Peptidic antagonists of class iii semaphorins/neuropilins complexes

Abstract

The present invention concern a peptidic antagonist of class III semaphorins / neuropilins complexes comprising an amino acid sequence, which is derived from the transmembrane domain of a protein selected in the group consisting of neuropilin-1, neuropilin-2, plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrin Beta 1 and integrin beta 2, and including at least a GxxxG motif, eventually fused to an heterologous sequence; a nucleic acid encoding for said peptidic antagonist, a pharmaceutical composition comprising such a peptidic antagonist or a nucleic acid encoding thereof and uses thereof.

Description

RELATED APPLICATIONS[0001]This application is a divisional application of U.S. application Ser. No. 11 / 993,509, filed Oct. 10, 2008, which is a national stage of International Application No. PCT / IB2006 / 002334, filed Jun. 28, 2006, which claims priority to European Application No. 05291392.8 filed Jun. 28, 2005. The contents of each of the foregoing are incorporated herein by reference.[0002]The present invention relates to peptidic antagonists of class III semaphorins / neuropilins complexes, and uses thereof.BACKGROUND OF THE INVENTION[0003]Next to its structural and trophic roles, the extra cellular matrix (ECM) defines an ideal environment for cell to cell communication and determines all the cellular behaviors, including proliferation, migration, differentiation or apoptosis. The molecular mechanisms controlling these processes are getting better understood. In the nervous system, 3 major families of diffusible or transmembrane signals (netrins, semaphorins and ephrins) ensure th...

AI Technical Summary

Benefits of technology

[0019]As used herein an “heterologous sequence” relates to any amino acid sequence which is not derived from neuropilin-1, neuropilin-2, plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrin beta 1 or integrin beta 2. This heterologous sequence can for example allows a specific cellular location or a better purification yield (e.g. His tag) of the peptidic antagonist of the invention.

[0043]According to a preferred embodiment, the nucleic acid encoding the peptidic antagonist is operatively linked to a gene expression sequence, which directs the expression of nucleic acid within a prokaryotic or an eukaryotic cell, preferably an eukaryotic cell. The “gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the peptidic antagonist nucleic acid to which it is operatively linked and any signal sequence ensuring appropriate targeting of the peptidic antagonist to the plasma membrane. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, beta.-actin promoter, muscle creatine kinase promoter, human elongation factor promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), cytomegalovirus (CMV), Rous sarcoma virus (RSV), hepatitis B virus (HBV), the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Others constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Others inducible promoters are known to those of ordinary skill in the art.

[0048]Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0051]According to a first specific embodiment, the nucleic acid vector of the present invention corresponds to “naked DNA” like plasmids, cosmids or phagemids. Such naked DNA can be associated with non-lipid cationic polymers (WU and WU, J. Biol. Chem., vol. 263, p: 14621-4, 1988) or liposomes (BRIGHMAN et al., Am. J. Med. Sci., vol. 298, p: 278-81, 1989) to form complexes enhancing cellular uptake.

[0057]There are numerous causes of peptide instability or degradation, including hydrolysis and denaturation. Hydrophobic interaction may cause clumping of molecules together (i.e. aggregation). This result may entail diminution of the induction of a Treg response. Stabilizers may be added to lessen or prevent such problems.

[0068]Advantageously, said medicament allows the release of a concentration of said peptidic antagonist of more than 10−12 M, preferably more than 10−11 M and most preferably more than 10−10 M.

Problems solved by technology

27, p: 237-249, 2000) and mutations in the extracellular domain of L1 or the complete absence of L1 in gene-targeted mice result in the disruption of Sema 3A signaling leading to guidance errors.

Nevertheless, these antagonists, which are not located in the transmembrane domain, have an IC50 of more than 1 μM, said concentration being too important to enable the use of such an antagonist in therapy.

Images