Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the field of this invention is methods for modulating nerve cell function.
• commissural growth cones initially orient toward the midline but then fail to cross it and instead recoil and extend on their own side
• robo mutant embryos display the opposite phenotype in that too many axons cross the midline; many growth cones that normally extend only on their own side instead now project across the midline and axons that normally cross the midline only once instead appear to cross and recross multiple times (Seeger et al, 1993; present disclosure).
• Double mutants of comm and robo display a rob ⁇ -like phenotype.
• Drosophila Drosophila.
• robo encodes a new class of guidance receptor with 5 immunoglobulin (Ig) domains, 3 fibronectin type III domains, a transmembrane domain, and a long cytoplasmic domain.
• Ig immunoglobulin
• Robo defines a new subfamily of Ig superfamily proteins that is highly conserved from fruit flies to mammals.
• the Robo ectodomains, and in particular the first two Ig domains, are highly conserved from fruit fly to human, while the cytoplasmic domains are more divergent. Nevertheless, the cytoplasmic domains contain three highly conserved short proline-rich motifs which may represent binding sites for SH3 or other binding domains in linker or signaling molecules.
• the invention provides methods and compositions for modulating the amount of active Robo expressed on a cell.
• the general method involves modulating the effective amount of a
• the Robo polypeptide in contact with a cell expressing an amount of active Robo polypeptide, whereby the amount of expressed active Robo is modulated inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell. For example, where the effective amount of the Comm polypeptide is increased, the amount of expressed Robo is decreased.
• the Robo polypeptide is preferably a human, mouse, C. elegans or Drosophila
• the Comm polypeptide specifically modulates Robo expression and (a) comprises SEQ ID NO: 14 or a deletion mutant thereof which specifically modulates Robo expression and/or (b) is encoded by a nucleic acid comprising SEQ ID NO: 13 or a nucleic acid which hybridizes with SEQ ID NO: 13, preferably under stringent conditions.
• the Comm polypeptide is provided to the cell exogenously in a pharmaceutically acceptable composition.
• the invention provides methods of screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo- expressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell.
• the subject methods involve modulating the effective amount of a Comm polypeptide in contact with a cell expressing an amount of active Robo polypeptide, whereby the amount of expressed active Robo is modified inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell.
• Robo expression is found to regulate a wide variety of cell functions, including cell-cell interactions, cell mobility, morphology, etc. Accordingly, the invention provides methods for modulating targeted cell function comprising the step of modulating Robo expression by contacting the cell with a Comm polypeptide.
• the targeted Robo polypeptide is generally naturally expressed on the targeted cells.
• C. elegans, human 1, human 2 and mouse 1 Robo polypeptides are shown as SEQ ID NOS:l,
• the targeted Robo polypeptides comprise at least a functional domain of SEQ ID NOS:2, 4, 6, 8, 10 and 12, which domain has Robo-specific amino acid sequence and binding specificity or function.
• Preferred Robo domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of one of these SEQ ID NOS.
• the domains comprise one or more structural/functional Robo immunoglobulin, fibronectin or cytoplasmic motif domains described herein.
• the subject domains provide Robo-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to Robo-
• D2 sequence was predicted by the gene-finder program Grail.
• Ig immunoglobulin domains
• FN fibronectin domains
• TM transmembrane domain
• conserved cytoplasmic motifs are indicated.
• the extracellular domain of rat robo 1 is nearly identical to HI.
• KSvellDasAinAsavrlE M HvSADEkyveg RiHyK..DaSVPSAQYHSITvMDAsa 725 DI DwelSnasvVDstsMKlTwQI... INGkyvegFyVYArQLpNPLNTKyRMLTILNGGGa D2
• species-specific antigenic and/or immunogenic peptides are readily apparent as diverged extracellular or cytosolic regions in Table 1.
• Human Robo-specific antibodies are characterized as uncross-reactive with non-human Robo polypeptides (SEQ ID NOS:2, 4, 6 and 12). Exemplary such human specific peptides are shown in Table 3.
• the subject domains provide Robo domain specific activity or function, such as Robo- specific cell, especially neuron modulating or modulating inhibitory activity, Robo-ligand- binding or binding inhibitory activity.
• Robo-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.
• the binding target may be a natural intracellular binding target, a Robo regulating protein or other regulator that directly modulates Robo activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a Robo specific agent such as those identified in screening assays such as described below.
• Robo-binding specificity may be assayed by binding equilibrium constants (usually at least about 10 7 M “ ', preferably at least about 10 8 M 1 , more preferably at least about 10 9 M "1 ), by the ability of the subject polypeptide to function as negative mutants in Robo-expressing cells, to elicit Robo specific antibody in a heterologous host (e.g a rodent or rabbit), etc.
• binding equilibrium constants usually at least about 10 7 M " ', preferably at least about 10 8 M 1 , more preferably at least about 10 9 M "1
• the Comm polypeptide is conveniently selected from Comm polypeptides which specifically modulate Robo expression.
• Comm polypeptides (a) comprise SEQ ID NO: 14 or a deletion mutant thereof which specifically modulates Comm expression and/or (b) are encoded by a nucleic acid comprising SEQ ID NO: 13 or a nucleic acid which hybridizes with SEQ ID NO: 13 under stringent conditions. Suitable deletion mutants are readily screened in Robo down-regulations assays as described below.
• Preferred Comm domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of SEQ ID NO: 14 and provide a Comm specific activity, such as Comm-specific antigenicity and/or immunogenicity, especially when coupled to carrier proteins as described above for Robo.
• Comm specific immunogenic and/or antigenic peptides are shown in Table 4.
• the subject domains provide Comm domain specific activity or function, such as
• Comm-specific cell especially neuron modulating or modulating inhibitory activity, Comm- ligand-binding or binding inhibitory activity.
• Comm-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.
• the binding target may be a natural intracellular binding target, a Comm regulating protein or other regulator that directly modulates Comm activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a Comm specific agent such as those identified in screening assays such as described below.
• Comm-binding specificity may be assayed by binding equilibrium constants (usually at least about 10 7 M “1 , preferably at least about 10 8 M "1 , more preferably at least about 10 9 M '1 ), by the ability of the subject polypeptide to function as negative mutants in Comm-expressing cells, to elicit Comm specific antibody in a heterologous host (e.g a rodent or rabbit), etc.
• binding equilibrium constants usually at least about 10 7 M "1 , preferably at least about 10 8 M "1 , more preferably at least about 10 9 M '1
• a heterologous host e.g a rodent or rabbit
• the Comm polypeptides are encoded by a nucleic acid comprising SEQ ID NO: 13 or a nucleic acid which hybridizes with a full-length strand of SEQ ID NO: 13, preferably under stringent conditions.
• nucleic acids are at least 36, preferably at least 72, more preferably at least 144 and most preferably at least 288 bases in length.
• Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M NaCl, 0.01 M NaPO 4 , pH7.7, 0.001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2 x SSPE (Conditions I); preferably hybridizing in a buffer comprising 50% formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2 x SSPE buffer at 42°C (Conditions II).
• Exemplary nucleic acids which hybridize with a strand of SEQ ID NO: 13 are shown in Table 5.
• SEQ ID NO:13 nucleotides 261-299 + SEQ ID NO 13, nucleotides 274-315 + SEQ ID NO 13, nucleotides 351-389 + SEQ ID NO 13, nucleotides 450-593 + SEQ ID NO 13, nucleotides 524-546 + SEQ ID NO 13, nucleotides 561-608 + SEQ ID NO 13, nucleotides 689-727 + SEQ ID NO 13, nucleotides 708-737 SEQ ID NO 13, nucleotides 738-801 + SEQ ID NO 13, nucleotides 805-854 + SEQ ID NO 13, nucleotides 855-907 + SEQ ID NO 13, nucleotides 910-953 + SEQ ID NO 13, nucleotides 1007-1059 +
• a wide variety of cell types express Robo polypeptides subject to regulation by the disclosed methods, including many neuronal cells, transformed cells, infected (e.g. virus) cells, etc. Ascertaining Robo expression is readily effected by .antibody staining. Accordingly, indications for the subject methods encompass a wide variety of cell types and function, including axon outgrowth, tumor cell invasion or migration, etc.
• the target cell may reside in culture or in situ, i.e. within the natural host. For in situ applications, the compositions are added to a retained physiological fluid such as blood or synovial fluid.
• Comm polypeptides may also be amenable to direct injection or infusion, topical, intratracheal/nasal administration e.g. through aerosol, intraocularly, or within/on implants e.g. fibers e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc.
• a particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic polypeptides.
• Other useful approaches are described in Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-
• the amount administered will be empirically determined, typically in the range of about 10 to 1000 ⁇ g/kg of the recipient and the concentration will generally be in the range of about 50 to 500 ⁇ g/ml in the dose administered.
• Other additives may be included, such as stabilizers, bactericides, etc. will be present in conventional amounts.
• the invention provides administering the subject Comm polypeptides in combination with a pharmaceutically acceptable excipient such as sterile saline or other medium, gelatin, an oil, etc. to form pharmaceutically acceptable compositions.
• a pharmaceutically acceptable excipient such as sterile saline or other medium, gelatin, an oil, etc.
• the compositions and/or compounds may be administered alone or in combination with any convenient carrier, diluent, etc. and such administration may be provided in single or multiple dosages.
• Useful carriers include solid, semi-solid or liquid media including water and non- toxic organic solvents.
• the invention provides the subject compounds in the form of a pro-dmg, which can be metabolically converted to the subject compound by the recipient host.
• pro-dmg formulations for polypeptide-based therapeutics are known in the art.
• compositions may be provided in any convenient form including tablets, capsules, troches, powders, sprays, creams, etc.
• the compositions in pharmaceutically acceptable dosage units or in bulk, may be incorporated into a wide variety of containers.
• dosage units may be included in a variety of containers including capsules, pills, etc.
• the compositions may be advantageously combined and/or used in combination with other therapeutic or prophylactic agents, different from the subject compounds.
• administration in conjunction with the subject compositions enhances the efficacy of such agents, see e.g. Goodman & Gilman 's The Pharmacological Basis of Therapeutics, 9 th Ed., 1996, McGraw-Hill.
• the invention provides methods of screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo-expressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell.
• the methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.
• Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
• Cell and animal based neural guidance/repulsion assays are described in detail in the experimental section below. The following experimental section and examples are offered by way of illustration and not by way of limitation.
• 1D4 MAb anti-Fas II which at stage 13 stains a subset of growth cones (including aCC, pCC, vMP2, MP1, dMP2) and from stages 14-17 stains three major longitudinal axon tracts, including (from medial to lateral) the pCC pathway (pioneered by the pCC growth cone), the MP1 pathway (pioneered by the MP1 growth cone), and a 3rd lateral pathway (Lin et al., 1994; Hidalgo and Brand, 1997).
• the vMP2 cell body lies embedded at the edge of the midline.
• the pCC growth cone extends anterior to a point just lateral to vMP2's cell body.
• the pCC growth cone is then met by the lateral extension of vMP2's growth cone, and as pCC extends anteriorly and a bit laterally, the vMP2 growth cone wraps around pCC's axons and extends right behind it (Lin et al., 1994).
• Fasciclin II Fas II
• CAM homophilic cell adhesion molecule
• pCC and vMP2 neurons express Fas II, and their growth cones and axons are so attracted to each other in a Fas Il-mediated fashion, why does not pCC's growth cone initially extend more medially toward vMP2's cell body which is a short distance away?
• pCC's initial trajectory is directly toward vMP2's cell body, where it adheres to vMP2; pCC's growth cone then crosses the midline, fasciculating with its contralateral homologue at the midline.
• Connectin Nose et al., 1992
• Connectin a CAM expressed on a more restricted subset of CNS axons than is Fas II.
• Connectin is also expressed on motor axons in the segmental nerve.
• C 1.427 MAb to follow Connectin expression (Meadows et al., 1994).
• Connectin is expressed on the SP1 neuron whose cell body lies near the midline just anterior to the anterior commissure, and just medial to the longitudinal tracts.
• SPl's growth cone normally projects across the midline, fasciculating with the axon of its contralateral homologue. The growth cone then appears to adhere to the cell body of its contralateral homologue, grows around that cell body, and turns to project anteriorly in a medial sub-fascicle of the pCC pathway.
• SPl's growth cone extends across the midline, adheres to the axon and then cell body of its contralateral homologue, and turns to project anteriorly. However, as it extends anteriorly into the next segment, it typically moves toward the midline, apparently attracted towards and adhering to the axon of its contralateral homologue just on the other side of the midline.
• the two SP1 axons typically join together around the posterior commissure of the next anterior segment. Sometimes they extend together on the left side of the midline and sometimes on the right side, freely crossing and recrossing the midline while fasciculating with the SP1 axons originating from both sides of neighboring segments.
• the Ap neurons have lateral cell bodies and their growth cones initially project towards the midline. Upon nearing the midline, these growth cones then turn to project anteriorly on their own side along the edge of the midline, fasciculating with each other and with their homologues from neighboring segments; in wild type embryos, they never cross the midline in abdominal segments.
• the Ap axons cross the midline in every segment, join up with their contralateral homologues, and often project anteriorly in one discrete longitudinal fasicle.
• the Ap fascicle displays two behaviors, usually crossing and recrossing the midline multiple times as a single bundle, or occasionally separating into different bundles of axons which project on one side or the other and independently cross the midline.
• the commissureless (comm) mutant has a complementary phenotype to that of robo in that too few axons cross the midline (Seeger et al., 1993).
• the axon commissures are noticeably absent.
• certain hypomorphic comm alleles e.g., comm ; Tear et al., 1996), the commissures are not completely absent, but rather partial and highly abnormal axon commissures do form in a few segments (particularly in the thorax).
• GAL4 system Brain and Perrimon, 1993 to change the pattern of comm expression.
• the robo phenotype can be mimicked by mutants causing inappropriate migration or cell death of the midline glia, both of which result in fuzzy commissures (Klambt et al., 1991).
• such phenotypes are not visible until midway through axonogenesis, and are easily detected by examining early axon behavior.
• Fas II positive axons suich as pCC
• pCC Fas II positive axons
• Comm overexpressed, the pCC growth cone extends towards the vMP2 cell body, and then across the midline, just as it does in a robo mutant.
• the pCC fascicle freely crosses and midline and forms the same circles or whirls as it does in the robo loss-of- function.
• the elav-GAL4 line also expresses pan-neurally but only in post-mitotic neurons; it begins driving expression of UAS transgenes during stage 12 and remains expressed throughout the rest of embryogenesis. Ectopic expression of comm by elav-GAL4 led to a less severe version of the robo phenotype. We interpret this weaker phenotype as being due to either a weaker overall level of Comm expression or because increased Comm initiates after the pioneers have already established the initial pathways.
• Comm Only a small amount of Comm is normally expressed at the midline.
• the midline also expresses high levels of a putative repellent that is the ligand for the Robo receptor.
• Growth cones that express high levels of Robo such as ipsilaterally projecting growth cones from the outset or commissural growth cones once they cross the midline, are relatively immune to significant down-regulation by the normally low levels of midline Comm and thus are prevented from crossing the midline. Only abnormally high levels of Comm (using transgenes that drive overexpression) are sufficient to down-regulate this Robo expression to a level that allows these growth cones to cross the midline.
• growth cones that normally express lower levels of Robo are highly sensitive to Comm, in that the normal low levels of Comm can further reduce their levels of Robo and thus allow them to cross the midline. In the absence of Comm, these growth cones can not cross the midline, due to their low levels of Robo; in the robo; comm double mutant they all freely cross.
• MAb 13C9 was diluted 1:10 in PBS with 0.1% Tween- 20, and the embryos were fixed and cracked so as to minimize exposure to methanol. The presence of triton and storage of embryos in methanol were both found to destroy the activity of MAb 13C9.
• MAb C1.427 the embryos were fixed in
• the comm cDNA was inserted as a 1.7kb Xhol-Xbal fragment into the Xhol and Xba sites of pUAST (Brand and

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