WO2000064473A1 - Composition pour la regeneration neuronale, comprenant des anticorps specifiques de la myeline et des complements - Google Patents

Composition pour la regeneration neuronale, comprenant des anticorps specifiques de la myeline et des complements Download PDF

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Publication number
WO2000064473A1
WO2000064473A1 PCT/CA2000/000440 CA0000440W WO0064473A1 WO 2000064473 A1 WO2000064473 A1 WO 2000064473A1 CA 0000440 W CA0000440 W CA 0000440W WO 0064473 A1 WO0064473 A1 WO 0064473A1
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myelin
complement
composition
fragments
antibody
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PCT/CA2000/000440
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English (en)
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John D. Steeves
Jason K. Dyer
Hans S. Keirstead
Jason Bourque
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University Of British Columbia
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Priority to AU40959/00A priority Critical patent/AU4095900A/en
Priority to JP2000613463A priority patent/JP2002542299A/ja
Priority to EP00920306A priority patent/EP1173200A1/fr
Priority to CA002371592A priority patent/CA2371592A1/fr
Priority to IL14619600A priority patent/IL146196A0/xx
Publication of WO2000064473A1 publication Critical patent/WO2000064473A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • This invention relates to compositions and their methods of use in promoting the growth and/or regeneration of neurological tissue within the central nervous system (CNS).
  • CNS central nervous system
  • the CNS (the brain and the spinal cord) is comprised of neurons and glia, such as astrocytes, microglia, and oligodendrocytes.
  • Neurons typically have two types of processes: dendrites, which receive synaptic contact from the axons of other neurons; and axons, through which each neuron communicates with other neurons and effectors.
  • the axon of a CNS neuron is wrapped in a myelin sheath.
  • axons within the CNS possess a limited capacity for repair after injury.
  • Axotomized neurons of the adult mammalian CNS fail to exhibit substantial axonal regeneration, in contrast to neurons within the embryonic or neonatal CNS or within the adult peripheral nervous system (PNS) (Saunders et al, (1992) Proc. R. Soc. Lond. B. Biol. 250:171-180; Schwab and Bartoldi (1996) Physiol Rev. 76:319-370; Steeves et al, (1994) Prog. Brain Res. 103:243-262).
  • PNS peripheral nervous system
  • Interventional therapies including opiate antagonists, thyrotropin-releasing hormone, local cord cooling, dextran infusion, adrenergic blockade, corticosteroids, and hyperbaric oxygen have been utilized, but are of questionable clinical value.
  • Peripheral nerve transplants have been suggested as bridges across CNS lesions (David and Aguayo (1981) Science 214:931-933; Houle (1991) Exp. Neurol. 113: 1-9; Richardson et al, (1984) J. Neurocytol. 13: 165-182; Richardson et al, (1980) Nature 284:264-265; Xu et al, (1995) Exp. Neurol 138:261-276; Ye and Houle (1997) Exp. Neurol. 143:70-81).
  • US Patents No. 5650148 and 5762926 describe a method for treating damage to the CNS by grafting donor cells into the CNS that have been modified to produce molecules such as neurotrophins.
  • transplanted neural cells are also of limited clinical value: although axons will be able to grow into the transplanted tissue, they will not be able to grow out of the transplanted tissue back into the CNS due to inhibitory matter in the CNS.
  • Myelin It has been suggested that the failure of CNS axons to regenerate after injury is associated with the presence of myelin.
  • the myelin sheath wrapping an axon is composed of compacted plasma membranes of Schwann cells and oligodendrocytes. Although its composition resembles that of any other plasma membrane in that it contains lipids, proteins, and water, the relative proportions and dispositions of these components are unique to myelin.
  • Myelin in the CNS is produced by oligodendrocytes and is characterized by the expression of myelin basic protein (MBP). MBP is only associated with myelin and is one of the first proteins expressed at the onset of myelination of CNS axonal fibers.
  • MBP myelin basic protein
  • GalC Galactocerebroside
  • GalC is the major sphingolipid produced by oligodendrocytes. GalC comprises approximately 15 percent of the total lipid in human myelin and is highly conserved across species. Although GalC is expressed on the surface of oliogodendrocyte cell bodies, it is expressed in greater concentration on the surface of myelin membranes (Ranscht et al, (1982) Proc. Natl. Acad. Sci. USA 79:2709-2713).
  • CNS myelin can retard or inhibit the regenerative growth of some severed CNS axons (Schwab and Bartoldi (1996) Physiol Rev. 76:319-370), including a number of examples from widespread vertebrate families (Schwegler et al, (1995) J. Neurosci. 15:2756-2767; Steeves et al, (1994) Prog. Brain Res. 103:243-262). Both the lower vertebrate CNS (e.g. lamprey) and the developing CNS of higher vertebrates (e.g. birds and mammals) exhibit substantial axonal regeneration after injury (Davis and McClellan (1994) J. Comp. Neurol. 344:65-82;
  • the common phenotype for all these positive examples of regeneration is either a C ⁇ S that lacks compact myelin (lamprey) or incomplete myelin development (embryonic chick, neonatal opossum and rat) at the time of injury.
  • the developmental appearance of myelin temporally correlates with the loss of regeneration by injured C ⁇ S axons.
  • the robust growth of transplanted fetal neurons in the adult C ⁇ S (Bregman et al, ( ⁇ 993) Exp. Neurol 123:3-16; Li and Raisman (1993) Brain Res. 629: 115-127;
  • Yakovleff et al, (1995) Exp. Brain Res. 106:69-78) may be partially attributed to either a lack of receptors for myelin inhibitors at that stage of their differentiation and/or an ability to override any inhibitory signals from myelin.
  • myelin-associated glycoprotein MAG
  • MAG myelin-associated glycoprotein
  • myelin-deficient strains of mice and rats are readily available, but are of limited experimental value due to a shortened life span: most do not survive beyond a couple of weeks after birth.
  • the complement system is the primary humoral mediator of antigen-antibody reactions. It consists of at least 20 chemical and immunologically distinct serum proteins capable of interacting with one another, with antibody, and with cell membranes (see, for example, J. Klein, Immunology: The Science of Self _Nonself Discrimination (New
  • the classical pathway is activated by an antigen-antibody reaction: when an antibody binds with an antigen, a specific reactive site on the constant portion of the antibody becomes activated, which in turn binds directly with the Cl molecule of the complement system. This sets into motion a cascade of sequential reactions, beginning with the activation of the Cl proenzyme. Only a few antigen-antibody combinations are required to activate many molecules in this first stage of the complement system.
  • the Cl enzymes then activate successively increasing quantities of enzymes in the later stages of the complement system. Multiple end-products are formed, which cause important effects that help to prevent damage by an invading organism or toxin, including opsonization and phagocytosis, lysis, agglutination, neutralization of viruses, chemotaxis, activation of mast cells and basophils, and inflammatory effects.
  • the complement system can also be activated by an alternate pathway without the intermediation of an antigen-antibody reaction.
  • Certain substances react with complement factors B and D, forming an activation product that activates factor C3, setting off the remainder of the complement cascade; thus, essentially all the same final products of the system are formed as in the classical pathway, causing the same effects.
  • the alternate pathway does not involve an antigen-antibody reaction, it is one of the first lines of defense against invading microorganisms.
  • C3 is a 195 kD protein, which comprises two disulfide bridged chains of 105 and 75 kD.
  • the enzymatically active C4-C2 complex activated in the classical pathway by the binding of Clq to an antigen-antibody complex, cleaves C3 into two fragments, C3a and C3b.
  • the larger fragment, C3b binds covalently to the surface of a target cell where it acts as a protease to catalyze the subsequent steps in the complement cascade.
  • membrane-immobilized C3b triggers a further cascade of reactions that leads to the assembly of membrane attack complexes from the late components.
  • the onset of myelination in the embryonic chick spinal cord at E13 coincides with the transition from a permissive to a restrictive period for the functional repair of transected spinal cord.
  • the first appearance of chick oligodendrocytes on the tenth and eleventh embryonic day of development (E10-E11) precedes the initial formation of myelin by 2-3 embryonic days and is characterized by the expression of galactocerebroside (GalC), the major sphingolipid produced by oligodendrocytes.
  • GalC galactocerebroside
  • the purpose of the present invention is to provide a means of promoting regrowth, repair, and regeneration of neurons in the mammalian CNS. Accordingly, the invention provides compositions and methods of use for promoting regrowth, repair, and/or regeneration of neurons in the CNS of a mammalian subject, such as a human, in both chronic and acute disorders
  • a composition comprising therapeutically effective amounts of the following:
  • the antibodies may be monoclonal and/or polyclonal.
  • the complement proteins or fragments thereof may be derived from a species different from that species to which it is administered. In a preferred embodiment, the complement proteins or fragments thereof are human.
  • the complement component may be a physically distinct component from the antibody component, or it may be covalently or noncovalently attached directly to the antibody component, such that binding of the antibody to the surface of the myelin triggers the endogenous immune system attack.
  • One or more growth factors may be added (in an appropriate sequence) to facilitate regrowth and regeneration.
  • the epitope of myelin is a myelin sheath epitope, such as galactocerebroside (GalC), O4, Myelin Oligodendrocyte Glycoprotein (MOG), or Myelin Associated Glycoprotein (MAG), NOGO, NI22, NI-35/250, or arretin, or fragments thereof.
  • the epitope of myelin is GalC.
  • Another preferred embodiment is MOG.
  • the complement proteins or fragments thereof include the C3 component or a fragment, variant, analog, or chemical derivative thereof.
  • the component C3b is used.
  • the composition further comprises neurotrophins and growth factors, such as NT-3, CNTF, FGF-1, BDNF, PDGF, GDNF, CT-l, or BMP.
  • neurotrophins and growth factors such as NT-3, CNTF, FGF-1, BDNF, PDGF, GDNF, CT-l, or BMP.
  • the present invention also relates to the use of these compositions to promote regrowth, repair, and/or regeneration of neurons in a subject by the transient disruption and/or transient demyelination of myelin.
  • the compositions are used in subjects requiring neuron repair and/or regeneration due to neuron dysfunction.
  • This neuron dysfunction may be a result of acute or chronic injury to the CNS. It may also be a result of degenerative disease, such as Alzheimer's or Parkinson's disease.
  • the compositions are used in subjects to generate an environment within the CNS that is relatively permissive to growth of transplanted cells.
  • the present invention also relates to a method of promoting regrowth, repair, and regeneration of neurons in mammalian CNS, wherein the damage resulted from either a chronic or acute disorder.
  • the method entails delivery of one or more complement- fixing antibodies or fragments thereof, which specifically bind to an epitope of myelin and delivery of one or more complement proteins or fragments thereof, delivered either together or separately to effect transient disruption and/or transient demyelination of myelin in the neuronal zone requiring regeneration.
  • Table 1 presents rubrospinal neuronal cell counts obtained from individual control and experimental animals with retrograde Fluorogold labeling from the lumbar cord of an adult rat.
  • Figure 1 presents (A) Photomicrograph of a transverse section of spinal cord of an adult rat at the level of T10 left side hemisection lesion, stained with cresyl violet. All lesions were assessed and always resulted in severing the funiculi through which the rubrospinal tract traverses. The contralateral dorsal (dh) and ventral (vh) horns were always left undamaged; the central canal (cc) is labeled for orientation. (B) Assessment of visible Fluorogold diffusion in the control treated and immunologically disrupted hemisected spinal cord. Diffusion of the retrograde tracer was measured at the light microscope level at the time points indicated after injection into the lumbar spinal cord (see methods for details).
  • FIG. 2 shows electron photomicrographs of transverse sections through the dorsolateral funiculus after continuous intraspinal infusion of immunological reagents for 7 days.
  • A Within one spinal segment ( ⁇ 2mm) of the infusion site, large regions of naked, demyelinated axons were visible. Some axons were observed to be associated with monocyte cells (M, e.g. infiltrating macrophage) and or endogenous microglia, some of which also contained myelin ovoids (arrow) or myelin debris.
  • M monocyte cells
  • endogenous microglia some of which also contained myelin ovoids (arrow) or myelin debris.
  • monocytes and invading polymorphonucleocytes could also be seen in close association with demyelinated axons. Macrophages and/or microglia were identified on the basis of their high density endoplasmic reticulum (arrow-heads), and "finger-like" processes. Some monocytes have laid down basal lamina components such as collagen (Col), which distinguishes them from astrocytes. Multi-lobed nuclei are characteristic of PMNs and facilitate their identification.
  • C Example of myelin- disruption.
  • Figure 3 presents demonstrations of regeneration of rubrospinal neurons after left-side thoracic hemisection and subsequent immunological myelin suppression treatment.
  • Panels A and B are photomicrographs of rubrospinal neurons from the same experimentally-treated animal (14 days infusion of serum complement with anti-GalC);
  • A is from the uninjured Red nucleus while B is from the injured Red nucleus.
  • Panels C and D are also from same control-treated animal (14 days infusion of serum complement only): C is the uninjured Red nucleus and D is the injured Red nucleus.
  • Fluorogold injection within the rostral lumbar cord 28 days after injury resulted in the retrograde labeling of uninjured rubrospinal neurons (A and C) as well as those rubrospinal neurons that had regenerated from the injured Red nucleus (B and D).
  • Figure 4 shows a relative quantitative assessment of regeneration of rubrospinal neurons after thoracic injury and immunological treatment. Regeneration was assessed by counting FG-labeled cells in alternating tissue sections: those with both multipolar neuronal morphology and FG labeling were deemed to be positive. Percentage regeneration was calculated by comparison of the retrograde labeled cell counts from the injured Red nucleus with the control uninjured Red nucleus within the same animal. For each animal, the degree of lesion was assessed. Filled bar: myelin suppressed; hatched bar: pooled control treated groups. Data shown ⁇ s.d.
  • Figure 5 demonstrates effects of removal of a single complement protein on immunological demyelination.
  • A Control uninjured spinal cord. Electron photomicrographs of transverse sections through the dorsolateral funiculus indicating the ultrastructure of adult myelin sheaths.
  • B 7 day infusion with myelin-specific antibody and human complement sera results in a profound myelin suppression.
  • C The removal of the C3 component of complement results in a lack of myelin-removal, indicating the fundamental role of this protein in either (i) opsonization, or (ii) the propagation of the cascade to the lytic membrane attack complex (MAC), the final lytic pathway complex. It is believed that it is a fundamental and essential requirement of a myelin specific cell surface binding antibody to activate the classical complement pathway for effective transient demyelination.
  • MAC lytic membrane attack complex
  • Figure 6 shows a relative quantitative assessment of regeneration of lateral vestibulospinal neurons after thoracic injury and delayed immunological treatment. Immunological demyelination treatment was delayed for 1 or 2 months after injury as indicated. Regeneration was assessed by counting FG-labeled cells in alternating tissue sections: those with both multipolar neuronal morphology and FG labeling were deemed to be positive. Percentage regeneration was calculated by comparison of the retrograde labeled cell counts from the injured lateral vestibulospinal nucleus with the control uninjured lateral vestibulospinal nucleus within the same animal. For each animal, the degree of lesion was assessed. Filled bar: myelin suppressed; open bar: pooled control treated groups. Data shown ⁇ s.d.
  • Figure 7 presents A) Drawing of a dorsal view of the rat central nervous system, indicating the relative origins and course of the rubrospinal tract (RN) and lateral vestibular tract (LNe). Also illustrated (solid line) is the left-side thoracic hemisection lesion ( ⁇ T10, line), the immunological infusion site ( ⁇ Tl 1, vertical hatching), and the site of the Fluorogold injection ( ⁇ L1, diagonal hatching). B) composite photomicrograph of parasagittal sections through the lower thoracic and rostral lumbar spinal cord (T9- LI, rostral is up).
  • Figure 8 shows regeneration of lateral vestibulospinal neurons after left-side thoracic hemisection and subsequent immunological myelin suppression treatment.
  • Panels A and B are photomicrographs of lateral vestibulospinal neurons from the same experimentally-treated animal (14 days infusion of serum complement with anti-GalC);
  • A is of the injured lateral vestibular nucleus and B is from the uninjured lateral vestibular nucleus and.
  • Panels C and D are also from same control-treated animal (14 days infusion of serum complement only); where C is the injured lateral vestibulospinal nucleus and D is the uninjured lateral vestibulospinal nucleus.
  • Fluorogold injection within the rostral lumbar cord 28 days after injury resulted in the retrograde labeling of uninjured lateral vestibulospinal neurons (B and D) as well as those lateral vestibulospinal neurons that had regenerated from the injured lateral vestibulospinal nucleus (A and C), please see results for further details.
  • Figure 9 shows relative quantitative assessment of regeneration of rubrospinal and lateral vestibulospinal neurons after thoracic injury and immunological treatment. Regeneration was assessed by counting FG-labeled cells in alternating tissue sections; those with both multipolar neuronal morphology and FG labeling, were deemed to be positive. Percentage regeneration was calculated by comparison of the injured nucleus with the contralateral (uninjured) nucleus within the same animal. For each animal the degree of lesion was assessed. Filled bars, experimental; open bars, pooled control groups.
  • Figure 10 shows a quantitative assessment of regeneration of descending brainstem- spinal axons after chronic lateral hemisection & delayed immunological treatment. Percentages of retrogradely labeled red nucleus (red) and lateral vestibular (green) neurons vs. Contralateral uninjured, after control (PBS, Ab, GpC) treatment (open bars) or immunological disruption/demyelination (filled bars). Expressed as percentage labeled cells in the injured nucleus vs. Uninjured contralateral.
  • Figure 11 shows the factors known to be involved in the classical and alternative pathways involved in immunological demyelination.
  • Figure 12 shows the effects of removal of a single complement protein from the composition on immunological demyelination. Electron photomicrographs are of transverse sections through the dorsolateral funiculus indicating the ultrastructure of adult myelin sheaths.
  • A demonstrates the effect of 7-day infusion with myelin-specific antibody and human complement sera results in a profound myelin suppression.
  • B The removal of C3 component of the complement results in a lack of myelin removal.
  • C The removal of Factor B from the complement indicates that the alternative pathway is not involved in immunological demyelination.
  • Figure 13 presents the effects of removal of a single complement protein, C5, from the composition on immunological demyelination.
  • the electron photomicrograph is of a transverse section through the dorsolateral funiculus indicating the ultrastructure of adult myelin sheaths.
  • the removal of C5 component indicates that this component is not necessary to be included in the composition for immunological demyelination.
  • antibodies or fragments thereof includes recombinant, chimeric, and affinity modified forms made by techniques of molecular biology well known in the art;
  • CNS central nervous system
  • complement protein or fragment thereof refers to any of 13 whole serum proteins or any of more than 20 intermediates and complexes of the complement system, the primary humoral mediator of antigen-antibody reactions, and includes variants, analogs, and chemical derivatives thereof.
  • composition is used to indicate more than one component.
  • the elements of the composition can be mixed together, however, it is not necessary that they be combined in the same solution. In an alternative embodiment, they do not need to be packaged, stored or even mixed together.
  • the elements (antibody-type and complement-type) can be delivered to the site of nerve damage sequentially, or at the same time. The need for a therapeutically effective temporal sequence is understood by one skilled in the art.
  • the concept of at least one complement fixing antibody or fragment thereof, plus at least complement protein or active fragment thereof equates with the concept of the composition.
  • These elements are delivered to the site of damage to form a complex with an appropriate epitope present in myelin to be transiently demyelinated.
  • the first two types of elements (of which there can be more than one member of each type of element, for example, two or more antibody or component proteins or fragments) are delivered to the site targeted for transient demyelination to form a complex in situ, in vivo with the epitope(s) on myelin.
  • Demyelination refers to the removal or breakdown of myelin in neurological tissue. Demyelination consists of the removal of the myelin sheath, such as that surrounding neurons or neuronal projections (e.g., the axons). This process may be chemical or immunological in both the experimental and pathological states. This invention effects transient demyelination in order to promote repair and regrowth.
  • disruption refers to delamination or disruption of the three-dimensional conformation of myelin
  • disfunction when used to describe the therapeutic use of the invention encompasses any type of trauma to the nervous system and resulting loss of function. Such trauma can arise from either physical injury or disease;
  • Fab means an antibody fragment that is obtained by cleaving an antibody in the hinge region yielding two Fab fragments, each having the heavy and light chain domains of the antibody, along with an Fc region;
  • Fc means the constant region of the antibody, which may activate complement;
  • Fv fragment means a heterodimer of the heavy and light chain variable domain of an antibody. These variable domains may be joined by a peptide linker or by an engineered disulphide bond;
  • Growth factors are extracellular polypeptide signaling molecules that stimulate a cell to grow or proliferate. Examples are epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Most growth factors have other actions besides the induction of cell growth or proliferation. Growth factors can be divided into broad- and narrow- specificy classes. The broad-specificity factors, like PDGF and EGF affect any classes of cells. At the opposite extreme lie narrow-specificity factors. In intact animals proliferation of mot cell types depends on a specific combination of growth factors rather than a single growth factor. Thus a fairly smal number of growth factor families may serve, in different combinations, to regulate selectively the proliferation of each of the many types of cells in a higher animal.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • Fibroblast Growth Factor is any one of a group of proteins, usually intracellular, that have important angiogenic function and enhance would healing and tissue repair. Over-activity of these factors has been associate with neoplasia.
  • Neurotrophic factors are a family of substances that promote growth and regeneration of neurons. While growth factors elsewhere in the body promote and support cell division, neurons cannot divide; but they can regenerate after injury and neurotrophic factors promote this regeneration. They also promote the growth of axons and dendrites, the neuron branches that form connections with other neurons.
  • GalC refers to galactocerebroside
  • MAG refers to myelin-associated glycoprotein
  • MBP myelin basic protein
  • MOG refers to myelin oligodendrocyte glycoprotein
  • neurological tissue refers to neurons and other cells typically situated in the region of the nervous system, such as the spinal cord of the CNS;
  • PNS peripheral nervous system
  • recombinant antibodies or fragments thereof collectively includes chimeric or recombinant forms of the antibodies or fragments thereof wherein the Fc domain is substituted for an Fc domain of another species or isotype, affinity modified forms of the antibodies or fragments thereof wherein the binding sites are altered, avidity modified forms of the antibodies or fragments thereof wherein the hinge regions are altered, immunoreactive fragments thereof, and combinations thereof; and
  • the term "regeneration of neurological tissue” includes the regrowth of neurons that results in the reformation of neuronal connections, both anatomically and/or functionally.
  • the present invention resides in the unexpected discovery that a combination of both antibody, which binds an epitope on a myelin-producing glial cell, and complement can be used for disruption and demyelination of the myelin sheath, such that repair and regeneration of mammalian neurological tissue is enhanced.
  • the composition of this invention is valuable as a therapeutic agent in cases in which there is injury or disease of the mammalian nervous system such that there is a need to facilitate neuronal plasticity and the regrowth of neural connections.
  • the neurological tissue is exposed to the myelin disrupting composition, according to the invention, as soon as possible following the injury, trauma, or disease.
  • the present invention provides compositions and methods of their use for promoting regeneration of neurological tissue in a mammalian subject, such as a human, with a nervous system dysfunction by contacting the neurological tissue with a therapeutically effective amount of a composition comprising a complement fixing antibody, which binds to myelin, and complement.
  • a composition comprising a complement fixing antibody, which binds to myelin, and complement.
  • Uses of the composition in the field of veterinary medicine are also an embodiment of the present invention.
  • compositions of the present invention are comprised of one or more antibodies or fragments thereof, which bind myelin, and one or more serum complement proteins or fragments thereof.
  • the antibodies used in this invention can be any antibodies or fragments thereof that specifically bind to myelin, wherein said antibodies activate the complement system.
  • the preferred antibodies of the present invention specifically bind a myelin sheath epitope, such as galactocerebroside (GalC), 04, Myelin Oligodendrocyte Glycoprotein (MOG), or Myelin Associated Glycoprotein (MAG).
  • GalC galactocerebroside
  • MOG Myelin Oligodendrocyte Glycoprotein
  • MAG Myelin Associated Glycoprotein
  • NOGO (formerly NI 35/250) and NI220 and arretin.
  • the antibodies of the present invention can be: a) naturally occurring; b) antibodies obtained from disease states such as B-cells from multiple- sclerosis patients; c) produced by recombinant DNA technology; d) produced by biochemical or enzymatic fragmentation of larger molecules; e) produced by methods resulting from a combination of a) to c); or f) produced by any other means for producing antibodies.
  • Human antibodies can be generated by a number of techniques known to those skilled in the art, including the use of insect cells and transgenic plants such as tobacco or corn seed (Cramer, C.L., CropTech Development Corp; Reno, J., NeoRx - IVC's IV Annual
  • the antibodies of the present invention can also be made by traditional technqiues such as monoclonal or polyclonal, although monoclonal antibodies are preferred.
  • antibodies may be obtained by injecting the desired immunogen into a wide variety of vertebrates or invertebrates in accordance with conventional techniques. While rodents, particularly mice, are preferred, other species may be employed, such as members of the bovine, ovine, equine, porcine, or avian families. Immunization of these animals can be readily performed and their lymphocytes, particularly splenocytes, may be obtained for fusions.
  • Immunization protocols are well known and can vary considerably yet remain effective (Goding, Monoclonal Antibodies: Principles and Practice (2nd ed.) (Academic Press, 1986).
  • Isolated proteins, synthetic peptides, and bacterial fusion proteins which contain antigenic fragments of the myelin molecule may be used as immunogens.
  • the immunogen of peptides or recombinant proteins will be enriched for proteins or fragments thereof containing the epitopes to which antibody-producing B cells or splenocytes are desired.
  • the proteins or peptides thereof may be suspended or diluted in an appropriate physiological carrier for immunization, or may be coupled to an adjuvant.
  • Immunogenic amounts of antigenic preparations enriched in myelin, or antigenic portions thereof are injected, generally at concentrations in the range of 1 ug to 100 mg/kg of host. Administration may be by injection, such as intramuscularly, peritoneally, subcutaneously, or intravenously. Administration may be one or a plurality of times, usually at one to four week intervals.
  • Immunized animals are monitored for production of antibody to the desired antigens, then the spleens are removed and splenic B-lymphocytes isolated and fused with a myeloma cell line or transformed.
  • the B-lympocytes can also be isolated from the blood.
  • the transformation or fusion can be carried out in conventional ways, the fusion technique being described in an extensive number of patents, such as U.S. Patent Nos. 4,172,124; 4,350,683; 4,363,799; 4,381,292; and 4,423,147.
  • the manner of immortalization is not critical, but the most common method is fusion with a myeloma fusion partner.
  • Human monoclonal antibodies may be obtained by fusion of the spleen cells with an appropriate human fusion partner, such as WI-L2, described in European Application No. 82.301103.6.
  • WI-L2 human fusion partner
  • a detailed technique for producing mouse X-mouse monoclonal antibodies has been taught (Oi and Herzenberg (1980) in Mishell and Shiigi (eds.) Selected Methods in Cellular
  • the resulting hybridomas are screened to isolate individual clones, each of which secretes a single antibody species to the antigen.
  • the immortalized cell lines may be cloned and screened in accordance with conventional techniques, and antibodies in the cell supernatants detected that are capable of binding to myelin.
  • the appropriate immortalized cell lines may then be grown in vitro or injected into the peritoneal cavity of an appropriate host for production of ascites fluid.
  • Immortalized hybridoma cell lines can be readily produced from a variety of sources. Alternatively, these cell lines may be fused with other neoplastic B-cells, where such other B-cells may serve as recipients for genomic DNA coding for the antibody.
  • the monoclonal antibody secreted by the transformed or hybrid cell lines may be of any of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgGi--., or IgE. As IgG is the most common isotype utilized in diagnostic assays, it is often preferred.
  • chimeric antibodies may be constructed.
  • the antigen binding fragment of an immunoglobulin molecule (variable region) may be connected by peptide linkage to at least part of another protein not recognized as foreign by humans, such as the constant portion of a human immunoglobulin molecule. This can be accomplished by fusing the animal variable region exons with human kappa or gamma constant region exons.
  • Various techniques are known to the skilled artisan, such as those described in PCT 86/01533, EP171496, and EP 173494.
  • US Patent No. 5627052 describes methods of producing proteins that replicate the binding characteristics and desired function of particular antibodies.
  • An example of application of this method includes the isolation and characterization of a human B-lymphocyte cell, producing a specific anti- myelin antibody, for example from the blook of a patient with Multiple Sclerosis.
  • the antibodies may be used intact, or as fragments, such as Fv, Fab, and F(ab') 2 as long as there is an Fc region present to bind complement. Such antibody fragments provide better diffusion characteristics in vivo than the whole antibody, due to their smaller size.
  • the means for engineering antibodies by recombinant DNA and chemical modification methods are considered well-known in the art.
  • the antibodies may be fragmented to obtain highly immunoreactive F(ab') 2 , F(ab'), and Fab fragments using the enzyme pepsin by methods well known in the art (see Colcher et al, (1983) Cancer Res. 43:736-742).
  • Antibodies or fragments thereof are also made into recombinant forms by techniques of molecular biology well known in the art (see Rice et al, (1982) Proc. Natl Acad Sci. USA 79:7862-7865; Kurokawa et ⁇ b, (1983) Nucleic Acids Res. 11:3077-3085; Oi et al, (1983) Proc. Natl. Acad. Sci. USA 80:825-829; Boss et al, (1984) Nucleic Acids Res. 12:3791-3806; Boulianne et al, (1984) Nature (London) 312:643-646; Cabily et al, (1984) Proc. Natl. Acad. Sci.
  • the antibodies and fragments thereof may be altered to a chimeric form by substituting antibody fragments of another species, e.g., human constant regions (Fc domains) for mouse constant regions by recombinant DNA techniques known in the art as described in the above cited references.
  • Fc domains can be of various human isotypes, i.e., IgGi, IgG 2 , IgG 3 , IgG 4 , or IgM.
  • antibodies and fragments thereof may be altered to an affinity modified form, avidity modified form, or both, by altering binding sites or altering the hinge region using recombinant DNA techniques well known in the art as described in the above cited references.
  • the recombinant antibody forms may also be fragmented to produce immunoreactive fragments F(ab') 2 , F(ab'), and Fab in the same manner as described.
  • Antibody fragments may also include Fv fragments, the smallest functional modules of antibodies required to maintain the binding and specificity of the whole antibody.
  • Fv fragments are heterodimers composed of a variable heavy chain and a variable light chain domain. Proteolytic digestion of antibodies can yield isolated Fv fragments, but the preferred method of obtaining Fvs is by recombinant technology (See Skerra and Pluckthun (1988) Science 240: 1038-1041).
  • Fvs can be noncovalently-associated VH and V L domains, although these tend to dissociate from one another.
  • Stable Fvs can be produced by making recombinant molecules in which the VH and VL domains are connected by a peptide linker so that the antigen-combining site is regenerated in a single protein. These recombinant molecules are termed single chain Fvs (scFvs).
  • the means for preparing scFvs are known in the art (See: Raag and Whitlow (1995) FASEB 9:73; Bird et al, (1988) Science 242:423- 426; Huston et al, (1988) Proc. Natl Acad. Sci. USA 85:5879-5883).
  • the two variable domains may be joined and stabilized by an engineered disulphide bond; these are termed disulfide Fvs (dsFvs) (Reiter and Pastan (1996) Clin. Cancer Res. 2:245-252).
  • the Fc domain of an antibody is required for the activation of complement.
  • Fv fragments which lack the Fc domain, cannot activate complement.
  • Fv fragments would have to be designed with a novel activator of the complement cascade.
  • the Fv fragment could be designed to include the C H 2 domain of an IgG antibody.
  • a wholly synthetic molecule may be linked to the Fv fragment to activate complement, or an activator of complement familiar to those in the field may be linked to the Fv fragment.
  • the antibody may also be modified by the addition of such molecules as polyethylene glycol (as described in U.S. Patent 5766897) as to prolong its biological half-life, potency, or the diffusion of the molecule in situ (U.S. Patent 5747446, Chinol et al., 98
  • the antibodies of this invention, or fragments thereof, may be used without modification or may be modified in a variety of ways, for example, by labeling.
  • Labeling is intended to mean joining, either covalently or non-covalently, a label which directly or indirectly provides for a means of detection of the antibody to enable monitoring of the progress of therapeutic treatment using the composition.
  • a label can comprise any material possessing a detectable chemical or physical property.
  • labels including radionuclides, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), fluorescers, chromophores, luminescers, and magnetic particles. These labels are detectable on the basis of either their own physical properties (eg., fluorescers, chromophores and radioisotopes), or their reactive or binding properties (eg., enzymes, substrates, cofactors and inhibitors). These materials are well known to one skilled in the art.
  • U.S. Patent 4,671,958 teaches methods that can be used for labelling antibodies or attaching complement to antibodies.
  • the complement portion of the composition may be comprised of one or more complement proteins, fragments, variants, analogs, and/or chemical derivatives.
  • a fragment of a complement protein refers to any subset of the C molecule.
  • fragments of C3 include C3b, iC3b, C3a, C3c, C3dg, and C3d.
  • a “variant" of a complement protein or fragments thereof refers to a molecule substantially similar to either the entire protein or a fragment thereof, which possesses biological activity that is substantially similar to a biological activity of the complement protein or fragments thereof.
  • a molecule is said to be “substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity.
  • Variants of C3b include C3b dimers, and higher oligomers.
  • C activation occurs at the cell_surface, multiple cycles of enzyme reactions result in the deposition on the surface of C3b in multimeric form.
  • C3b dimers or higher oligomers indeed have higher affinity for the cell than do C3b monomers.
  • variants of complement protein or fragments thereof are produced by chemical or recombinant means well-known in the art. Such variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the amino acid sequence. For example, at least one amino acid residue may be removed and a different residue inserted in its place. Substantial changes in functional or immunological properties are made by selecting substitutions that are less conservative, ie. that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions that in general are expected to induce greater changes are those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side
  • deletions, insertions, and substitutions are not expected to produce radical changes in the characteristics of the protein molecule; however, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. For example, a change in the immunological character of the protein molecule, such as binding to a given antibody, is measured by an immunoassay such as a competitive type immunoassay.
  • an "analog" of a complement protein or fragment thereof refers to a non-natural molecule substantially similar to either the entire protein or a fragment thereof.
  • a "chemical derivative" of a complement protein or fragment thereof contains additional chemical moieties that are not normally part of the protein or fragment.
  • Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with organic derivatizing agents that are capable of reacting with selected side chains or terminal residues, as is well-known in the art (T. E.
  • Creighton Proteins Structure and Molecule Properties (San Francisco: W. H. Freeman, 1983) at 70-86 ).
  • the complement portion of the composition may be a physically distinct component from the antibody component.
  • the complement proteins or fragments thereof may be covalently or noncovalently attached directly to the antibody component, such that binding of the antibody to the surface of the myelin triggers the endogenous immune system attack.
  • the complement components may be fractions that have been purified as well as those that have been enriched in the proteins which comprise the complement system. Such preparations should take into account the relative lability of complement and provide a sufficient combination of factors to allow complete activation of the complement cascade to allow transient demyelination to occur.
  • the complement portion of the composition may be comprised of one or more complement proteins, fragments, variants, analogs, and/or chemical derivatives. It should be noted, however, that the C3 component of complement plays a fundamental role either in opsonization or in the propagation of the cascade to the lytic MAC. In a preferred embodiment, the C3 component or a fragment, variant, analog, or chemical derivative thereof should be included in the complement portion of the composition. In situations targeted for demyelination, the C3 component should certaintly be present for optimal results. In situations targeted for regeneration, it is less certaintly required.
  • the invention also includes modifications of the complement proteins. Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polyp eptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., changing glycosylation patterns, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
  • modifications of the complement proteins include in vivo, or in vitro chemical derivatization of polyp eptides, e.g., acetylation, or carboxylation.
  • modifications of glycosylation e.g., changing glycosylation patterns, e.g., those made by modifying the glycosylation patterns of a
  • the complement portion of the composition may be derived from a subject's own serum, from the serum of a donor, or from the pooled sera of a number of donors, such as those available commercially, which are produced to consistent, approved standards.
  • the complement components may be derived from species different from that species to which it is administered due to the fact that the compositions are introduced directly to the neural tissue (e.g., intrathecally).
  • the complement portion is a depleted complement, wherein one or more components of the normal complement have been removed.
  • Example V demonstrates that removal of certain components allows the complement portion of the composition to act according to the present invention. While, removal of the C3 protein or the C4 protein results in a composition with reduced efficacy, the use of complement depleted in Factor B, C5 or C6 protein resulted in effective demyelination. In each case, after removal of the complement component, the resulting composition is tested in vivo, for efficient demyelination ability.
  • the advantages to the use of depleted complement rather than whole complement are the reduction of potential side effects and the capacity to tailor the composition to each situation. In this way, the composition of the present invention can be designed to meet the requirements of treatment of various disorders and individuals.
  • the complement portion is prepared in combination with one or more inhibitors of one or more components of the normal complement.
  • one or more inhibitors of one or more components of the normal complement For example, several regulatory proteins of the complement system have been identified. Their primary functions are to regulate the activity of C3/C5 convertases for prevention of excessive complement activation and autolytic destruction of host tissues.
  • These complement regulators are either soluble plasma proteins or integral membrane proteins expressed on a variety of cell types.
  • the former include C4b binding protein (C4bp) and Factor H.
  • C3b/C4b receptor include C3b/C4b receptor
  • Complement receptor 1, CR1, CD35 membrane cofactor protein (MCP, CD46), and decay accelerating factor (DAF, CD55).
  • SCRs short consensus repeats
  • the genes encoding these proteins have been localized to chromosome 1 and are collectively known as the regulators of complement activation (RCA) gene cluster (Hourcade, D. et al., 1989, Adv. Immunol. 45:381).
  • RCA complement activation
  • erythrocyte CR1 In addition to its role in regulating complement activation, erythrocyte CR1 also functions as a receptor for circulating immune complexes to promote their clearance from plasma (Cornacoff, J. et al., 1983, J. Clin. Invest. 71:236).
  • U.S. Patent No. 5,851,528 discloses chimeric proteins in which a first polypeptide which inhibits complement activation is linked to a second polypeptide which inhibits complement activation, and the polynucleotides encoding such chimeric proteins.
  • the invention also features a method of reducing inflammation characterized by excessive complement activation by administering the chimeric protein of the invention to a patient afflicted with such a condition.
  • a worker skilled in the art would be able to incorporate the chimeric proteins of U.S. Patent No. 5,851,528 in the compositions of the present invention in order to selectively inhibit one or more of the complement components.
  • the composition may optionally include other chemicals or drugs such as growth factors and neurotrophins. It is known that the beneficial effects of blocking CNS myelin-associated inhibitors on axonal regeneration can be augmented by the concomitant application of neurotrophins, such as NT-3 (Bregman et al, (1995) Nature 378:498-501; Schnell et al, (1994) Nature 367: 170-173). FGF-1 can also be used (Chang et al., 1996, supra).
  • PCT Publication Number W ⁇ 93/06116 suggests the use of GDNF for preventing and treating nerve damage and nerve related diseases, such as Parkinson's disease, by implanting, into the brains of patients, cells that secrete GDNF.
  • PCT Publication Number WO 93/088208 teaches the intravenous administration of certain nerve growth factors in the treatment of neuronal damage associated with ischemia, hypoxia or neurodegeneration.
  • PCT Publication Number WO 90/07341 states that nerve growth factor (NGF) has been demonstrated to be a neurotropic factor for the forebrain cholinergic nerve cells that die during Alzheimer's disease. NGF increases growth of said nerve cells in culture.
  • NGF nerve growth factor
  • BDNF Brain Derived Neurotrophic Factor
  • compositions of the present invention are designed to reduce the inhibitory components of the CNS to, thereby, facilitate axonal growth. Therefore, the addition of trophic factors, as described above, in combination with the compositions of the present invention is an even more effective tool to treat neurodegenerative disorders due to disease or traumatic damage of nervous tissues.
  • another embodiment is the preparation of compositions of the present invention in combination with neurotrophic factors in any amount, concentration or level deemed to be effective in the treatment of neurological diseases in a manner familiar to one skilled in the art.
  • peripheral nerve Schwann cells Kerman et al, (1999) Exp. Neurol. 159:225-236
  • This technique enhances regeneration of injured spinal cord axons by providing scaffolding for growth and support and cell filled guidance channels which induce ingrowth and elongation of regenerating CNS axons (Smith and Stevenson (1988), Exp. Brain Res.; Xu et al., (1995) Exp.
  • Schwann cells express a variety of cell adhesion molecules including N-CAM, LI, and N-cadherin, synthesize extracellular matrix molecules and release trophic factors including the neurotrophins NGF, BDNF, and NT3 (Venstrom and Reichardt (1993)
  • compositions of the present invention may additionally comprise neurotrophic factors, and may optionally be administered prior to, simultaneously with, or subsequent to Schwann cell transplantation.
  • the demyelination renders the environment more permissive to cellular growth following Schwann cell transplantation.
  • the composition shall be prepared and delivered by a method previously known to someone skilled in the art, for example by injection, transplantation, or perfusion.
  • compositions of the present invention can be prepared comprising Tumour Necrosis Factor (TNF), which is used in a quantity and purity sufficient to facilitate regeneration of CNS axons in mammals, particularly humans.
  • TNF Tumour Necrosis Factor
  • compositions of the present invention can be prepared comprising mononuclear phagocytes.
  • U.S. Patent No. 5, 800,812 demonstrates that allogenic mononuclear phagocytes can be effective in the promotion of axonal regeneration following injury or disease of the CNS.
  • composition of this invention can be used in combination with cell grafting techniques such as those taught in U.S. Patent No. 5,750,103, which provides a method for grafting a cell in the brain of a mammalian subject comprising allowing the cell to attach to the surface of a support matrix in vitro, preferably by culturing the cell with the matrix, such that the cell is not encapsulated by the matrix, and implanting the support matrix with the attached cell into the brain.
  • cell grafting techniques such as those taught in U.S. Patent No. 5,750,103, which provides a method for grafting a cell in the brain of a mammalian subject comprising allowing the cell to attach to the surface of a support matrix in vitro, preferably by culturing the cell with the matrix, such that the cell is not encapsulated by the matrix, and implanting the support matrix with the attached cell into the brain.
  • the techniques include support matrices made of glass or other silicon oxides, polystyrene, polypropylene, polyethylene, polyacrylamide, polycarbonate, polypentene, acrylonitrile polymer, nylon, amylases, gelatin, collagen, natural or modified polysaccharides, including dextrans and celluloses (e.g. nitrocellulose), hyaluronic acid, extracellular matrix, agar, or magnetite.
  • Preferred support matrices are beads, porous or nonporous, in particular microbeads having a diameter from about 90 to about 150 ⁇ m.
  • composition of the present invention may be used with methods that employ cells of many different types, preferably either cells of neural or paraneural origin, such as adrenal chromaffin cells. Also useful are cell lines grown in vitro. Cells not of neural or paraneural origin, such as fibroblasts, may also be used following transfection with DNA encoding a neuropeptide or an enzyme or set of enzymes which results in production of neurotransmitter, or a neuronal growth factor.
  • a cell will be selected based on its ability to provide a missing substance to the recipient brain. Missing substances can be neurotransmitters or other neurally-active molecules, the absence of which results in neurological disease or dysfunction. It is important that the transplanted cell not grow as a tumor once it is inserted into the recipient.
  • Neural lines may express a tremendous amount of genetic information that corresponds to the genetic expression seen in CNS neurons.
  • Such cells are described in the following references: Kimhi, Y. et al., Proc. Natl. Acad. Sci. USA 73: 462-466 (1976); In: Excitable Cells in Tissue Culture, Nelson, P. G et al., eds., Plenum Press, New York, 1977, pp. 173-245); Prasad, K. M. et al., In: Control of Proliferation of Animal Cells, Clarkson, B. et al., eds., Cold Spring Harbor Laboratory
  • composition of the present invention may be used with another important source of potential graft material which are cells engineered by somatic cell hybridization, a process which can immortalize single neurons. Fusing cells which differ in the expression of specific genes allows for the exploration of the mechanisms controlling gene expression while chromosome alterations occur at rates to generate genetically different cell lines.
  • Hybrid cells can be formed which retain the properties of differentiated cells. Hybrids derived from fusion of sympathetic ganglia and neuroblastoma cells can synthesize dopamine (Greene, L. A. et al., Proc. Natl. Acad. Sci. USA 82: 4923-4927 (1975) while brain cell hybrids express choline acetyltransferase (Minna, J. D.
  • embryonic precursors to dopaminergic neurons from the CNS can be fused with mitotic cells to incorporate both genomes into a single one that loses extra chromosomes over time and results in a new hybrid line. It is within the skill of the art to produce such hybrid neural or paraneural cells without undue experimentation, screen them for the desired traits, such as dopamine secretion, and select those having the best potential for transplantation.
  • the composition of the present invention may be used with cells for transplantation derived from another source of cells which is the adrenal medulla.
  • This neural crest- derived tissue has been involved in clinical trials to treat Parkinson's disease.
  • Adult monkey adrenal medulla can be cultured in vitro for at least about three weeks as single cells (Notter, M. F. et al., Cell Tiss. Res. 244: 69-76 (1986)).
  • retinal pigment epithelial cells secrete dopamine and other factors and may be used for brain implants according to the present invention (Li, L. et al., Exp. Eye Res. 47: 771-785 (1988); Lui, G. M. et al., Proc. Int'l. Soc. Eye Res.
  • an additional embodiment is directed to transplantation of cells attached to a support matrix combined with the treatment, either in vitro prior to transplant, in vivo after transplant, or both, with the appropriate growth differentiation factor and the composition of the present invention.
  • Grafted glial cells may play an important role in functional recovery of neurons and may be an important source of trophic factors (Doering, L. C. et al., J. Neurolog. Sci. 63: 183-196 (1984); Gumple, J. et al., Neurosci. Lett. 37: 307-311 (1984)). Therefore, another embodiment involves co-culture of neural or paraneural cells with glial cells, their co-incubation with a support matrix, followed by implantation of the support matrix carrying both cell types into the CNS following or concurrent with use of the composition of the present invention.
  • olfactory ensheathing glia (Ram ⁇ n-Cueto , et al (1998) J Neurosci., 18:3803-15), which are helper cells found only in nerves that carry odor sensations to the brain.
  • Olfactory ensheathing glia share some characteristics with Schwann cells, including some of their growth-promoting properties, but they also express traits that resemble astrocytes, a helper cell in the CNS that can inhibit axon growth. Unlike either cell type, EG also migrate extensively within the CNS.
  • the present invention involves use of the composition with cells which are not of neural or paraneural origin, but which have been altered to produce a substance of neurological interest.
  • a preferred cell type is a human foreskin fibroblast which is easily obtained and cultured, and survives upon transplantation into the rat brain using the methods of the present invention.
  • the cells are genetically altered, using methods known in the art, to express neuronal growth factors, neurotransmitters, neuropeptides, or enzymes involved in brain metabolism. (See, for example, Gage, F. H. et al., Neuroscience 23 : 795-807 (1987); Rosenberg, M. B.
  • the transplanted cells may be attached to, or mixed with, a support matrix, which are frozen and stored in a frozen state using methods well known in the art. Following thawing, the matrix-bound cells are implanted into a recipient brain.
  • Materials of which the support matrix can be comprised include those materials to which cells adhere following in vitro incubation, and on which cells can grow, and which can be implanted into a mammalian brain without producing a toxic reaction, or an inflammatory or gliosis reaction which would destroy the implanted cells or otherwise interfere with their biological or therapeutic activity.
  • Such materials may be synthetic or natural chemical substances or substances having a biological origin.
  • the matrix-materials include, but are not limited to, glass and other silicon oxides, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride, polyurethane, polyalginate, polysulphone, polyvinyl alcohol, acrylonitrile polymers, polyacrylamide, polycarbonate, polypentene, nylon, amylases, gelatin, collagen, natural and modified polysaccharides, including dextrans and celluloses (e.g. nitrocellulose), agar, and magnetite. Either resorbable or non-resorbable materials may be used. Also intended are extracellular matrix materials, which are well-known in the art (see below). Extracellular matrix materials may be obtained commercially or prepared by growing cells which secrete such a matrix, removing the secreting cells, and allowing the cells which are to be transplanted to interact with and adhere to the matrix.
  • the solid matrix may optionally be coated on its external surface with factors known in the art to promote cell adhesion, growth or survival.
  • factors include cell adhesion molecules, extracellular matrix, such as, for example, fibronectin, laminin, collagen, elastin, glycosaminoglycans, or proteoglycans (see: Albers, B. supra, pp. 802-834) or growth factors, such as, for example, NGF.
  • the growth- or survival-promoting factor or factors may be incorporated into the matrix material, from which they would be slowly released after implantation in vivo.
  • cells growing on, or mixed with, resorbable matrices such as, for example, collagen
  • resorbable matrices such as, for example, collagen
  • cells attached to the matrix of the invention may be implanted into the spinal cord, or placed in, or adjacent to, the optic nerve.
  • the matrix material on which the cells to be implanted grow, or with which the cells are mixed, may be an endogenous product of the implanted cells themselves.
  • the matrix material may be extracellular matrix or basement membrane material which is produced and secreted by the very cells to be implanted.
  • the composition of the present invention may be used with a number of methods for treating various human neurological diseases, wherein it would be useful to transplant cells into the CNS.
  • the composition can be used either prior to, or concurrent with cellular transplantation.
  • Parkinson's Disease can be treated according to the present invention by implanting dopamine-producing cells in the recipient's striatum, either concurrent or following use of the composition.
  • Alzheimer's disease involves a deficit in cholinergic cells in the nucleus basalis.
  • a subject having Alzheimer's disease or at risk therefor may be implanted with cells producing acetylcholine.
  • Huntington's disease involves a gross wasting of the head of the caudate nucleus and putamen, usually accompanied by moderate disease of the gyrus.
  • a subject suffering from Huntington's disease can be treated by implanting cells producing the neurotransmitters gamma amino butyric acid (GABA), acetylcholine, or a mixture thereof.
  • GABA neurotransmitters gamma amino butyric acid
  • acetylcholine acetylcholine
  • the support matrix material to which such cells are attached is preferably implanted into the caudate and ptamen.
  • Epilepsy is not truly a single disease but rather is a symptom produced by an underlying abnormality.
  • each epileptic subject will have damage or epileptic foci which are unique for the individual. Such foci can be localized using a combination of diagnostic methods well-known in the art, including electroencephalography, computerized axial tomography and magnetic resonance imaging.
  • a patient suffering from epilepsy can be treated according to the present invention by implanting the support matrix material to which GABA-producing cells are attached into the affected site. Since blockers of glutamate receptors and NMD A receptors in the brain have been used to control experimental epilepsy, cells producing molecules which block excitatory amino acid pathways may be used according to the invention. Thus implantation of cells which have been modified as described herein to produce polyamines, such as spermidine, in larger than normal quantities may be useful for treating epilepsy.
  • composition of the present invention is intended for use with any mammal which may experience the beneficial effects of the methods of the invention. Foremost among such mammals are humans, although the invention is not intended to be so limited, and is also applicable to veterinary uses.
  • the composition can also be used with certain agents identified herein as "CNS neural growth modulators" (CNGMs), and particularly to a class of neural cell adhesion molecules as defined herein, to promote neurite outgrowth in the central nervous system (CNS).
  • CNS neural growth modulators CNGMs
  • CNGMs central nervous system
  • neurons in the adult central nervous system have been considered incapable of regrowth, due to inhibitory molecular cues present on glial cells.
  • the agents and methods of the present invention can be used to overcome this inhibition and promote CNS neurite outgrowth.
  • Such agents may be selected from any cell adhesion molecule which is capable of modulating or promoting CNS neurite outgrowth, and particularly to molecules belonging to the immunoglobulin superfamily. More particularly, the molecules are selected from the members of the immunoglobulin superfamily which mediate Ca 2+ - independent neuronal cell adhesion, including LI, N-CAM and myelin-associated glycoprotein.
  • the invention also contemplates fragments of these molecules, and analogs, cognates, congeners and mimics of these molecules which have neurite- promoting activity.
  • Particularly preferable structural motifs for these fragments and analogs include domains similar to the fibronectin type III homologous repeats particularly repeats 1-2) and immunoglobulin-like domains (particularly domains I-II,
  • composition of the present invention may be used with ectopic expression of CNS neural growth modulators (CNGMs) or neural cell adhesion molecules on differentiated astrocytes in vivo.
  • CNGMs CNS neural growth modulators
  • the inhibitory action of astroglial and oligodendroglial cells may be overcome, at least in part, by the neurite outgrowth promoting properties of the agents defined herein, and as particularly illustrated by the activity of ectopically expressed
  • the present invention extends to the promotion of neural growth in the CNS, including such growth as is desired to regenerate structures lost due to injury or illness, as well as those structures and tissues exhibiting incomplete or immature formation.
  • the composition of the present invention may be used in combination with CNGM- secreting cells for the modulation of neural outgrowth, regeneration, and neural survival in the CNS.
  • certain soluble CNGMs and fragments thereof, and cognate molecules thereof are also within the invention.
  • composition of the present invention can be prepared in combination with inhibitors of myelination such as metalloproteases (U.S. Patent No. 6,025,333), inhibitors of apoptosis and/or necrosis (U.S. Patent Nos. 6,004,579, 6,015,665, and 6,046,007), inhibitors of proinflammatory cytokines, activators antiinflammatory cytokines, (U.S. Patent No. 5,650,396), antiinflammatory cytokines, activators and generators of antioxidants (U.S. Patent Nos. 5,747,532, 5,747,459, and 5,667,776), or any combination thereof.
  • the compositions of the present invention may also be administered in conjunction with methods that increase the production of phagocytosis (U.S. Patent No. 5,800,812).
  • the composition is comprised of a GalC-specific monoclonal antibody and human serum complement.
  • the composition is comprised of a MOG-specific monoclonal antibody and human serum complement.
  • compositions of the present invention can be used to promote regrowth, repair, and/or regeneration of neurons in the CNS of a subject by stimulating transient immunological disruption of myelin or transient demyelination of axons.
  • the transient demyelination process of the present invention occurs in the CNS, most preferably in the spinal cord.
  • the subject may be any mammal. In a preferred embodiment, the subject is human.
  • compositions of the present invention can be used to promote regrowth, repair, and/or regeneration of dysfunctional neurons in the CNS that have been damaged as a result of injury, such as a spinal cord injury.
  • the method can be used following immediate or chronic injury.
  • compositions of the present invention can also be used to promote regrowth, repair, and/or regeneration of dysfunctional neurons in the CNS that have been damaged as a result of disease, such as degenerative diseases including Alzheimer's and Parkinson's disease.
  • compositions of the present invention can also be used to generate an environment within the mammalian CNS that is relatively permissive to growth of transplanted cells.
  • compositions of the present invention can be used to disrupt the myelin in the CNS to allow the axons to extend into this area.
  • compositions of the present invention comprise administering a therapeutically effective amount of such a composition to the subject.
  • therapeutically effective amount refers to an amount of composition sufficient to effectively and transiently disrupt and/or demyelinate the CNS so that repair and regeneration of neurological tissue and neuronal connections is enhanced.
  • the therapeutic composition is administered at a range from about 0.03 mg antibody to about 0.6 mg antibody in a 20% to 30% complement solution per kg body weight.
  • the range is from 0.05 mg antibody to 0.4 mg antibody in a 20% to 30% complement solution per kg body weight.
  • the range is from 0.1 mg antibody to 0.3 mg antibody in a 20% to 30% complement solution per kg body weight.
  • the exact ratio of antibody to complement will vary depending on the circumstances; however, since the amount of complement activated is directly proportional to the number of bound antibody molecules, it is possible to administer relatively high concentrations of complement in excess of the relative concentration of antibody.
  • the particular concentration of antibody administered will vary with the particular dysfunction and its severity, as well as with such factors as the age, sex, and medical history of the patient. Those of skill in the clinical arts will know of such factors and how to compensate the dosage ranges of the composition accordingly.
  • the majority of spinal cord injuries result from damage to the vertebral column surrounding the spinal cord. This damage includes fractures, dislocations, or both. Much of the damage to the spinal cord is due to secondary phenomena that occur within hours following the injury. At this point, the resultant damage may be reversible; consequently, a critical factor for recoverable CNS function is the amount of time that evolves between injury and the institution of therapy. Most preferably, when the nervous system dysfunction is a result of injury, administration of the composition to the subject will be as close in time to the time of the injury as possible.
  • a composition according to the method of the invention can be administered to a subject parenterally by injection or by gradual infusion over time.
  • the composition can be administered intrathecally or injected directly into the spinal cord.
  • compositions of the present invention can be administered in any manner which is calculated to bring the therapeutically effective components to the vicinity of the injured axons to be regenerated.
  • the composition is injected in a pharmaceutically acceptable liquid carrier directly to the site.
  • an implant bearing components of the composition may be surgically inserted.
  • Such an implant may consist of any material, such as nitrocellulose, which can absorb these components and slowly release it at site of implantation.
  • U.S. Patent No. 5,360,610 which makes use of polymeric microspheres as injectable, drug- delivery systems for use to deliver bioactive agents to sites within the CNS.
  • bioactive agents are contained within a compatible biodegradable polymer which is then administered to a patient in need of therapy.
  • the compositions of the present invention can be prepared in a series of microspheres which can be implanted at the site of injury or disorder, thereby allowing a sustained release of the therapeutically active components.
  • U.S. Patent Nos. 4,883,666 and 5,601,835 also disclose polymeric drug delivery systems for controlled release of any substance to the CNS. A worker skilled in the art can easily adapt such polymeric drug delivery systems to facilitate administration of the compositions of the present invention to a patient in need of such therapy.
  • Preparations for parenteral administration are contained in a pharmaceutically acceptable carrier that is compatible with both the components of the composition and the patient.
  • a pharmaceutically acceptable carrier that is compatible with both the components of the composition and the patient.
  • Such carriers include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents include propylene glycol, polyethylene glycol, metabolizable oils such as olive oil or squalane, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/acqueous solutions, and emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and
  • kits may comprise a carrier means compartmentalized to receive in close confinement one or more container means, such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • container means such as vials, tubes, and the like
  • each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a GalC-specific antibody.
  • the antibody and complement may be present in the same container.
  • the constituents may be present in liquid or lyophilized form, as desired.
  • Needles and/or other equipment that facilitates delivery of the complement and antibody to the site of damage may include: a) silastic, Polyethylene, Tygon (Norton Performance Plastics) tubing; b) subcutaneous pumps, (such as the Medtronic pump system known for the administration of baclofen intrathecally); c) spinal needle for direct intraspinal administration, or for short-term intrathecal administration.
  • a 14-gauge Tuohy needle is inserted into the lumbar subarachnoid space.
  • a 5-F catheter is coaxially placed with the tip at L10 and tunneled to the flank (or appropriate location). This type of instruction would be understood by one familiar with the technique.
  • Tubing is placed intrathecally, and connected to the pump.
  • the pump, containing a finite folume of the reagents is placed under the skin this can be refilled in the Doctor's office, via a needle inserted into a septa in the pump. Or the Infusaid pump may be used in the alternative.
  • compositions and their uses of the present invention have a number of advantages over methods currently available for the regeneration of neuronal growth in the CNS.
  • Interventional therapies including opiate antagonists, thyrotropin-releasing hormone, local cord cooling, dextran infusion, adrenergic blockade, corticosteroids, and hyperbaric oxygen, are targeted at reducing secondary inflammatory damage after a traumatic injury to the spinal cord in order to prevent the spread of damage to uninjured neurons. Unlike the present invention, however, they do not promote regeneration of the damaged neurons.
  • Peripheral nerve transplants and the grafting of donor cells into the CNS are useful in that axons can grow into them; however, the axons cannot grow out of them into the surrounding CNS due to the inhibitory myelin present.
  • the present invention disrupts the inhibitory myelin to allow regrowth of neurons in the CNS.
  • EXAMPLE I REGENERATION OF BRAINSTEM-SPINAL AXONS
  • the following example illustrates that the transient developmental suppression of myelination or the disruption of mature myelin by local intraspinal infusion of serum complement proteins along with a complement-fixing, myelin-specific antibody facilitates brainstem-spinal axonal regeneration after spinal transection in a mammalian subject.
  • Surgical Spinal Transection and Transient Immunological Myelin Disruption Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in weight, were anaesthetized with Ketamine/Xylazine (60mg/kg and 7.5mg/kg, respectively). After a limited dorsolateral laminectomy at T 10, a left-side spinal cord hemisection lesion was made with micro-scissors. The extent of the lesion was then confirmed by passing a sharp scalpel through the lesion site three times (Fig. 1 A).
  • Cannulae were held in place by means of dental acrylic applied to the vertebral bone. Muscle layers were then sutured over the dental acrylic, and the superficial tissue and skin were closed. The extent of the hemisection lesion was always confirmed histologically at the end of the 5-week treatment and recovery period.
  • Tissue for ultrastructural analysis was obtained from 10-12 week old adult female Sprague-Dawley rats sacrificed 7 days after infusion of serum complement along with a complement-fixing IgG antibody to GalC (see above for details) via an osmotic pump. Animals were lethally anaesthetised with Ketamine/Xylazine (120mg/kg and 15mg/kg, respectively), then perfused intracardially with 200 ml of 0. IM PBS (pH 7.4) followed by 100 ml of 4% glutaraldyhyde in 0.1M PB, (pH 7.3) and subsequently postfixed overnight in the same fixative.
  • the infusion site and surrounding cord was cut into lmm transverse blocks and processed to preserve rostral-caudal sequence.
  • Blocks were washed in 0.1M sodium cacodylate buffer (24 hours), postfixed in 2% OsO4, dehydrated through ascending alcohols, and embedded in Spurrs' resin according to standard protocols.
  • Tissue blocks from experimental and untreated-control animals were processed in parallel. Thin sections (l ⁇ m) were cut from each block, stained with alkaline Toluidine Blue, and examined under a light microscope. For electron microscopic examination, blocks were trimmed then sections were cut at 80-100nm, mounted on copper grids, stained with uranyl acetate and lead citrate, and viewed under a Ziess EM 10C electron microscope (at 80k V).
  • Retrograde Neuronal Labeling If a retrograde tracer (single label) is injected into the rostral lumbar cord (1 cm caudal to the injury site), it should be incorporated and transported back to the cell bodies of origin by both intact axons, as well as regenerated projections. Consequently, it is essential that the retrograde tracer reliably and extensively label most, if not all, descending spinal projection neurons.
  • An equally important parameter is that the tracer must be injected in a controlled and reproducible manner at a distance sufficiently caudal to the spinal injury to prevent any direct diffusion of the tracer to the level of the hemisection injury.
  • Fluorogold Surfactant dextran amines, such as RDA, require a recent axonal injury to facilitate axonal uptake (Heimer and Zaborszky Neuroanatomical Tract-tracing Methods 2: Recent Progress (New
  • each adult rat was anaesthetized with Ketamine/Xylazine (60mg/kg and 7.5mg/kg, respectively).
  • Fluorogold (FG, 100-150nl total volume, 5% w/v in sterile dH 2 O; Fluorochrome Inc.
  • Englewood, CO, USA was injected (50-75nl) bilaterally into the middle of the spinal tissue at the LI level, approximately 1cm caudal to the lesion site.
  • Rats (n 8) were experimentally treated as described above; however, animals were killed at 12, 24, 72, and 120 hours after injection of FG into the LI cord.
  • the hemisection site was packed with gel-foam soaked with 12% (w/v in sterile dH 2 O) rhodamine-conjugated dextran amine (RDA, 10,000MW FluoroRuby, Molecular Probes) for 30 minutes. The gel-foam was then removed, and the remaining surgical procedures were completed (as outlined above). After 28 days survival, all animals were anaesthetized with Ketamine/Xylazine (60mg/kg and
  • Coronal or parasagital sections were cut at 25 ⁇ m thickness on a cryostat.
  • the brainstem and spinal cord tissue sections were examined under a Zeiss Axioskop with a 100W mercury bulb (excitation/emission wavelengths: FG, 365/420nm; RDA, 546/590nm).
  • the brainstem-spinal nucleus used to assess the axonal regenerative abilities of experimentally treated animals was the Red Nucleus (RN, origin is contralateral to the hemisection).
  • RN Red Nucleus
  • Spinal-projecting axons from each RN cross to the opposite side of the midbrain and descend throughout the spinal cord within the contralateral dorsolateral funiculus.
  • This contralateral spinal projection pathway is known to be a completely lateralized trart with the possible exception of 2-5 % of the axons, which may project to the cord via an ipsilateral route (Brown (1974) J. Comp. Neurol. 154: 169-188; Huisman etal, (1981) Brain Res. 209:217-286; Shieh et /., (1983) J Comp. Neurol. 214:79-86; Waldron and Gwyn (1969) J. Comp. Neurol. 137:143-154).
  • Nucleus were counted in every other tissue section throughout the nucleus to avoid counting the same neuron twice. Only those cells exhibiting a nucleus and a neuronal morphology (i.e. multi-polar in appearance), and that were specifically labeled with FG (i.e. not visible using other fluorescent filters; see above) extending into the proximal processes, were deemed to be positively labeled spinal-projecting neurons.
  • the percentage of regenerating neurons was then determined in comparison to the number of labeled neurons within the contralateral (uninjured) control nucleus within the same animal.
  • the immunological demyelination and disruption of myelin within the experimentally- treated adult rat spinal cord is an active process extending throughout the entire cross- sectional profile of the cord.
  • Immunological myelin disruption commences within 1 day and is associated with an invasion of macrophages or resident microglia and polymorphonuclear cells (e.g. leukocytes such neutrophils, eosinophils and basophils). Many macrophages/microglia contain myelin fragments and complete their phagocytic activity within 7 days (Fig. 2B). This pattern of demyelination and myelin disruption can be maintained for as long as the serum complement and myelin-specific antibody are infused.
  • macrophages or resident microglia and polymorphonuclear cells e.g. leukocytes such neutrophils, eosinophils and basophils.
  • Many macrophages/microglia contain myelin fragments and complete their phagocytic activity
  • IA severed the contralaterally-projecting magnocellular neurons of the right red nucleus (RN), but left the projections from the left RN undamaged (as they project through the intact right dorsolateral funiculus of the thoracic cord).
  • the Fluorogold label (100-150nl) was injected bilaterally within the rostral lumbar cord (1cm or 3 spinal segments caudal to the hemisection injury site).
  • Random 25 ⁇ m sections of experimental and control-treated spinal cords (extending from L2 to T8) were examined under a fluorescent microscope using the highest intensity setting of the 100W mercury lamp.
  • Fig. 1 A The extent of the hemisection lesion was assessed in every animal. In all but one experimental and one control-treated animal, the left thoracic spinal cord was hemisected (Fig. 1 A). Most importantly, the region of the rubrospinal trart (dorsolateral funiculus) was severed. The right side white matter tracts were always remained intact and undamaged; usually the gray matter of the contralateral side was also undamaged.
  • the labeling of some neurons within the injured right RN nucleus may represent the small number of RN that do not project to the opposite side of the midbrain and descend within the ipsilateral (uninjured) cord (Shieh et al, (1983) J Comp. Neurol. 214:79-86). No retrograde- labeling of cells was observed within the parvocellular region of the RN. This was expected since this RN region predominantly projects only as far as the cervical region of the cord.
  • Double retrograde labeling of the injured and myelin-suppressed rubrospinal tract was also qualitatively assessed (Fig. 3E and F).
  • Large numbers of RDA-positive (first label) magnocellular RN neurons were observed after direct labeling of the lesion site at the time of hemisection injury to the thoracic spinal cord. After intraspinal myelin- suppression and subsequent injection of Fluorogold caudal to the lesion site, a small overlapping population of FG-positive neurons was observed (i.e. some neurons were labeled with both RDA and FG). Cells labeled exclusively by the first or the second tracer were also present in every brainstem analysed. The low number of double labeled brainstem-spinal neurons may in part be due to the failure of a severed axon to take up
  • the present invention is demonstrated using a hemisection model for this study so that each animal could serve as its own internal control (i.e. axonal regeneration from injured brainstem-spinal projections could be readily compared to the uninjured contralateral homologue).
  • the present invention strove to minimize the degree of cyst cavity formation that often occurs with larger spinal lesions, as well as the amount of animal discomfort over the relatively long recovery periods required for this study.
  • a limited dorsolateral laminectomy was performed at T10, and connected to an Alzet osmotic pump (14 day) to subsequently deliver a continuous intraspinal infusion (@ 0.5 ⁇ l/hr) of C3-depleted serum complement (Sigma S8788, 33% v/v) along with a complement-fixing IgG antibody to galactocerebroside (either our own polyclonal antibody or Chemicon Intl. Ltd., #AB142, 25% v/v).
  • Cannulae were held in place by means of dental acrylic applied to the vertebral bone. Muscle layers were then sutured over the dental acrylic, and the superficial tissue and skin were closed.
  • All control animals were intraspinally infused via an osmotic pump, for the same time period, with whole human serum complement (Sigma SI 764, 33% v/v) along with a complement-fixing IgG antibody to galactocerebroside (either our own polyclonal antibody or Chemicon Intl. Ltd., #AB142, 25% v/v). All surgical procedures and subsequent animal care protocols were in accordance with Canadian and University of British Columbia Animal Care Committee guidelines.
  • Electron microscopy was performed as described in Example I.
  • mice 6 experimental and 5 control ) were subjected to a left-side lateral hemisection of the T10 spinal cord as follows: 10 to 12 week old adult female rats
  • Cannulae were held in place by means of dental acrylic applied to the vertebral bone. Muscle layers were then sutured over the dental acrylic, and the superficial tissue and skin were closed.
  • the osmotic pump delivered a continuous intraspinal infusion (0.5 ⁇ l/hr) of guinea-pig serum complement (33% v/v) along with a complement-fixing IgG antibody to galactocerebroside (either our own polyclonal antibody or Chemicon Intl. Ltd., #AB142, 0.25 mg/mL).
  • All control animals received an identical hemisection lesion and were then intraspinally infused via an osmotic pump for the same time period with whole guinea-pig serum complement (33% v/v) alone.
  • Results The extent of the hemisection lesion was assessed in every animal. In all animals the region of the vestibulospinal tract was severed. The right side white matter tracts always remained intact and undamaged while the gray matter of the contralateral side usually remained undamaged.
  • compositions of the present invention are useful for promoting regrowth, repair, and regeneration of chronically injured neurons in the CNS of a mammalian subject.
  • Cannulae were held in place by means of dental acrylic applied to the vertebral bone. Muscle layers were then sutured over the dental acrylic, and the superficial tissue and skin closed. The extent of the hemisection lesion was always confirmed histologically at the end of the 5-week treatment and recovery period.
  • Tissue for ultrastructural analysis was obtained from 10-12 week old adult female Sprague-Dawley rats sacrificed 7 days after infusion of serum complement along with a complement-fixing IgG antibody to GalC (see above for details) via an osmotic pump. Animals were lethally anaesthetised with Ketamine/Xylazine (120mg/kg, 15mg/kg respectively), then perfused intracardially with 200 ml of 0. IM PBS (pH 7.4) followed by 100 ml of 4% glutaraldyhyde in 0.1M PB, (pH 7.3) and subsequently postfixed overnight in the same fixative.
  • the infusion site and surrounding cord was cut into lmm transverse blocks and processed to preserve rostral-caudal sequence. Blocks were washed in 0.1M sodium cacodylate buffer (24 hours), post fixed in 2% OsO4, dehydrated through ascending alcohols and embedded in Spurrs' resin according to standard protocols. Tissue blocks from experimental and untreated-control animals were processed in parallel. Thin sections (l ⁇ m) were cut from each block, stained with alkaline Toluidine Blue and examined under a light microscope. For electron microscopic examination blocks were trimmed and sections cut at 80-100nm, mounted on copper grids, stained with uranyl acetate and lead citrate and viewed under a Ziess EM 10C electron microscope (at 80kV).
  • each adult rat was anaesthetized with Ketamine/Xylazine (60mg/kg, 7.5mg/kg respectively).
  • Fluorogold (FG, 100-150nl total volume, 5% w/v in sterile dH 2 O; Fluorochrome Inc. Englewood,
  • Double Label Studies At the time of lesion, the hemisection site was packed with gel-foam soaked with 12%
  • mice Seven days following the injection of the FG tracer into the lumbar cord, animals were lethally anaesthetised with Ketamine/Xylazine (120mg/kg, 15mg/kg respectively) and then perfused intracardially with 200 ml of 0. IM PBS (pH 7.4) followed by 100 ml of 4% paraformaldehyde in 0. IM PBS, (pH 7.3).
  • the brain and spinal cord were then removed and postfixed overnight in the same fixative. Subsequently, each brain and spinal cord was cleared of fixative and cryo-preserved by placing the tissue in a series of sucrose solutions (15% followed by 21%). Coronal or parasagital sections were cut at 25 ⁇ m thickness on a cryostat.
  • the brainstem and spinal cord tissue sections were examined under a Zeiss Axioskop with a 100W mercury bulb (excitation/emission wavelength at: FG, 365/420nm; RDA, 546/590nm; fluorescein, 490/515nm)
  • the two brainstem-spinal nuclei used to assess the axonal regenerative abilities of experimentally treated animals were the Red Nucleus (RN) (origin is contralateral to the hemisection) and the Lateral Vestibular (LVe) Nucleus (origin is ipsilateral to the hemisection).
  • RN Red Nucleus
  • LVe Lateral Vestibular Nucleus
  • Spinal-projecting axons from each RN cross to the opposite side of the midbrain and descend throughout the spinal cord within the contralateral dorsolateral funiculus.
  • This contralateral spinal projection pathway is known to be a completely lateralized trart with the possible exception of 2-5 % of the axons which may project to the cord via an ipsilateral route (Waldron and Gwyn 1969; Brown, 1974; Huisman et al., 1981; Shieh et al., 1983).
  • the LVe trart projects from the dorsolateral pontine hindbrain, maintaining an exclusive ipsilateral course throughout the brainstem and the ventrolateral white matter of the spinal cord (Zemlan et al., 1979; Shamboul, 1980).
  • Nucleus (RN) (contralateral to the hemisection) and the Lateral Vestibular (LVe) Nucleus (ipsilateral to the hemisection) were counted in every other tissue section (throughout these brainstem nuclei) to avoid counting the same neuron twice. Only those cells exhibiting a nucleus, a neuronal morphology (i.e. multi-polar in appearance) and specifically labeled with FG (i.e. not visible using other fluorescent filters; see above) extending into the proximal processes, were deemed to be positively labeled spinal-projecting neurons. The percentage of regenerating neurons for each brainstem- spinal projection was then determined in comparison to the number of labeled neurons within the contralateral (uninjured) control nucleus within the same animal.
  • the immunological demyelination and disruption of myelin within the experimentally- treated adult rat spinal cord was an active process extending throughout the entire cross- sectional profile of the cord.
  • Immunological myelin disruption commenced within 1 day and was associated with an invasion of macrophages or resident microglia and polymorphonuclear cells (e.g. leukocytes such neutrophils, eosinophils and basophils).
  • macrophages/microglia contained myelin fragments and completed their phagocytic activity within 7 days (Fig. 2D). This pattern of demyelination and myelin disruption could be maintained for as long as the serum complement and myelin- specific antibody were infused.
  • a left thoracic hemisection severed the contralaterally-projecting magnocellular neurons of the right red nucleus (RN), but left the projections from the left RN undamaged (as they project through the intact right dorsolateral funiculus of the thoracic cord).
  • a left thoracic hemisection severed the ipsilateral projecting neurons of the left lateral vestibulospinal nucleus (LVe), but left the axons from the right LVe nucleus undamaged (as they also project through the intact right side of the thoracic cord via the ventrolateral white matter).
  • a retrograde tracer (single label) is injected into the rostral lumbar cord (1 cm caudal to the injury site), it should be incorporated and transported back to the cell bodies of origin by both intact axons, as well as regenerated projections. Consequently, it is essential that the retrograde tracer reliably and extensively label most, if not all, descending spinal projection neurons.
  • An equally important parameter is the tracer must be injected in a controlled and reproducible manner at a distance sufficiently caudal to the spinal injury to prevent any direct diffusion of the tracer to the level of the hemisection injury.
  • the retrograde label that best satisfied all these conditions was Fluorogold (Sahibzada, et al., 1987).
  • Fluorescent dextran amines such as RDA, require a recent axonal injury to facilitate axonal uptake (c.f. Heimer and Zaborszky, 1989), and were therefore better suited for use in the double label retrograde-tracing studies (see description below).
  • the Fluorogold label 100-150nl was injected bilaterally within the rostral lumbar cord (1 cm or 2-3 spinal segments caudal to the hemisection injury site, Fig. 7).
  • the extent of the hemisection lesion was assessed in every animal. In all but one experimental and one control-treated animal, the left thoracic spinal cord was hemisected (Fig. 7). Most importantly, the regions of the rubrospinal trart (dorsolateral funiculus) and the lateral vestibulospinal tract (ventrolateral funiculus) were severed. The right side white matter tracts were always remained intact and undamaged and usually the gray matter of the uninjured side was also undamaged.
  • the 2 pairs of brainstem-spinal nuclei examined for evidence of retrograde labeling were the RN and the LVe.
  • These brainstem-spinal nuclei were chosen for their unilateral projection patterns within the thoracic and lumbar cord, enabling comparisons to be made between the retrograde-labeling within an injured nucleus and the uninjured contralateral homologue. Comparing "blind" counts of the number of labeled neurons within each RN (Fig.
  • the labeling of some neurons within the injured right RN nucleus may represent the small number of RN that do not project to the opposite side of the midbrain and descend within the ipsilateral (uninjured) cord (Shieh et al., 1983). No retrograde-labeling of cells was observed within the parvocellular region of the RN.
  • Retrograde-labeling of regenerating LVe neurons was also observed, but only after experimental demyelination and disruption of spinal cord myelin (Fig. 8).
  • Double retrograde labeling of the injured and myelin-suppressed rubrospinal tract was also qualitatively assessed (Fig. 9E and F).
  • Large numbers of RDA-positive (first label) magnocellular RN neurons were observed after direct labeling of the lesion site at the time of hemisection injury to the thoracic spinal cord. After intraspinal myelin- suppression and subsequent injection of Fluorogold caudal to the lesion site (see above for details) a small overlapping population of FG-positive neurons was observed (i.e. some neurons were labeled with both RDA and FG). Cells labeled exclusively by the first or the second tracer were also present in every brainstem analysed.
  • the present invention is demonstrated using a hemisection model so that each animal could serve as its own internal control (i.e. axonal regeneration from injured brainstem-spinal projections could be readily compared to the uninjured contralateral homologue).
  • the present invention strove to minimize the degree of cyst cavity formation that often occurs with larger spinal lesions, as well as the amount of animal discomfort over the relatively long recovery periods required.
  • the present invention also illustrates that the demyelination produced by the intraspinal infusion of serum complement and a myelin-specific antibody (e.g. GalC) produced a rapid and active demyelination over 1-2 segments of the cord with myelin disruption extending a further 2 segments, either side of the infusion site. Resident microglia and/or invading macrophages were observed to contain myelin debris.
  • the immunological suppression of spinal cord myelin surrounding the thoracic hemisection facilitated significant axonal regeneration by 2 unilaterally projecting brainstem-spinal pathways, the rubrospinal and lateral vestibulospinal (RN and LVe, respectively) tracts. Control treated animals (hemisection injury plus local intraspinal infusion of PBS alone, GalC antibody alone, or serum complement alone) showed little or no retrograde labeling within the injured RN or Lve.
  • a myelin-specific antibody e.g. GalC
  • EXAMPLE V EFFECTS OF REMOVAL OF SINGLE COMPLEMENT PROTEINS ON IMMUNOLOGICAL DEMYELINATION
  • Surgical Spinal Transection and Transient Immunological Myelin Disruption Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in weight, were anaesthetized with Ketamine/Xylazine (60mg/kg and 7.5mg/kg, respectively). A limited dorsolateral laminectomy was performed at T10, and connected to an Alzet osmotic pump (14 day) to subsequently deliver a continuous intraspinal infusion (@ 0.5 ⁇ l/hr) of depleted serum complement (Sigma S8788, 33% v/v) along with a complement-fixing IgG antibody to galactocerebroside (either our own polyclonal antibody or Chemicon Intl. Ltd., #AB142, 25% v/v). Cannulae were held in place by means of dental acrylic applied to the vertebral bone. Muscle layers were then sutured over the dental acrylic, and the superficial tissue and skin were closed.

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Abstract

L'invention concerne des nouvelles compositions ainsi que l'administration combinée de compléments sériques et d'anticorps fixant lesdits compléments. Les anticorps se lient spécifiquement à un ou plusieurs épitopes de myéline et aux compléments. Ces compositions sont utiles pour favoriser la repousse, la réparation et la régénération des neurones dans le système nerveux central d'un sujet mammifère. Les compositions et la méthode de l'invention peuvent être utilisés immédiatement après une lésion chronique.
PCT/CA2000/000440 1999-04-28 2000-04-28 Composition pour la regeneration neuronale, comprenant des anticorps specifiques de la myeline et des complements WO2000064473A1 (fr)

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AU40959/00A AU4095900A (en) 1999-04-28 2000-04-28 Composition for neuronal regeneration comprising myelin-specific antibodies and complement proteins
JP2000613463A JP2002542299A (ja) 1999-04-28 2000-04-28 ミエリン特異性抗体および補体タンパク質を含むニューロン再生用組成物
EP00920306A EP1173200A1 (fr) 1999-04-28 2000-04-28 Composition pour la regeneration neuronale, comprenant des anticorps specifiques de la myeline et des complements
CA002371592A CA2371592A1 (fr) 1999-04-28 2000-04-28 Composition pour la regeneration neuronale, comprenant des anticorps specifiques de la myeline et des complements
IL14619600A IL146196A0 (en) 1999-04-28 2000-04-28 Composition of neuronal regeneration comprising myelin specific antibodies and complement proteins

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US8414888B2 (en) 2008-11-06 2013-04-09 University Health Network Therapeutic use of IgG as a neuroprotective agent
US8703136B2 (en) 2006-10-10 2014-04-22 Regenesance B.V. Complement inhibition for improved nerve regeneration

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US8414888B2 (en) 2008-11-06 2013-04-09 University Health Network Therapeutic use of IgG as a neuroprotective agent

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