Frederick W. Jacobsen

Stem cell factor is encoded at the SI locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.

Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor MpI

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

Parkinson's Disease-associated α-Synuclein Is More Fibrillogenic than β- and γ-Synuclein and Cannot Cross-seed Its Homologs

Parkinsons disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in α-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes β- and γ-synuclein. α-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for β- or γ-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between α- and β-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of α-, β-, and γ- synuclein show the same natively unfolded structure. While over time α-synuclein forms the previously described fibrils, no fibrils could be detected for β- and γ-synuclein under the same conditions. Most importantly, β- and γ-synuclein could not be cross-seeded with α-synuclein fibrils. However, under conditions that drastically accelerate aggregation, γ-synuclein can form fibrils with a lag phase roughly three times longer than α-synuclein. These results indicate that β- and γ-synuclein are intrinsically less fibrillogenic than α-synuclein and cannot form mixed fibrils with α-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to α-synuclein and are also abundant in brain.

A bispecific antibody targeting sclerostin and DKK-1 promotes bone mass accrual and fracture repair

Inhibition of the Wnt antagonist sclerostin increases bone mass in patients with osteoporosis and in preclinical animal models. Here we show increased levels of the Wnt antagonist Dickkopf-1 (DKK-1) in animals treated with sclerostin antibody, suggesting a negative feedback mechanism that limits Wnt-driven bone formation. To test our hypothesis that co-inhibition of both factors further increases bone mass, we engineer a first-in-class bispecific antibody with single residue pair mutations in the Fab region to promote efficient and stable cognate light–heavy chain pairing. We demonstrate that dual inhibition of sclerostin and DKK-1 leads to synergistic bone formation in rodents and non-human primates. Furthermore, by targeting distinct facets of fracture healing, the bispecific antibody shows superior bone repair activity compared with monotherapies. This work supports the potential of this agent both for treatment and prevention of fractures and offers a promising therapeutic approach to reduce the burden of low bone mass disorders.

Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

The physiological role of Dickkopf‐1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1‐Ab) that blocked Dkk1 binding to both low density lipoprotein receptor‐related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1‐Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.

Development of a novel mammalian cell surface antibody display platform

Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500 fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.

Rapid LC-MS screening for IgG Fc modifications and allelic variants in blood.

A new method for simultaneously screening allelic variants and certain Fc modifications on endogenous human IgG1 and IgG2 directly from blood samples is described. The IdeS endoproteinase was used to cleave IgG in serum to generate Fc, which, after denaturation, was analyzed directly as monomeric Fc (Fc/2) by LC-MS to identify the haplotype(s) present in each individual. The relative levels of IgG isotype and haplotype ratios were generated along with the profile of the major Fc glycans and several other modifications associated with each IgG1 or IgG2 haplotype. Since only minute quantities (5 μL) of blood are required and analysis can be highly automated, this approach lends itself to screening large populations. We demonstrate its utility in examining possible correlations between Fc properties and allelic variants. IgG1 core fucosylation, which significantly impacts antibody dependent cellular cytotoxicity (ADCC), showed an asymmetric distribution, with a small number of individuals showing unexpectedly high core afucosylation levels. In these individuals, IgG2 afucosylation levels were normal. Finally, a new IgG1 allotype, previously not characterized, was identified using this analytical methodology.

Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability

IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn297 mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fcγ receptors.

Defining dose-response relationships in the therapeutic blockade of B7RP-1-dependent immune responses

The ICOS (Inducible T cell Co-Stimulator)/B7RP-1 (B7-related protein 1) interaction is critical for the proper activation of a T lymphocyte. In this manuscript we describe a systematic in vivo approach to determine the level of blockade required to impair the generation of a T cell-dependent antibody response. We have developed an overall strategy for correlating drug exposure, target saturation, and efficacy in a biological response that can be generalized for most protein therapeutics. Using this strategy, we determined that low levels of B7RP-1 blockade are still sufficient to inhibit the immune response. These data suggest that contact between the T cell and the antigen-presenting cell during antigen presentation is much more sensitive to inhibition than previously believed and that ICOS/B7RP-1 blockade may be efficacious in the treatment of autoimmune diseases.