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

Cheung, Catherine; Zhu, Zhongyu; Lui, Julian C.; Dimitrov, Dimiter S.; Baron, Jeffrey

doi:10.1007/s11095-015-1636-z

Cited by 2 publications

(5 citation statements)

References 48 publications

(75 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We previously used yeast display to identify scFv antibody fragments with high affinity and specificity toward the cartilage-specific protein matrilin-3, and we showed that these antibody fragments can home to growth plate cartilage when administered in vivo. 3 In the current study, we created fusion proteins combining these antibody fragments with human IGF-1, and we showed that at least two of these fusion proteins (IGF1-c22 and -c26) retained both cartilage-binding ability and IGF-1 biological activity. Using an ex vivo metatarsal bone culture system, we found that these IGF-1 fusion proteins can stimulate whole-bone growth.…”

Section: Discussionmentioning

confidence: 74%

“…We previously identified three clones of cartilage-targeting singlechain variable (scFv) antibody fragments (c13, c22, and c26). 3 In the current study, we used these cartilage-targeting scFvs to generate fusion proteins of cartilage-targeting IGF-1 (hereafter referred to as IGF1-c13, IGF1-c22, and IGF1-c26; Figure 1A), and we tested their potential to deliver IGF-1 to the growth plate cartilage. To this end, we first used ELISA to assess the ability of our fusion proteins to bind to matrilin-3 (both human and mouse).…”

Section: Igf-1 Fusion Proteins Retained Cartilage-binding Abilitymentioning

confidence: 99%

“…These antibody fragments had previously been derived using yeast display technology. 3 To generate fusion proteins, these antibody fragments were each placed on the C-terminal side of a sequence encoding the human Fc antibody region and cloned into plasmid pSecTAG designed for the secretion and purification of fusion proteins. The DNA sequence for mature human IGF-1, which is 70 amino acids long and differs from the mouse IGF-1 by 2 amino acids at the C terminus, was constructed by overlapping PCR and inserted into pSecTAG at the N terminus of the human Fc, separated by a short linker Gly-Gly-Gly-Ser)x3 (G4S).…”

Section: Construction Of Igf-1 Fusion Proteinsmentioning

confidence: 99%

“…We hypothesized that targeting chondrogenesis-promoting factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues. To develop cartilage-targeting therapy, we recently identified several antibody fragments 3 that bind with high affinity to human and mouse matrilin-3, which is an extracellular matrix protein expressed with high tissue specificity in cartilage. 4 In this study, we generated fusion proteins conjugating these antibody fragments with IGF-1, and then we tested the hypothesis that the fusion protein would target the growth plate and, thereby, increase therapeutic efficacy and decrease off-target actions on other tissues.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth

et al. 2019

Self Cite

View full text Add to dashboard Cite

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy, particularly in severe, non-GH-deficient conditions such as chondrodysplasias, and potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we developed a cartilage-targeting single-chain human antibody fragment (CaAb) aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. To this end, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor 1 (IGF-1), an endocrine and/or paracrine factor that positively regulates chondrogenesis. These CaAb-IGF-1 fusion proteins retained both cartilage binding and IGF-1 biological activity, and they were able to stimulate bone growth in an organ culture system. Using a GH-deficient (lit) mouse model, we found that subcutaneous injections of these CaAb-IGF-1 fusion proteins increased overall growth plate height without increasing proliferation in kidney cortical cells, suggesting on-target efficacy at the growth plate and less off-target effect on the kidney than IGF-1 alone. Alternate-day injections of these fusion proteins, unlike IGF-1 alone, were sufficient to produce a therapeutic effect. Our findings provide proof of principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders.

show abstract

Section: Discussionmentioning

confidence: 74%

Section: Igf-1 Fusion Proteins Retained Cartilage-binding Abilitymentioning

confidence: 99%

Section: Construction Of Igf-1 Fusion Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…Wile-type SPIN1, wild-type SPIN4, or SPIN4 mutant were cloned into pSecTag plasmid containing a secretary peptide sequence so that the expressed proteins are secreted into the culture media. An antibody Fc region was inserted at the carboxyl terminus, connected with a G4S linker (37) to allow protein purification using protein A resin as previously described (38). Briefly, Protein A resin (GenScript, Piscataway, NJ) slurry (1-5 ml) was packed into a glass Econo-column (Bio-RAD), and equilibrated with 50 ml of binding/ washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 8.0).…”

Section: Purification Of Fc-tagged Spin4 Proteinsmentioning

confidence: 99%

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome

Lui¹,

Wagner²,

Zhou³

et al. 2023

JCI Insight

Self Cite

View full text Add to dashboard Cite

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes.Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth, and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth. 4like domains, with SPIN1, an epigenetic reader which recognizes histone H3 trimethylated at lysine-4 (8, 9) and H3 asymmetrically dimethylated at arginine-8 (10). SPIN1 has also been shown to stimulate Wnt signaling and promote cell proliferation and cancer progression (11,12).We evaluated a male individual with generalized overgrowth of prenatal onset, which, at the time of clinical presentation, included extreme tall stature, enlarged liver and spleen, and macrocephaly but no developmental delay. Exome sequencing identified a protein-truncating frameshift variant in SPIN4. In mice, ablation of Spin4 resulted in overgrowth of multiple organs and overall body size, recapitulating the human phenotype. We found that wild-type SPIN4 was able to bind specifically modified histone H3, promote canonical WNT signaling, and inhibit cell proliferation in vitro, but that the variant of SPIN4, which lacks two of the three Tudor-like domains, had lost all of these functions. The findings indicate that SPIN4 normally serves as an epigenetic reader which negatively regulates body size in mice and humans, and, therefore, loss of function of Spin4/SPIN4 increases somatic growth. Results Proband and the familyThe proband was a 13-year-old boy who was referred for evaluation of an overgrowth syndrome. He was born large for gestational age, with a birth weight of 5.85 kg (+4.3 SDS) and a length of 62 cm (+4.8 SDS). The mother had no history of gestational diabetes. He has been growing remarkably with height tracking between +4.5 and +5 SDS, although his mid-parental height was +1.2 SDS (Figure 1A). His bone age was advanced by one year; 13 years...

show abstract

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

Cited by 2 publications

References 48 publications

Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth

Cartilage-Targeted IGF-1 Treatment to Promote Longitudinal Bone Growth

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome

Contact Info

Product

Resources

About