA

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