WO2020006298A2 - Polypeptides à base de cyclotide pour le ciblage thérapeutique de récepteurs baff dans le lupus érythémateux systémique les - Google Patents

Polypeptides à base de cyclotide pour le ciblage thérapeutique de récepteurs baff dans le lupus érythémateux systémique les Download PDF

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WO2020006298A2
WO2020006298A2 PCT/US2019/039605 US2019039605W WO2020006298A2 WO 2020006298 A2 WO2020006298 A2 WO 2020006298A2 US 2019039605 W US2019039605 W US 2019039605W WO 2020006298 A2 WO2020006298 A2 WO 2020006298A2
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baff
receptor
cell
candidate
cells
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WO2020006298A3 (fr
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Julio Camarero PALAO
Chaim JACOB
William STOHL
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University of Southern California USC
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University of Southern California USC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics

Definitions

  • BAFF B-cell -activating factor
  • BAFF B-cell -activating factor
  • the first (and, so far, the only) BAFF antagonist approved by the FDA is belimumab, a human IgG m Ab that binds and neutralizes soluble BAFF.
  • belimumab has demonstrated only modest efficacy for the treatment of SLE in two independent clinical trials.
  • Applicant presents an alternative approach for effective BAFF antagonist therapy, i.e. to target the BAFF receptor rather than soluble BAFF.
  • selective antagonists for specific pairs BR3/TACI or BR3/BCMA
  • Applicant developed unique cyclotide-based molecular scaffolds for generating genetically-encoded molecular libraries can be screened and selected for the ability to selectively antagonize the interaction between soluble BAFF and BR3 and BCMA (but not TAC ) or BR3 and TACI (but not BCMA).
  • C yclotides are a family of large plant-derived hackbone-cyclized polypeptides (—30 amino acids long) that share a 3’ disulfide-stabilized core characterized by an unusual knotted structure. They have several characteristics that make them ideal drug development tools. They are remarkably stable to chemical, thermal and proteolytic degradation due to the cyclic topology and cysteine knot. They are small, making them readily accessible by chemical synthesis. Cyclotides can be encoded within standard cloning vectors, expressed in bacteria or animal cells, and are amenable to substantial sequence variation. Cyclotides can be also engineered to target intracellular and extracellular protein interactions. Cyclotides have been shown to be orally bioavailable.
  • the disclosure provides an isolated peptide comprising, or alternatively consisting essentially of, or yet further consisting of a protease recognition leading signal, fused or grafted to a cyclotide, optionally comprising a linker and a candidate BAFF receptor antagonist.
  • the candidate BAFF receptor antagonist comprises a CDR of an antibody that specifically binds a BAFF receptor.
  • first reporter polypeptides comprising a BAFF ligand fused to a reporter element.
  • second BAFF receptor polypeptides that bind to the BAFF ligand fused to a reporter element that when in contact with the first reporter polypeptide, emit a detectable signal.
  • peptides for use as a linker comprises, or alternatively consists essentially of, or yet further consists of a peptide selected from the group consisting of GXXGXP, wherein X is any amino acid; GGSGGF; GASGPG; GSGAPG; ASKAPG; and ASRAPG.
  • polynucleotides encoding the polypeptides described above or a complement thereof.
  • the polynucleotides can be incorporated and operatively linked to an expression or replication vector, which can further comprise the appropriate regulatory sequences for expression or duplication of the sequences, that in one aspect are inducible and distinct.
  • the polynucleotides and vectors can further comprise a label or tag, e.g., a fluorescent label or tag.
  • the peptides, polynucleotides, vectors can be incorporated into host cells, such as a prokaryotic (E. coli) or eukaryotic cells.
  • host cells such as a prokaryotic (E. coli) or eukaryotic cells.
  • the peptides, polynucleotides, vectors, host cells can be combined with a carrier, such as a pharmaceutically acceptable carrier.
  • the peptides and compositions can be used to identify candidate BAFF receptor antagonists, as described herein.
  • the methods can be performed in vitro (cell free) or in a cellular environment. Further provided are the candidate agents identified by this method.
  • kits comprises, or alternatively consists essentially of, or yet further consisting of, any of one or more of the isolated polypeptide as disclosed above, an isolated polynucleotide as disclosed herein, an isolated vector or host cell as described above, or the compositions, and instructions for use.
  • cell-based libraries E. coli cell libraries
  • FACS fluorescence-activated cell sorting
  • In-cell FRET -based screening reporters can be used to select cyclotides comprising antagonists able to inhibit BAFF binding to its individual receptors.
  • Selected cyclotides encoding candidate receptor antagonists are tested in vitro using a combination of fluorescence assays and nuclear magnetic resonance (NMR). Biological activity is assayed using cell lines expressing the different individual BAFF receptors and primary murine B cells derived from genetically-engineered mice that express only one of the three BAFF receptors.
  • the selected cyclotides are also evaluated for in vivo activity.
  • the cyclotides with the best in vitro activity are evaluated for biologic and therapeutic activities in vivo using SLE- prone NZM 2328 mice.
  • cyclotides prepared by the method that in one aspect demonstrate in vitro and/or in vivo BAFF selective activity.
  • polypeptide comprising, or consisting essentially of, or yet further consisting of the disclosed cyclotide polypeptides, and equivalents thereof, which retain the ability to inhibit the interaction between BAFF and its receptor BR3.
  • polynucleotides encoding the polypeptides, and their complements as well as vectors and host cells comprising the polynucleotides, as well as methods for recombinant
  • FIG. 1 Soluble BAFF signaling and the corresponding cellular effects on B cells, plasmablasts and plasma cells.
  • FIG. 2 Primary and tertiary structures of cyclotides from the Mobius (kalata B 1), bracelet (cycloviolacin 01) and trypsin inhibitor (MCoTI-II) subfamilies. conserveed cysteine residues are marked in yellow and disulfide connectivities in red. The circular backbone topology is shown with a line.
  • FIGS. 3A-3B Clinical disease and renal immunopathology among NZM mice deficient in two BAFF receptors.
  • Kidney sections from 6- to 7-month-old NZM WT, BCMA/TACI, BR3/TACI, and BR3/BCMA mice were stained for IgG immunofluorescence (top row) or C3 immunofluorescence (middle row) or with hematoxylin and eosin (H&E) for histologic evaluation.
  • FIGS. 4A-4B Genetically-encoded libraries based on the MCoTI cyclotide scaffold.
  • FIG. 4A Molecular diversity of libraries based on loop 2, loop 5 and loops 2 & 5 of MCoTII.
  • FIG. 4B E. coli expression of selected members of the MCoTI cyclotide library based on loop 2. Cyclotides were captured with trypsin (and therefore correctly folded) and characterized by ES-MS.
  • FIG. 5 Principle for the cell-based screening of biosynthesized genetically-encoded cyclotide libraries using a FRET -based approach.
  • Protein domains A refers to soluble BAFF and B refers to the corresponding Cys-rich domains of receptors BR3, TACI or BCMA.
  • FIGS. 6A-6C Cell-based FRET reporter to screen for RING-mediated Hdm2- HdmX interactions as depicted in FIG. 5.
  • FIG. 6A Coexpression of several RING domains fused to fluorescent proteins YPet and CyPet.
  • FIG. 6B FACS analysis of cells with FRET- ON and FRET-OFF phenotypes.
  • FIG. 6C Enrichment of FRET-OFF phenotype by performing multiple rounds of FACS.
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like.
  • Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.
  • nucleic acids such as DNA or RNA
  • isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule.
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an“isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides are meant to encompass both purified and recombinant polypeptides.
  • polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.
  • A“subject,”“individual” or“patient” is used interchangeably herein and refers to a vertebrate, for example a primate, a mammal or preferably a human. Mammals include, but are not limited to equines, canines, bovines, ovines, murines, rats, simians, humans, farm animals, sport animals and pets.
  • Cells “host cells” or“recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • “Amplify”“amplifying” or“amplification” of a polynucleotide sequence includes methods such as traditional cloning methodologies, PCR, ligation amplification (or ligase chain reaction, LCR) or other amplification methods. These methods are known and practiced in the art. See, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202 and Innis et al. (1990) Mol. Cell Biol. 10(11):5977-5982 (for PCR); and Wu et al. (1989) Genomics 4:560- 569 (for LCR).
  • the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.
  • Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular region are preferably complementary to, and hybridize specifically to sequences in the target region or in its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art.
  • grafted intends replaced or inserted, e.g., the phrase “grafted between” means that a peptide replaces the amino acid residues between the two indicated amino acids.
  • the term“fused” intends the attachment of two amino acids to each other by a peptide bond.
  • the term“genotype” refers to the specific allelic composition of an entire cell, a certain gene or a specific polynucleotide region of a genome, whereas the term“phenotype’ refers to the detectable outward manifestations of a specific genotype.
  • the term“gene” or“recombinant gene” refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence.
  • a gene may also refer to a polymorphic or a mutant form or allele of a gene.
  • “Homology” or“identity” or“similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position.
  • a degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • An “unrelated” or“non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present invention.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %,
  • “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment.
  • One alignment program is BLAST, using default parameters. In particular, programs are
  • Bioly equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.
  • nucleic acid, polynucleotide or peptide refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof.
  • a homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof.
  • homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi -stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.
  • lx SSC formamide concentrations of about 55% to about 75%
  • wash solutions of about lx SSC, O.lx SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • oligonucleotide refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine.
  • a nucleotide of a nucleic acid which can be DNA or an RNA
  • the terms“adenosine”,“cytidine”,“guanosine”, and “thymidine” are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.
  • polynucleotide and“oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. ETnless otherwise specified or required, any embodiment of this invention that is a
  • polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polymorphism refers to the coexistence of more than one form of a gene or portion thereof.
  • a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a“polymorphic region of a gene”.
  • a polymorphic region can be a single nucleotide, the identity of which differs in different alleles.
  • “Overexpression” or“underexpression” refers to increased or decreased expression, or alternatively a differential expression, of a gene in a test sample as compared to the expression level of that gene in the control sample.
  • the test sample is a diseased cell, and the control sample is a normal cell.
  • the test sample is an experimentally manipulated or biologically altered cell, and the control sample is the cell prior to the experimental manipulation or biological alteration.
  • the test sample is a sample from a patient, and the control sample is a similar sample from a healthy individual.
  • the test sample is a sample from a patient and the control sample is a similar sample from patient not having the desired clinical outcome.
  • the differential expression is about 1.5 times, or alternatively, about 2.0 times, or alternatively, about 2.0 times, or alternatively, about 3.0 times, or alternatively, about 5 times, or alternatively, about 10 times, or alternatively about 50 times, or yet further alternatively more than about 100 times higher or lower than the expression level detected in the control sample.
  • the gene is referred to as“over expressed” or“under expressed”.
  • the gene may also be referred to as“up regulated” or“down regulated.”
  • the term“carrier” encompasses any of the standard carriers, such as a phosphate buffered saline solution, buffers, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see below.
  • the carrier is a buffered solution such as, but not limited to, a PCR buffer solution.
  • A“gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • Gene delivery,”“gene transfer,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a“transgene”) into a host cell, irrespective of the method used for the introduction.
  • exogenous polynucleotide sometimes referred to as a“transgene”
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection, sometimes called transduction), transfection, transformation or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of“naked” polynucleotides (such as electroporation,“gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • transfected, transduced or transformed may be used interchangeably herein to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a cell.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • a cell that“stably expresses” an exogenous polypeptide is one that continues to express a polypeptide encoded by an exogenous gene introduced into the cell either after replication if the cell is dividing or for longer than a day, up to about a week, up to about two weeks, up to three weeks, up to four weeks, for several weeks, up to a month, up to two months, up to three months, for several months, up to a year or more.
  • the term“express” refers to the production of a gene product.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.
  • A“gene product” or alternatively a“gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
  • Under transcriptional control is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription.“Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
  • the term“encode” as it is applied to polynucleotides refers to a polynucleotide which is said to“encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the
  • a“vector” is a vehicle for transferring genetic material into a cell. Examples of such include, but are not limited to plasmids and viral vectors.
  • a viral vector is a virus that has been modified to transduct genetic material into a cell.
  • a plasmid vector is made by splicing a DNA construct into a plasmid.
  • the appropriate regulatory elements are included in the vectors to guide replication and/or expression of the genetic material in the selected host cell.
  • A“viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • “retroviral mediated gene transfer” or“retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
  • the integrated DNA form is called a provirus.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • A“lentiviral vector” is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral Vectors, New York: Spring-Verlag Berlin Heidelberg.
  • a vector construct refers to the adenovirus (Ad) or adeno-associated virus (AAV).
  • Ads Adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell’s genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470 and
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5’ and/or 3’ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5’ of the start codon to enhance expression.
  • Gene delivery vehicles also include several non-viral vectors, including
  • A“plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.
  • Plasmids used in genetic engineering are called“plasmic vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
  • MCS multiple cloning site
  • Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for.
  • Eukaryotic cells comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
  • a eukaryotic host including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples include simian, bovine, ovine, porcine, murine, rats, canine, equine, feline, avian, reptilian and human.
  • Prokaryotic cells that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to prokaryotic Cyanobacteria, bacillus bacteria, E. coli bacterium, and Salmonella bacterium.
  • the term“propagate” means to grow a cell or population of cells.
  • the term “growing” also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.
  • the term“culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
  • A“probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
  • a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels are described and exemplified herein.
  • A“primer” is a short polynucleotide, generally with a free 3’ -OH group that binds to a target or“template” potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target.
  • A“polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a“pair of primers” or a“set of primers” consisting of an“upstream” and a“downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme.
  • a primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook et al., supra.
  • the primers may optionally contain detectable labels and are exemplified and described herein.
  • the term "detectable label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition.
  • the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the labels can be suitable for small scale detection or more suitable for high-throughput screening.
  • suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • the label may be simply detected or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
  • Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal.
  • Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
  • luminescent probes include, but are not limited to, aequorin and luciferases.
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red.
  • suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker.
  • Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to,
  • antigens/antibodies e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavidin.
  • the term“discrete marker” intends an entity or molecule that allows for the selective identification of a peptide of interest.
  • Non-limiting examples of such include, isotope-coded affinity tags, isobaric tags, radioactively labeled amino acids, and bioorthogonial noncanonical amino acid tags. Dieterich et al. (2006) PNAS, l03(25):9482- 9487.
  • solid support refers to non-aqueous surfaces such as“culture plates” “gene chips” or“microarrays.”
  • Gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to one of skill in the art.
  • oligonucleotides are attached and arrayed on a gene chip for determining the DNA sequence by the hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041.
  • Polynucleotides and probes can be used for detection of a genetic sequence.
  • Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659.
  • a probe also can be attached or affixed to an electrode surface for the
  • Various“gene chips” or“microarrays” and similar technologies are known in the art. Examples of such include, but are not limited to, LabCard (ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (Caliper Technologies Corp); a low-density array with electrochemical sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput, automated mass spectrometry systems with liquid-phase expression technology (Gene Trace Systems, Inc.); a thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq,
  • “gene chips” or“microarrays” containing probes or primers homologous to p53 are prepared.
  • a suitable sample is obtained from the patient, extraction of genomic DNA, RNA, protein or any combination thereof is conducted and amplified if necessary.
  • the sample is contacted to the gene chip or microarray panel under conditions suitable for hybridization of the gene(s) or gene product(s) of interest to the probe(s) or primer(s) contained on the gene chip or microarray.
  • the probes or primers may be detectably labeled thereby identifying the sequence(s) of interest.
  • a chemical or biological reaction may be used to identify the probes or primers which hybridized with the DNA or RNA of the gene(s) of interest.
  • the genotypes or phenotype of the patient is then determined with the aid of the aforementioned apparatus and methods.
  • A“composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
  • A“pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term“pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).
  • An“effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of
  • Treatment dosages generally may be titrated to optimize safety and efficacy.
  • dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration.
  • Studies in animal models generally may be used for guidance regarding effective dosages for treatment of diseases.
  • a compound is found to demonstrate in vitro activity, for example as noted in the Tables discussed below one can extrapolate to an effective dosage for administration in vivo.
  • “treating” or“treatment” of a disease in a patient refers to (1) preventing the symptoms or disease from occurring in an animal that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition
  • A“control” is an alternative subject or sample used in an experiment for comparison purpose.
  • a control can be“positive” or“negative”.
  • the purpose of the experiment is to determine a correlation of a mutated allele with a particular phenotype
  • it is generally preferable to use a positive control a sample from a subject, carrying such mutation and exhibiting the desired phenotype
  • a negative control a subject or a sample from a subject lacking the mutated allele and lacking the phenotype.
  • intein intends a segment of a protein that is able to excise itself and rejoin the remaining portions (the exteins) with a peptide bond by protein splicing. Inteins have also been called“protein introns.” Inteins are known in the art and sequences are publicly available, see for example, InBase, The Intein Database and Registry, available at the web addresss: neb.com/extemal-links/inbase (accessed on March 5, 2013) and Inteins - protein introns, available at the web address: bioinformatics.weizmann.ac.il/ ⁇ pietro/inteins/ (accessed on March 5, 2013).
  • B AFF a member of the TNF ligand superfamily
  • B AFF is a 285-amino acid type-II transmembrane precursor protein that is cleaved by a furin protease to yield a soluble, biologically active ligand [6] that binds to three receptors, BCMA, TACI and BR3 (also known as BAFF-R) on the surface of B cells (FIG.
  • BAFF is essential for B cell survival and maturation [10-12] and contributes importantly to the differentiation of immature B cells [13] and to Ig class switching and Ig production [14]
  • B cells Given the importance of B cells to SLE, it is not surprising that BAFF has become a therapeutic target in this disorder [2] Indeed, the anti -BAFF mAh belimumab was recently approved by the FDA for the treatment of SLE.
  • belimumab demonstrated only modest efficacy in two independent clinical trials [4, 5, 15] Given the limited therapeutic capacity effected through global neutralization of BAFF
  • BCMA-deficient mice display a near-normal phenotype. These mice harbor normal numbers and subsets of lymphocytes and do not manifest any signs of
  • mice mount impaired antibody responses to T cell-independent antigens but, at the same time, harbor increased number of T helper and B cells. Indeed, as TACI-deficient mice age, they develop circulating autoantibodies, Ig deposition in their kidneys, and histological glomerulonephritis and premature death [22-24]
  • mice deficient in a single BAFF receptor it is only BR3-deficient mice that manifest deficiencies in mature B cells and antibody responses to degrees similar to those manifest by BAFF-deficient mice [25, 26]
  • B cells do not expand in BR3- deficient mice after treatment with exogenous BAFF.
  • B cells bearing a mutated BR3 gene also display decreased in vivo survival.
  • NZM mice genetic ablation of the Br3 gene in SLE-prone NZM 2328 mice
  • NZM mice singly-deficient in any of the BAFF receptors (i.e., NZM Br3 ! mice, NZM. Taci 1 mice, and ZM. cmci 1 mice), despite manifesting their expected dramatic differences in B cell profiles, all developed clinical disease with time courses indistinguishable from that of NZM wild-type (WT mice) [27] That is, any therapeutic approach that eliminates (or, presumably, neutralizes) only any single BAFF receptor is not efficacious in SLE-prone NZM mice and would likely not be efficacious in human SLE as well. This surprising result prompted additional experiments, described herein.
  • Cyclotides are spectacular micro proteins ( ⁇ 30 residues long) present in plants from the Violaceae, Rubiaceae, Cucurbitaceae and more recently Fabaceae families [30] They display various biological properties such as protease inhibitory, anti-microbial, insecticidal, cytotoxic, anti-HIV and hormone-like activities [31] They share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology (FI
  • Cyclotides belong to the family of knottins, a group of microproteins that also includes conotoxins and spider toxins. Cyclotides can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head- to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot [32-34]
  • the main features of cyclotides are a remarkable stability due to the cystine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations.
  • kalata Bl is an orally effective uterotonic [35]
  • other cyclotides have been shown to cross the cell membrane [36, 37]
  • Applicant and other groups have reported engineered cyclotides that target extracellular [38-40] and intracellular [41] targets in animal models.
  • Some of these novel cyclotides are orally bioavailable [39] and are able to cross cellular membranes efficiently [36, 37] Cyclotides thus appear as highly promising leads or frameworks for peptide drug design [42, 43]
  • NZM .Bcma 1 .Taci 1 mice develop clinical disease at a more rapid pace than do NZM WT mice (FIG. 3A). Whereas >50% of NZM WT mice do not develop severe proteinuria even by 7 months of age, >50% of ZM.Bcma 1 .Taci 1 mice already develop severe proteinuria by 6 months of age.
  • Cyclotides are ribosomally produced in plants from precursors that comprise between one and three cyclotide domains.
  • the mechanism of excision of the cyclotide domains and ligation of the free N- and C-termini to produce the circular peptides has not yet been completely elucidated.
  • Applicant has developed bio-mimetic approaches for the biosynthesis of folded cyclotides inside bacterial cells by making use of modified protein splicing units [47-50] This important finding opens for the first time the interesting possibility for the generation of large genetically-encoded libraries of cyclotides (up to ⁇ l0 9 ) inside living bacterial cells (FIG. 4).
  • Genetically-encoded libraries can be readily screened inside the cell using appropriate cell-based reporters for the rapid selection of particular cyclotide sequences that antagonize specific soluble protein targets.
  • Applicant can use cell-based libraries ( E . coli cell libraries) where every single cell expresses a different cyclotide, in what could be labeled a single cell-single compound approach. These compounds are then screened in-cell and selected for their ability to inhibit a particular protein-protein interaction inside the bacterial cell.
  • Biologically-generated libraries can be screened inside the cell using appropriate cell-based reporters for the selection of particular cyclotide sequences that bind a specific protein target using high throughput methods.
  • Cell-based screening opens the possibility of using single cells as microfactories where the biosynthesis and screening of particular ligands can take place in a single process within the same cellular cytoplasm [51]
  • the use of a complex molecular environment, such as the cellular cytoplasm provides also the ideal background for the selection of highly specific inhibitors. This cell-based screening approach for the selection of particular cyclotide sequences able to inhibit individual BAFF receptors.
  • FRET -based reporter for the in-cell detection of cyclotides able to antagonize BAFF binding to individual receptors.
  • FACS FRET-based reporter
  • This screening approach is optimized for use in E. coli in combination with FACS, and it is designed to screen large libraries while minimizing the number of false positives.
  • FACS FACS
  • Applicant also uses a FACS protocol to separate mixtures of cells depending on their FRET-state when using the fluorescence reporter described above. This protocol can be easily used to enrich a population of E.
  • Cyclotides are small globular microproteins (ranging from 28 to 37 amino acids) with a unique head-to-tail cyclized backbone, which is stabilized by three disulfide bonds forming a cystine-knot motif.
  • This cyclic cystine-knot (CCK) framework provides a rigid molecular platform with exceptional stability towards physical, chemical and biological degradation.
  • These micro-proteins can be considered natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization, but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.
  • naturally-occurring cyclotides have shown to possess various pharmacologically-relevant activities, and have been reported to cross cell membranes.
  • cyclic backbone includes a molecule comprising a sequence of amino acid residues or analogues thereof without free amino and carboxy termini.
  • the cyclic backbone of the disclosure comprises sufficient disulfide bonds, or chemical equivalents thereof, to confer a knotted topology on the three-dimensional structure of the cyclic backbone.
  • cyclotide refers to a peptide comprising a cyclic cystine knot motif defined by a cyclic backbone, at least two but preferably at least three disulfide bonds and associated beta strands in a particular knotted topology.
  • the knotted topology involves an embedded ring formed by at least two backbone disulfide bonds and their connecting backbone segments being threaded by a third disulfide bond.
  • a disulfide bond may be replaced or substituted by another form of bonding such as a covalent bond.
  • the disclosure provides an isolated peptide comprising, or alternatively consisting essentially of, or yet further consisting of a protease recognition leading signal, fused or grafted to cyclotide, optionally comprising a linker and a candidate BAFF receptor antagonist.
  • the candidate BAFF receptor antagonist comprises a CDR of an antibody that specifically binds a BAFF receptor.
  • first reporter polypeptides comprising a BAFF ligand fused to a reporter element.
  • second BAFF receptor polypeptides that bind to the BAFF ligand fused to a reporter element that when in contact with the first reporter polypeptide, emit a detectable signal.
  • the linker is inserted at the junctions between the interacting proteins or protein domains and the corresponding fluorescent proteins.
  • peptides for use as a linker comprises, or alternatively consists essentially of, or yet further consists of a peptide selected from the group consisting of [GGS]n, wherein n is an integer from 1 to 20, GXXGXP, wherein X is any amino acid; GGSGGF; GASGPG; GSGAPG; ASKAPG; or ASRAPG.
  • the BAFF receptors are selected from the group of: BR3, TAC1, or BCMA.
  • the BAFF ligand comprises the soluble human BAFF (shBAFF) or a fragment thereof.
  • the fragment comprises the N-terminus of soluble hBAFF, optionally 134-285, and further optionally delta 217-224.
  • the shBAFF is a mutated shBAFF wherein eight residues at the flap region are replaced by 2 glycine amino acids.
  • the hBR3 comprises amino acids 1-62.
  • the hTACI comprises amino acids 68-109.
  • the hBCMA comprises amino acids 1-54. Examples of the amino acid sequence for these proteins are provided herein.
  • the BAFF receptor comprises extracellular cysteine-rich CDR BAFF receptor selected from the group of human or murine BR3 (hBR3 or mBR3), human or murine BCMA (hBCMA or mBCMA), or human or murine TACI (hTACI or mTACI), and optionally wherein the receptor is fused to the C-terminus to one of the fluorescent pair, optionally CyPet or YPet.
  • the paired reporters comprises the components of a FRET-based system, optionally a CyPet and a YPet fluorescent protein pair.
  • the flexible linker is inserted at the junctions between the interacting proteins or protein domains and the corresponding fluorescent proteins, and optionally, wherein the linker comprises the amino acid sequence [GGS]n, wherein n is an integer from 1 to 20.
  • the linker non-limiting examples of linkers comprise, or alternatively consist essentially of, or yet further consist of a peptide selected from the group consisting of GXXGXP, wherein X is any amino acid; GGSGGF; GASGPG;
  • the peptides can be linear or processed and in their cyclic form.
  • the candidate BAFF receptor antagonist can be grafted into any available loop, e.g., loop 1, or loop 2, or loop 3, or loop 4, or loop 5 or loop 6, of the cyclotide. In one aspect, it is loop 2 or loop 5.
  • the cyclotide comprises a molecular framework comprising a sequence of amino acids forming a cyclic backbone wherein the cyclic backbone comprises sufficient disulfide bonds to confer knotted topology on the molecular frameword or part thereof.
  • the cyclic backbone comprises the structure:
  • X a ], [X n i...Xb], [X m i...Xc], [X Iv i...Xd], [X v i...Xe], and [X VI i...Xf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20.
  • [X VI i...Xf] in the cyclotide comprise the target peptide.
  • the target peptide is grafted into loop 1 of the cyclotide.
  • the amino acid residues corresponding to [Xh.. X a ] in the cyclotide comprise the target peptide.
  • the target peptide is grafted into loop 2 of the cyclotide.
  • the amino acid residues corresponding to [X n i...Xb] in the cyclotide comprise the target peptide.
  • the target peptide is grafted into loop 3 of the cyclotide.
  • the amino acid residues corresponding to [X m i.. X C ] in the cyclotide comprise the target peptide.
  • the target peptide is grafted into loop 4 of the cyclotide.
  • [X Iv i...Xc] in the cyclotide comprise the target peptide.
  • the target peptide is grafted into loop 5 of the cyclotide.
  • the amino acid residues corresponding to [X v i.. X e ] in the cyclotide comprise the target peptide peptide.
  • this disclosure provides an isolated polynucleotide encoding one or more of the isolated cyclotide peptides described above, alone or in a gene delivery vehicle (e.g., liposome, biocompatible polymers, lipoproteins, artificial viral envelopes, plasmids, viral vectors) a replication or an expression vector, e.g., a viral vector or a plasmid.
  • a gene delivery vehicle e.g., liposome, biocompatible polymers, lipoproteins, artificial viral envelopes, plasmids, viral vectors
  • an expression vector e.g., a viral vector or a plasmid.
  • isolated complementary sequences to the isolated polynucleotide encoding one or more of the isolated peptides described above can further contain the necessary regulatory element(s) operatively linked to the coding sequences for expression of the polynucleotide in a host cell.
  • the isolated polynucleotides can further comprise a detectable label,
  • a first expression vectors encoding a cyclotide comprising the candidate BAFF receptor antagonist, and wherein the first expression vectors further comprise a first origin of replication.
  • a reporter plasmid encoding a BAFF ligand and a plasmid encoding one or more BAFF receptors, wherein each reporter plasmid is distinct from the first expression vector and comprises a distinct origin of replication from the first origin of replication.
  • the polynucleotide encoding the candidate BAFF receptor antagonist encodes a CDR of an antibody that specifically binds a BAFF receptor. Examples of BAFF receptors are selected from the group of: BR3, TAC1, or BCMA.
  • the candidate receptor antagonist specifically binds the receptors BR3 and TACI, but not BCMA.
  • the plasmid encodes a BAFF candidate receptor antagonist specifically binds the receptors BR3 and BCMA, but not TACI.
  • the linker is a flexible linker and the polynucleotide encoding such is inserted in the plasmid at the junctions between the polynucleotides encoding the interacting proteins or protein domains.
  • peptides for use as a linker comprises, or alternatively consists essentially of, or yet further consists of a peptide selected from the group consisting of GXXGXP, wherein X is any amino acid; GGSGGF; GASGPG;
  • the first expression vector is a pASK expression vector and/or the expression vector of the reporter plasmid comprises pRSF-det and/or pBAD33.
  • vectors other than plasmids can be substituted for plasmids and are selected for use in the selected host cell system, e.g., prokaryotic, eukaryotic, E. coli, mammalian cell, or for example, a yeast cell.
  • the plasmid encoding the BAFF ligand comprises a
  • polynucleotide encoding the soluble human BAFF (shBAFF) or a fragment thereof.
  • the polynucleotide encodes a fragment that comprises the N-terminus of soluble hBAFF, optionally 134-285, and further optionally delta 217-224.
  • the polynucleotide encodes a shBAFF that is a mutated shBAFF wherein eight residues at the flap region are replaced by 2 glycine amino acids.
  • the at least one reporter plasmid encodes an extracellular cysteine-rich CDR BAFF receptor selected from the group of human or murine BR3 (hBR3 or mBR3), human or murine BCMA (hBCMA or mBCMA), or human or murine TACI (hTACI or mTACI).
  • hBR3 comprises amino acids 1-62.
  • hTACI comprises amino acids 68- 109.
  • the hBCMA comprises amino acids 1-54.
  • isolated peptides and polynucleotides of this invention can be produced by chemical synthetic methods or by recombinant expression of isolated polynucleotides as described herein or using methods known to those of skill in the art.
  • This disclosure also provides an isolated host cell comprising one or more of: the peptides as described above, an isolated polynucleotide, a gene delivery vehicle or vector containing the isolated polynucleotides.
  • the isolated host cell is a prokaryotic or a eukaryotic cell. In one particular aspect, the host cell is an E. coli cell.
  • “Host cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Examples of such include, prokaryotic cells such as E. coli cells.
  • Examples of eukaryotic cells include, but are not limited to cells from animals, e.g., murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human.
  • the cells can be cultured cells or they can be primary cells. Cultured cell lines can be purchased from vendors such as the American Type Culture Collection (ATCC), U.S.A.
  • ATCC American Type Culture Collection
  • the host cells can be used for recombinant production of the polynucleotides or peptides of this invention or to provide a screen for therapeutic agents. Further provided is a method for recombinantly producing the peptides of this disclosure by growing an isolated host cell as described above under conditions that favor the expression of the polynucleotide. In one aspect, the peptides are isolated from the host cells.
  • compositions can be combined with a carrier, such as a pharmaceutically acceptable carrier.
  • This disclosure also provides methods for recombinantly producing peptides and polynucleotides of this invention by culturing a host cell the isolated polynucleotides, the plasmid or vector containing the polynucleotide encoding the peptide, under conditions for the expression of the polynucleotide to peptide.
  • the peptide is isolated from the cell.
  • suitable cells may be cultured in microtiter plates and several agents may be assayed at the same time by noting genotypic changes, phenotypic changes and/or cell death.
  • a method for isolating a candidate BAFF receptor antagonist comprising:
  • each reporter plasmid is distinct from the first expression vector and comprises a distinct origin of replication from the first origin of replication;
  • the cells of steps a) and/or b) are prokaryotic or eukaryotic, optionally E. coli cells.
  • the reporter of step b) comprises the components of a FRET- based system, optionally a CyPet and a YPet fluorescent protein pair, one member on the receptor and one member on the ligand.
  • a linker such as a flexible linker is inserted at the junctions between the interacting proteins or protein domains and the corresponding fluorescent proteins, and optionally, wherein the linker comprises the amino acid sequence [GGS]n, wherein n is an integer from 1 to 20.
  • Any appropriate expression vector can be used.
  • a non-limiting example of such comprises the expression vector of step a) being a pASK expression vector and/or the expression vector of the reporter of step b) comprises pRSF-det and pBAD33.
  • the candidate BAFF receptor antagonist encodes CDR of an antibody that specifically binds a BAFF receptor.
  • step b) is performed before step a).
  • the one or more BAFF receptors are selected from the group of: BR3, TAC1, or BCMA.
  • cell of step b) comprises a vector encoding a BR3 receptor and a vector encoding a TAC1 receptor.
  • the cell of step b) comprises a vector encoding a BR3 receptor and a BCMA receptor.
  • the cell of step b) comprises one or more vectors encoding a BR3 receptor, a TAC1 receptor or a BCMA receptor.
  • the method selects for a candidate receptor antagonist specifically binds the receptors BR3 and TACI, but not BCMA.
  • the method selects for a candidate receptor antagonist specifically binds the receptors BR3 and BCMA, but not TACI.
  • the BAFF ligand comprises the soluble human BAFF (shBAFF) or a fragment thereof.
  • the fragment comprises the N- terminus of soluble hBAFF, optionally 134-285, and further optionally delta 217-224.
  • the shBAFF is a mutated shBAFF wherein eight residues at the flap region are replaced by 2 glycine amino acids.
  • At least one reporter vector encodes an extracellular cysteine-rich CDR BAFF receptor selected from the group of human or murine BR3 (hBR3 or mBR3), human or murine BCMA (hBCMA or mBCMA), or human or murine TACI (hTACI or mTACI), and optionally wherein the receptor is fused to the C-terminus to one of the fluorescent pair, optionally CyPet or YPet.
  • hBR3 or mBR3 human or murine BCMA
  • TACI human or murine TACI
  • the hBR3 comprises amino acids 1-62.
  • the hTACI comprises amino acids 68-109.
  • the hBCMA comprises amino acids 1-54.
  • the method further comprises isolating BAFF receptor antagonist of step e) and further inserting the isolated candidate receptor antagonist into a second or further system comprising a vector encoding a BAFF receptor that is different from the BAFF receptor of step b) and repeating steps c) through e).
  • the methods can be performed in a cell or the proteins can be brought in contact with each other in a test tube, in vitro.
  • candidate receptor antagonist that prohibit the binding of the ligand and receptor will prohibit the recognition and binding of the elements of the reporter pair.
  • the candidate BAFF receptor antagonist that has the preferred binding affinity is isolated and further characterized by repeating the method with different ligand/receptor pairs or other possible binding partners.
  • the antagonist can be further characterized and sequenced.
  • the candidate is characterized by a method comprising mapping the molecular interaction, optionally by NMR.
  • the candidate receptor antagonists are isolated and further assayed for additional in vitro activity and isolating the candidate with the in vitro activity.
  • the receptor antagonist is used in a for inhibiting the interaction of BAFF with a BAFF receptor comprising contacting a cell or solution comprising the BAFF and the BAFF receptor with the receptor antagonist.
  • the contacting can be in vitro, ex vivo or in vivo.
  • Also provided is a method for treating SLE comprising administering to a subject suffering from SLE an effective amount of the BAFF receptor antagonist.
  • a method for treating a disease or condition that can be treated by inhibiting the binding of a BAFF ligand to a BAFF receptor comprising, or alternatively consisting essentially of, or yet further consisting of, administering to a subject in need of such treatment an effective amount of one or more of the receptor antagonist.
  • compositions can be administered to an animal or mammal by a treating veterinarian or to a human patient by a treating physician.
  • the antagonist is administered in an effective amount to treat the condition or disease and by any suitable means and with any suitable formulation as a composition and therefore includes a carrier such as a pharmaceutically acceptable carrier.
  • a formulation comprising the necessary antagonist is further provided herein.
  • the formulation can further comprise one or more preservatives or stabilizers. Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4. , 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,
  • Non-limiting examples include, no preservative, 0.1- 2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1.,
  • 0.001-0.5% thimerosal e.g., 0.005, 0.01
  • 0.001-2.0% phenol e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%)
  • 0.0005-1.0% alkylparaben(s) e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, and 1.0%).
  • the term carrier can include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-
  • polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-
  • quadrature. -cyclodextrin polyethylene glycols
  • flavoring agents e.g., polysorbates such as“TWEEN 20” and“TWEEN 80”
  • surfactants e.g., polysorbates such as“TWEEN 20” and“TWEEN 80”
  • lipids e.g., phospholipids, fatty acids
  • steroids e.g., cholesterol
  • chelating agents e.g., EDTA
  • An“effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of the antagonist as described herein and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18,
  • the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising the antagonist and/or a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a physician, technician or patient to reconstitute the therapeutic in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
  • Various delivery systems are known and can be used to administer an antagonist of the disclosure, e.g., direct delivery, encapsulation in liposomes, microparticles,
  • microcapsules are used in a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection or by means of a catheter.
  • kits comprises, or alternatively consists essentially of, or yet further consisting of, any of one or more of the isolated polypeptide as disclosed above, an isolated polynucleotide as disclosed herein, an isolated vector or host cell as described above, or the compositions, and instructions for use.
  • Cyclotide-based libraries are created at the DNA level using double stranded DNA inserts with degenerate sequences for some of the different loops of the cyclotide scaffold (i.e. loops 1, 2, 3 and/or 5, see FIG. 2). Briefly, a long degenerate synthetic oligonucleotide (which encodes the whole cyclotide, -100 nucleotide-long) template is PCR amplified using 5’- and 3’-primers corresponding to the non-degenerate flanking regions.
  • the resulting double-stranded degenerate DNA is double digested and then ligated to a linearized protein splicing unit (intein)-encoding expression vector to produce a library of plasmids. These libraries are then transformed into electrocompetent E. coli cells to finally obtain a library of cells containing typically up to -10 9 different clones (i.e. cyclotide sequences).
  • the degenerate synthetic oligonucleotide template is synthesized using a NN(G/T) codon scheme for the randomized loops. This scheme uses 32 codons to encode all 20 amino acids and encodes only 1 stop codon (amber codon).
  • the degenerate template can be also synthesized from mixtures of trinucleotide codons representing all 20 amino acids and no stop codons.
  • a FRET-based reporter can be used for the in-cell detection of cyclotides that inhibit a particular protein/protein interaction.
  • the CyPet and YPet fluorescent proteins can be used as a FRET-couple to monitor protein interactions inside the cell [52-54] The principle for this approach is depicted in FIG. 5.
  • addition of appropriate flexible polypeptide linkers i.e.
  • [GGS]n) are added at the junctions between the interacting proteins or protein domains and the corresponding fluorescent proteins.
  • N-terminus of soluble hBAFF 134-285, D217-224
  • this mutated version of soluble BAFF with eight residues at the flap region replaced by 2 Gly residues avoids oligomerization while still binding BAFF receptors [55]
  • the extracellular Cys-rich domains (CRDs) of BAFF receptors hBR3 (1-62), hTACI (68- 109) and hBCMA (1-54) are fused to either the C-terminus of CyPet and YPet, respectively.
  • FRET reporters for murine BAFF and BAFF-receptors also can be generated as described above.
  • BAFF and the BAFF receptor extracellular CRDs have shown to tolerate well the addition of protein domains (e.g., GST and/or thioredoxin) to their N or C-termini and can be expressed using bacterial expression systems without affecting their biological activity [55- 57]
  • protein domains e.g., GST and/or thioredoxin
  • the cyclotide-based library is cloned into a pASK expression vector, while the FRET- based reporters are cloned into plasmids pRSF-duet and pBAD33, respectively.
  • the library and reporter plasmids both contain different replication origins (ColEl, pl5A or pRSFl030) to prevent replication dominance of one plasmid [58]
  • These plasmids also have different inducible bacterial promoters (arabinose, tetracycline or T7 promoters) in order to control the expression level of the library and screening reporter-system.
  • the plasmids encoding the reporters are first transformed in Origami (DE3) competent cells to provide the corresponding FRET-based electrocompetent cell-line reporters.
  • the library plasmid (with complexities ⁇ 10 9 ) are then transformed to into the corresponding cell-based reporters. Transformation conditions that ensure from 10- to 1000-fold coverage of the library are used. This usually requires performing between 20-50 transformations.
  • FACS fluorescence-activate cell sorting
  • the selected pool of plasmids encoding cyclotides are re-transformed in fresh competent cells encoding the reporter.
  • FIG. 6 wherein this approach to select novel cyclotides able to antagonize the RING-mediated interaction between Hdm2 and HdmX.
  • the same protocol is used to enrich a population of E. coli cells from an initially predominant FRET-ON phenotype (complex BAFF-BAFF CRD receptor complex) into a FRET-OFF phenotype (complex BAFF-BAFF CRD receptor complex antagonized).
  • the enrichment process is also monitored by PCR and DNA sequencing. This approach is used to select antagonists against individual BAFF receptor CDRs.
  • an extra selection step is performed at the end of the evolution process to avoid sequences able to antagonize to the other two BAFF CDR receptors.
  • selected sequences able to antagonize BAFF-BR3 i.e. FRET-OFF phenotype
  • BAFF-BR3 i.e. FRET-OFF phenotype
  • BAFF-BCMA FRET- reporters to select cells expressing cyclotides with a FRET-ON phenotype, i.e. not able to cross- react with either TACI or BCMA.
  • FRET -based reporter can be also used in vitro using the purified fluorescent constructs for the characterization of antagonists.
  • Selected cyclotides can be recombinantly produced using protein splicing units [41, 47] or chemically synthesized by solid-phase peptide synthesis (SPPS) using a GSH-induced “one-pot” cyclizati on/folding process as described in the literature [37, 41, 47]
  • Initial biophysical characterization of the antagonists includes confirming their ability to inhibit the protein complexes using a standard FRET-based assay (for ICso values) and/or fluorescence anisotropy (Kd values) as previously described [41] It is important to highlight that the FRET-based reporter mentioned above can be also used in vitro using the purified fluorescent fusion constructs.
  • Fluorescence anisotropy assays are performed using fluorescent-labeled cyclotides obtained by either chemical synthesis [37, 41] or recombinant expression [47] as previously described by Applicant. Characterization of the mode of inhibition can be performed by high-field nuclear magnetic resonance (NMR). NMR is a very powerful technique to map the interacting site of a peptide/protein with its partner. This structural information can also be used to identify key residues in the molecular interaction to help in the design of new biased libraries during the ligand maturation process of active cyclotides. In vitro biological activity
  • ICso values are calculated as the concentration of cyclotide that inhibits BAFF binding to 50% levels.
  • Selected cyclotides are also tested in human or murine B cell proliferation assays as previously described in the literature [60] Briefly, B cell proliferation assays are performed using murine or human B cells isolated from peripheral blood mononuclear cells by positive selection using CD 19 MACS magnetic beads. To evaluate the antagonistic effects of selected cyclotides, B cells (1 c l0 5 /well) are incubated with soluble recombinant His-tagged BAFF (10 ng/mL) and in the presence and absence of various concentrations of active cyclotide.
  • B-cell proliferation is analyzed at day 6 by adding Celltiter Glo (Promega). The plates are then read in a luminometer after a lO-min incubation at room temperature.
  • Doses are determined based on the corresponding PK studies for the bioactive cyclotides. Without being bound by theory, doses are anticipated to range from 20 to 1 mg/kg. Health checks are performed daily to observe parameters such as body conditioning score, overall appearance and cleanliness, strength of grip, skin color and tone, mobility, gait, and activity level as indicators of potential drug related toxicities. Individual weights are recorded thrice weekly, comparing the control and treatment groups as an additional indicator of tolerance of drug treatment as well as providing the average weight for calculation of drug dosing. At the end of the treatment and prior to euthanasia, the animals are evaluated for proteinuria, and sera will be collected for levels of total IgG and IgG autoantibodies. Kidneys are harvested from the euthanized mice and evaluated by histology and immunofluorescence as previously described [27]
  • LymphoStat-B a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator, Arthritis Rheum 48, 3253-3265.
  • erythematosus a randomised, placebo-controlled, phase 3 trial, Lancet 377 , 721- 731.
  • BAFF-R a newly identified TNF receptor that specifically interacts with B AFF, Science 293 , 2108-2111.
  • BAFF is a survival and maturation factor for mouse B cells, European journal of immunology 32, 2004- 2010
  • BCMA is essential for the survival of long-lived bone marrow plasma cells, J Exp Med 199 , 91- 98.
  • TACI employs only a single cysteine-rich domain for high affinity ligand binding, J Biol Chem 280, 7218-7227.
  • BAFF/BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site, Biochemistry 42, 5977-5983.

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Abstract

La présente invention concerne une méthode à haut rendement pour identifier et isoler un antagoniste du récepteur BAFF candidat et des méthodes pour son utilisation in vitro et in vivo.
PCT/US2019/039605 2018-06-28 2019-06-27 Polypeptides à base de cyclotide pour le ciblage thérapeutique de récepteurs baff dans le lupus érythémateux systémique les Ceased WO2020006298A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021163989A1 (fr) * 2020-02-21 2021-08-26 Yinnuolai Biotech Ltd. Anticorps anti-récepteur baff et leurs utilisations
CN115120704A (zh) * 2022-07-25 2022-09-30 山东中医药大学 一种通过降低神经细胞氧化损伤来治疗抑郁症的药物
US12291008B2 (en) 2018-06-29 2025-05-06 Vitro Flat Glass Llc Burn-off protective coating

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BR0211614A (pt) * 2001-08-03 2006-10-31 Genentech Inc polipeptìdeo tacis e br3 e empregos dos mesmos
WO2013093045A2 (fr) * 2011-12-22 2013-06-27 Medizinische Universität Wien Cyclotides en tant qu'agents immunosuppresseurs

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12291008B2 (en) 2018-06-29 2025-05-06 Vitro Flat Glass Llc Burn-off protective coating
WO2021163989A1 (fr) * 2020-02-21 2021-08-26 Yinnuolai Biotech Ltd. Anticorps anti-récepteur baff et leurs utilisations
CN115120704A (zh) * 2022-07-25 2022-09-30 山东中医药大学 一种通过降低神经细胞氧化损伤来治疗抑郁症的药物

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