WO2011153416A2 - Variants d'anticorps anti-s1p inédits de lt1009 - Google Patents

Variants d'anticorps anti-s1p inédits de lt1009 Download PDF

Info

Publication number
WO2011153416A2
WO2011153416A2 PCT/US2011/039039 US2011039039W WO2011153416A2 WO 2011153416 A2 WO2011153416 A2 WO 2011153416A2 US 2011039039 W US2011039039 W US 2011039039W WO 2011153416 A2 WO2011153416 A2 WO 2011153416A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
amino acid
antibodies
replacement
binding
Prior art date
Application number
PCT/US2011/039039
Other languages
English (en)
Other versions
WO2011153416A3 (fr
Inventor
Jonathan Michael Wojciak
Original Assignee
Lpath, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lpath, Inc. filed Critical Lpath, Inc.
Publication of WO2011153416A2 publication Critical patent/WO2011153416A2/fr
Publication of WO2011153416A3 publication Critical patent/WO2011153416A3/fr

Links

Classifications

    • 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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to crystalline forms of anti-lipid antibodies, methods of making them, and methods of using data derived therefrom in antibody design and optimization.
  • Methods for designing antibodies or antibody fragments are provided, wherein the antibody target is a lipid, such as a bioactive lipid.
  • Lipids and their derivatives are now recognized as important targets for medical research, not as just simple structural elements in cell membranes or as a source of energy for ⁇ -oxidation, glycolysis or other metabolic processes.
  • certain bioactive lipids function as signaling mediators important in animal and human disease.
  • Most of the lipids of the plasma membrane play an exclusively structural role, a small proportion of them are involved in relaying extracellular stimuli into cells.
  • “Lipid signaling” refers to any of a number of cellular signal transduction pathways that use cell membrane lipids as second messengers, as well as referring to direct interaction of a lipid signaling molecule with its own specific receptor.
  • Lipid signaling pathways are activated by a variety of extracellular stimuli, ranging from growth factors to inflammatory cytokines, and regulate cell fate decisions such as apoptosis, differentiation and proliferation.
  • Research into bioactive lipid signaling is an area of intense scientific investigation as more and more bioactive lipids are identified and their actions characterized.
  • bioactive lipids include the eicosanoids (including the cannabinoids, leukotrienes, prostaglandins, lipoxins, epoxyeicosatrienoic acids, and isoeicosanoids) such as the hydroxyeicosatetraenoic acids (HETEs, including 5-HETE, 12-HETE, 15-HETE and 20-HETE), non-eicosanoid cannabinoid mediators, phospholipids and their derivatives such as phosphatidic acid (PA) and phosphatidylglycerol (PG), platelet activating factor (PAF) and cardiolipins as well as lysophospholipids such as lysophosphatidyl choline (LPC) and various lysophosphatidic acids (LPA).
  • HETEs hydroxyeicosatetraenoic acids
  • HETEs hydroxyeicosatetraenoic acids
  • HETEs hydroxyeicosatetraenoic acids
  • Bioactive signaling lipid mediators also include the sphingolipids such as sphingomyelin, ceramide, ceramide-1-phosphate, sphingosine, sphingosylphosphoryl choline, sphinganine, sphinganine-1 -phosphate (Dihydro-S1 P) and sphingosine-1-phosphate.
  • Sphingolipids and their derivatives represent a group of extracellular and intracellular signaling molecules with pleiotropic effects on important cellular processes.
  • bioactive signaling lipids include phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PEA), diacylglyceride (DG), sulfatides, gangliosides, and cerebrosides.
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PEA phosphatidylethanolamine
  • DG diacylglyceride
  • sulfatides gangliosides, and cerebrosides.
  • Sphingolipids are a unique class of lipids that were named, due to their initially mysterious nature, after the Sphinx. Sphingolipids were initially characterized as primary structural components of cell membranes, but recent studies indicate that sphingolipids also serve as cellular signaling and regulatory molecules (Hannun, et al., Adv. Lipid Res. 25:27-41 , 1993; Speigel ,et al., FASEB J. 10: 1388-1397, 1996; Igarashi, J. Biochem 122: 1080-1087, 1997; Hla, T. (2004). Semin Cell Dev Biol, 15, 513-2; Gardell, S.E., Dubin, A.E. & Chun, J. (2006).
  • Sphingolipids are primary structural components of cell membranes that also serve as cellular signaling and regulatory molecules (Hannun and Bell, Adv. Lipid Res. 25: 27-41 , 1993; Igarashi, J. Biochem 122: 1080-1087, 1997).
  • the sphingolipid signaling mediators, ceramide (CER), sphingosine (SPH) and sphingosine-1 -phosphate (S1 P) have been most widely studied and have recently been appreciated for their roles in the cardiovascular system, angiogenesis and tumor biology (Claus, et al., Curr Drug Targets 1 : 185-205, 2000; Levade, et al., Circ. Res.
  • S1P Sphingosine-1 -Phosphate
  • S1 P is a mediator of cell proliferation and protects from apoptosis through the activation of survival pathways (Maceyka, et al. (2002), BBA, vol. 1585): 192-201 , and Spiegel, et al. (2003), Nature Reviews Molecular Cell Biology, vol. 4: 397-407). It has been proposed that the balance between CER/SPH levels and S1 P provides a rheostat mechanism that decides whether a cell is directed into the death pathway or is protected from apoptosis.
  • S1 P has two fates: S1 P can be degraded by S1 P lyase, an enzyme that cleaves S1 P to phosphoethanolamine and hexadecanal, or, less common, hydrolyzed by S1 P phosphatase to SPH.
  • GPCRs G protein-coupled receptors
  • EDG-6 Endothelial Differentiation Genes
  • S1 PRs high- affinity S1 P receptors
  • S1 P is released from platelets (Murata et al., 2000) and mast cells to create a local pulse of free S1 P (sufficient enough to exceed the Kd of the S1 PRs) for promoting wound healing and participating in the inflammatory response.
  • the total S1 P in the plasma is quite high (300-500 nM); however, it has been hypothesized that most of the S1 P may be 'buffered' by serum proteins, particularly lipoproteins (e.g., HDL>LDL>VLDL) and albumin, so that the bio-available S1 P (or the free fraction of S1 P) is not sufficient to appreciably activate S1 PRs (Murata et al., 2000).
  • S1 P Widespread expression of the cell surface S1 P receptors allows S1 P to influence a diverse spectrum of cellular responses, including proliferation, adhesion, contraction, motility, morphogenesis, differentiation, and survival. This spectrum of response appears to depend upon the overlapping or distinct expression patterns of the S1 P receptors within the cell and tissue systems.
  • crosstalk between S1 P and growth factor signaling pathways including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and basic fibroblastic growth factor (bFGF) have recently been demonstrated (see, e.g., Baudhuin, et al. (2004), FASEB J, vol. 18: 341 -3).
  • S1 P The regulation of various cellular processes involving S1 P has particular impact on neuronal signaling, vascular tone, wound healing, immune cell trafficking, reproduction, and cardiovascular function, among others. Alterations of endogenous levels of S1 P within these systems can have detrimental effects, eliciting several pathophysiological conditions, including cancer, inflammation, angiogenesis, heart disease, asthma, and autoimmune diseases.
  • a recent novel approach to the treatment of various diseases and disorders involves reducing levels of biologically available S1 P, either alone or in combination with other treatments.
  • sphingolipid-based treatment strategies that target key enzymes of the sphingolipid metabolic pathway, such as SPHK, have been proposed, interference with the lipid mediator S1 P itself has not until recently been emphasized, largely because of difficulties in directly mitigating this lipid target, in particular because of the difficulty first in raising and then in detecting antibodies against the S1 P target.
  • a humanized antibody may be preferable to a murine antibody, particularly for therapeutic uses in humans, where human-anti-mouse antibody (HAMA) response may occur.
  • HAMA human-anti-mouse antibody
  • Such a response may reduce the effectiveness of the antibody by neutralizing the binding activity and/or by rapidly clearing the antibody from circulation in the body.
  • the HAMA response can also cause toxicities with subsequent administrations of mouse antibodies.
  • a first-in-class humanized anti-S1 P antibody (Sonepcizumab, LT1009) has now been developed and is described herein.
  • This antibody is expected to have all the advantages of the murine mAb in terms of efficacy in binding S1 P, neutralizing S1 P and modulating disease states related to S1 P, but with none of the potential disadvantages of the murine mAb when used in a human context.
  • this humanized antibody has in fact shown activity greater than that of the parent (murine) antibody in animal models of disease. Sonepcizumab is currently in clinical trials for cancer and age-related macular degeneration.
  • Lysolipids are low molecular weight lipids that contain a polar head group and a single hydrocarbon backbone, due to the absence of an acyl group at one or both possible positions of acylation. Relative to the polar head group at sn-3, the hydrocarbon chain can be at the sn-2 and/or sn-1 position(s) (the term "lyso," which originally related to hemolysis, has been redefined by lUPAC to refer to deacylation). See “Nomenclature of Lipids, www.chem.qmul.ac.uk/iupac/lipid/lip1 n2.html.
  • lipids are representative of signaling, bioactive lipids, and their biologic and medical importance highlight what can be achieved by targeting lipid signaling molecules for therapeutic, diagnostic/prognostic, or research purposes (Gardell, ef a/. (2006), Trends in Molecular Medicine, vol 12: 65-75).
  • LPA glycerol backbone
  • S1 P sphingoid backbone
  • Other lysolipids include sphingosine, lysophosphatidylcholine (LPC),
  • sphingosylphosphorylcholine lysosphingomyelin
  • ceramide ceramide-1 -phosphate
  • sphinganine sphingosylphosphorylcholine
  • dihydrosphingosine dihydrosphingosine-1 -phosphate and N-acetyl-ceramide-1-phosphate.
  • the plasmalogens which contain an O-alkyl (-O-CH2-) or O-alkenyl ether at the C-1 (sn1 ) and an acyl at C-2, are excluded from the lysolipid genus.
  • LPA is not a single molecular entity but a collection of endogenous structural variants with fatty acids of varied lengths and degrees of saturation (Fujiwara, et al. (2005), J Biol Chem, vol. 280: 35038-35050).
  • the structural backbone of the LPAs is derived from glycerol-based phospholipids such as phosphatidylcholine (PC) or phosphatidic acid (PA).
  • PC phosphatidylcholine
  • PA phosphatidic acid
  • S1 P lysosphingolipids
  • S1 P the fatty acid of the ceramide backbone at sn- 2 is missing.
  • S1 P, dihydro S1 P (DHS1 P) and sphingosylphosphorylcholine (SPC) is based on sphingosine, which is derived from sphingomyelin.
  • LPA and S1 P regulate various cellular signaling pathways by binding to the same class of multiple transmembrane domain G protein-coupled (GPCR) receptors (Chun J, Rosen H (2006), Current Pharm Des, vol. 12: 161 -171 , and Moolenaar, WH (1999), Experimental Cell Research, vol. 253: 230-238).
  • the S1 P receptors are designated as S1 Pi, S1 P 2 , S1 P 3 , S1 P 4 and S1 P 5 (formerly EDG-1 , EDG-5/AGR16, EDG-3, EDG-6 and EDG-8) and the LPA receptors designated as LPAi , LPA 2 , LPA 3 (formerly, EDG-2, EDG-4, and EDG-7).
  • LPA4 LPA receptor of this family has been identified for LPA (LPA4), and other putative receptors for these lysophospholipids have also been reported.
  • LPAs have long been known as precursors of phospholipid biosynthesis in both eukaryotic and prokaryotic cells, but LPAs have emerged only recently as signaling molecules that are rapidly produced and released by activated cells, notably platelets, to influence target cells by acting on specific cell-surface receptor (see, e.g., Moolenaar, ef a/. (2004), BioEssays, vol. 26: 870-881 , and van Leewen ef a/. (2003), Biochem Soc Trans, vol 31 : 1209-1212).
  • LPA can be generated through the hydrolysis of pre-existing phospholipids following cell activation; for example, the sn-2 position is commonly missing a fatty acid residue due to deacylation, leaving only the sn-1 hydroxyl esterified to a fatty acid.
  • autotoxin lysoPLD/NPP2
  • lysoPLD/NPP2 may be the product of an oncogene, as many tumor types up-regulate autotoxin (Brindley, D. (2004), J Cell Biochem, vol. 92: 900-12).
  • LPA concentrations in human plasma and serum have been reported, including determinations made using a sensitive and specific LC/MS procedure (Baker, ef a/. (2001 ), Anal Biochem, vol 292: 287-295).
  • LPA concentrations have been estimated to be approximately 1.2 ⁇ , with the LPA analogs 16:0, 18: 1 , 18:2, and 20:4 being the predominant species.
  • LPA concentrations have been estimated to be approximately 0.7 ⁇ , with 18: 1 and 18:2 LPA being the predominant species.
  • LPA influences a wide range of biological responses, ranging from induction of cell proliferation, stimulation of cell migration and neurite retraction, gap junction closure, and even slime mold chemotaxis (Goetzl, ef a/. (2002), Scientific World Journal, vol. 2: 324-338).
  • the body of knowledge about the biology of LPA continues to grow as more and more cellular systems are tested for LPA responsiveness. For instance, it is now known that, in addition to stimulating cell growth and proliferation, LPA promote cellular tension and cell-surface fibronectin binding, which are important events in wound repair and regeneration (Moolenaar, ef a/. (2004), BioEssays, vol. 26: 870-881 ).
  • LPA peroxisome proliferation receptor gamma is a receptor/target for LPA
  • LPA is now recognized as a key signaling molecule involved in the etiology of cancer.
  • LPA has proven to be a difficult target for antibody production, although there has been a report in the scientific literature of the production of polyclonal murine antibodies against LPA (Chen ef a/. (2000) Med Chem Lett, vol 10: 1691 -3).
  • Lpath has recently humanized a monoclonal antibody against LPA, disclosed in US Patent application US20080145360 (attorney docket no. LPT-3100-UT4).
  • the humanized anti-LPA antibody, LT3015 exhibits picomolar binding affinity as demonstrated using surface plasmon resonance and is highly specific for LPA.
  • Soluble antibodies of the Immunoglobin G (IgG) class consist of a pair of heavy and light chains that are held together by intra- and interchain disulfide bonds to generate the characteristic Y-shaped structure ( Figure 1 ).
  • immunoglobin domain a fold that is common to many effector molecules of the immune system.
  • Heavy chains begin with one variable domain (Vh) followed by three constant domains (Ch1 -3) while kappa light chains consist of one variable domain (Vk) followed by one constant domain (Ck).
  • Vh variable domain
  • Cho1 -3 constant domains
  • kappa light chains consist of one variable domain (Vk) followed by one constant domain (Ck).
  • Epitope binding specificity results from variability within the amino-terminal Vh and Vk domains, particularly within six loops (CDR H 1 , H2, H3, L1 , L2 and L3) also known as hypervariable regions.
  • Fab fragment consisting of both variable domains and the Ck and constant domains from the Fc domain, which contains a pair of Ch2 and Ch3 domains.
  • the Fab fragment retains one entire variable region and, therefore, serves as a useful tool for biochemical characterization of a 1 : 1 interaction between the antibody and epitope.
  • the Fab fragment is generally an excellent platform for structural studies via single crystal x-ray diffraction.
  • Lpath's lmmuneY2TM technology allows generation of monoclonal antibodies (mAb) against extracellular lipid signaling mediators.
  • Lpath has developed a first-in-class therapeutic agent, a humanized monoclonal antibody
  • SonepcizumabTM ( LT1009; the names Sonepcizumab and LT1009 are herein used interchangeably), which was derived from the murine form of the antibody, SphingomabTM.
  • Sonepcizumab neutralizes the bioactive lipid signaling mediator, sphingosine-1 -phosphate (S1 P). S1 P contributes to disease in cancer, multiple sclerosis, inflammatory disease and ocular diseases that involve dysregulated angiogenesis.
  • ASONEPTM is currently in Phase 1 trials for cancer while an ocular formulation of the same mAb, iSONEPTM, is in
  • Lpath has also recently developed the humanized mAb LpathomabTM (LT3015; the names Lpathomab and LT3015are herein used interchangeably), a mAb against the bioactive lipid mediator, lysophosphatidic acid (LPA).
  • LPA lysophosphatidic acid
  • LPA has been implicated in the pathogenesis and progression of severe diseases including cancer, fibrosis, neuropathic pain, and inflammatory diseases.
  • antibody refers to any form of a peptide, polypeptide derived from, modeled after or encoded by, an immunoglobulin gene, or fragment thereof, that is capable of binding an antigen or epitope. See, e.g., IMMUNOBIOLOGY, Fifth Edition, C. A. Janeway, P. Travers, M., Walport, M.J.
  • antibody is used herein in the broadest sense, and encompasses monoclonal, polyclonal or multispecific antibodies, minibodies, heteroconjugates, diabodies, triabodies, chimeric, antibodies, synthetic antibodies, antibody fragments, and binding agents that employ the complementarity determining regions (CDRs) of the parent antibody, or variants thereof that retain antigen binding activity.
  • CDRs complementarity determining regions
  • Antibodies are defined herein as retaining at least one desired activity of the parent antibody. Desired activities can include the ability to bind the antigen specifically, the ability to inhibit proleration in vitro, the ability to inhibit angiogenesis in vivo, and the ability to alter cytokine profile(s) in vitro.
  • Native antibodies are usually heterotetrameric glycoproteins of about 150,000 Daltons, typically composed of two identical light (L) chains and two identical heavy (H) chains.
  • the heavy chain is approximately 50 kD in size, and the light chain is approximately 25 kDa.
  • Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
  • the light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • the ratio of the two types of light chain varies from species to species. As a way of example, the average K to ⁇ ratio is 20:1 in mice, whereas in humans it is 2: 1 and in cattle it is 1 :20.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG 1 , lgG2, lgG3, lgG4, IgA, and lgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • an "antibody derivative” is an immune-derived moiety, i.e., a molecule that is derived from an antibody.
  • This comprehends, for example, antibody variants, antibody fragments, chimeric antibodies, humanized antibodies, multivalent antibodies, antibody conjugates and the like, which retain a desired level of binding activity for antigen.
  • antibody fragment refers to a portion of an intact antibody that includes the antigen binding site or variable regions of an intact antibody, wherein the portion can be free of the constant heavy chain domains (e.g., CH2, CH3, and CH4) of the Fc region of the intact antibody. Alternatively, portions of the constant heavy chain domains (e.g., CH2, CH3, and CH4) can be included in the "antibody fragment”.
  • Antibody fragments retain antigen-binding and include Fab, Fab', F(ab')2, Fd, and Fv fragments; diabodies; triabodies; single-chain antibody molecules (sc-Fv); minibodies, nanobodies, and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • a Fab fragment also contains the constant domain of a light chain and the first constant domain (CH1 ) of a heavy chain.
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.
  • variable domains interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • the six hypervariable regions confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three hypervariable regions specific for an antigen
  • Single-chain Fv or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH 1 domain including one or more cysteine(s) from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • an “antibody variant” refers herein to a molecule which differs in amino acid sequence from the amino acid sequence of a native or parent antibody that is directed to the same antigen by virtue of addition, deletion and/or substitution of one or more amino acid residue(s) in the antibody sequence and which retains at least one desired activity of the parent anti-binding antibody. Desired activities can include the ability to bind the antigen specifically, the ability to inhibit proliferation in vitro, the ability to inhibit angiogenesis in vivo, and the ability to alter cytokine profile in vitro.
  • the amino acid change(s) in an antibody variant may be within a variable region or a constant region of a light chain and/or a heavy chain, including in the Fc region, the Fab region, the CHi domain, the CH2 domain, the CH3 domain, and the hinge region.
  • the variant comprises one or more amino acid substitution(s) in one or more hypervariable region(s) of the parent antibody.
  • the variant may comprise at least one, e.g. from about one to about ten, and preferably from about two to about five, substitutions in one or more hypervariable regions of the parent antibody.
  • the variant will have an amino acid sequence having at least 50% amino acid sequence identity with the parent antibody heavy or light chain variable domain sequences, more preferably at least 65%, more preferably at 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
  • Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.
  • the variant retains the ability to bind LPA and preferably has desired activities which are superior to those of the parent antibody.
  • the variant may have a stronger binding affinity, enhanced ability to reduce angiogenesis and/or halt tumor progression.
  • desired properties for example les immunogenic, longer half-life, enhanced stability, enhanced potency
  • the variant antibody of particular interest herein can be one which displays at least about 10 fold, preferably at least about % 5, 25, 59, or more of at least one desired activity.
  • the preferred variant is one that has superior biophysical properties as measured in vitro or superior activities biological as measured in vitro or in vivo when compared to the parent antibody.
  • an "anti-LPA agent” refers to any therapeutic agent that binds LPA, and includes antibodies, antibody variants, antibody-derived molecules or non-antibody-derived moieties that bind LPA and its variants.
  • an "anti-LPA antibody” or an “immune-derived moiety reactive against LPA” refers to any antibody or antibody-derived molecule that binds LPA.
  • antibodies or immune- derived moieties may be polyclonal or monoclonal and may be generated through a variety of means, and/or may be isolated from an animal, including a human subject.
  • an "anti-S1 P agent” refers to any therapeutic agent that binds S1 P, and includes antibodies, antibody variants, antibody-derived molecules or non-antibody-derived moieties that bind LPA and its variants.
  • an "anti-S1 P antibody” or an “immune-derived moiety reactive against S1 P” refers to any antibody or antibody-derived molecule that binds S1 P.
  • antibodies or immune- derived moieties may be polyclonal or monoclonal and may be generated through a variety of means, and/or may be isolated from an animal, including a human subject.
  • a “bioactive lipid” refers to a lipid signaling molecule. Bioactive lipids are distinguished from structural lipids
  • bioactive lipids can be found in extracellular fluids, where they can be complexed with other molecules, for example serum proteins such as albumin and lipoproteins, or in "free" form, i.e., not complexed with another molecule species.
  • extracellular mediators some bioactive lipids alter cell signaling by activating membrane-bound ion channels or GPCRs or enzymes or factors that, in turn, activate complex signaling systems that result in changes in cell function or survival.
  • intracellular mediators bioactive lipids can exert their actions by directly interacting with intracellular components such as enzymes, ion channels or structural elements such as actin.
  • bioactive lipids examples include sphingolipids such as ceramide, ceramide-1 -phosphate (C1 P), sphingosine, sphinganine, sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1 P).
  • Sphingolipids and their derivatives and metabolites are characterized by a sphingoid backbone (derived from sphingomyelin). Sphingolipids and their derivatives and metabolites represent a group of extracellular and intracellular signaling molecules with pleiotropic effects on important cellular processes. They include sulfatides, gangliosides and cerebrosides.
  • bioactive lipids are characterized by a glycerol-based backbone; for example, lysophospholipids such as lysophosphatidyl choline (LPC) and various lysophosphatidic acids (LPA), as well as phosphatidylinositol (PI), phosphatidylethanolamine (PEA), phosphatidic acid, platelet activating factor (PAF), cardiolipin,
  • lysophospholipids such as lysophosphatidyl choline (LPC) and various lysophosphatidic acids (LPA), as well as phosphatidylinositol (PI), phosphatidylethanolamine (PEA), phosphatidic acid, platelet activating factor (PAF), cardiolipin,
  • LPC lysophosphatidyl choline
  • LPA lysophosphatidic acids
  • PI phosphatidylinositol
  • PEA phosphatidylethanol
  • bioactive lipids are derived from arachidonic acid; these include the eicosanoids (including the eicosanoid metabolites such as the HETEs, cannabinoids, leukotrienes, prostaglandins, lipoxins, epoxyeicosatrienoic acids, and isoeicosanoids), non-eicosanoid cannabinoid mediators.
  • eicosanoids including the eicosanoid metabolites such as the HETEs, cannabinoids, leukotrienes, prostaglandins, lipoxins, epoxyeicosatrienoic acids, and isoeicosanoids
  • Other bioactive lipids including other phospholipids and their derivatives, may also be used according to the instant invention.
  • glycerol-based bioactive lipids such as the LPAs
  • sphingosine-based bioactive lipids such as sphingoid backbone, such as sphingosine and S1 P
  • arachidonic acid-derived bioactive lipids for antibody generation, and in other embodiments arachidonic acid-derived and glycerol-derived bioactive lipids but not sphingoid-derived bioactive lipids are preferred.
  • the arachidonic acid-derived and glycerol-derived bioactive lipids may be referred to in the context of this invention as "non-sphingoid bioactive lipids.”
  • bioactive lipids Specifically excluded from the class of bioactive lipids according to the invention are phosphatidylcholine and phosphatidylserine, as well as their metabolites and derivatives that function primarily as structural members of the inner and/or outer leaflet of cellular membranes.
  • biologically active in the context of an antibody or antibody fragment or variant, refers to an antibody or antibody fragment or antibody variant that is capable of binding the desired epitope and in some ways exerting a biologic effect.
  • Biological effects include, but are not limited to, the modulation of a growth signal, the modulation of an anti-apoptotic signal, the modulation of an apoptotic signal, the modulation of the effector function cascade, and modulation of other ligand interactions.
  • a “biomarker” is a specific biochemical in the body which has a particular molecular feature that makes it useful for measuring the progress of disease or the effects of treatment.
  • S1 P is a biomarker for certain hyperproliferative and/or cardiovascular conditions.
  • cardiotherapeutic agent refers to an agent that is therapeutic to diseases and diseases caused by or associated with cardiac and myocardial diseases and disorders.
  • Cardiovascular therapy encompasses cardiac therapy (treatment of myocardial ischemia and/or heart failure) as well as the prevention and/or treatment of other diseases associated with the cardiovascular system, such as heart disease.
  • heart disease encompasses any type of disease, disorder, trauma or surgical treatment that involves the heart or myocardial tissue. Of particular interest are conditions associated with tissue remodeling.
  • cardiotherapeutic agent refers to an agent that is therapeutic to diseases and diseases caused by or associated with cardiac and myocardial diseases and disorders.
  • a “carrier” refers to a moiety adapted for conjugation to a hapten, thereby rendering the hapten immunogenic.
  • a representative, non-limiting class of carriers is proteins, examples of which include albumin, keyhole limpet hemocyanin, hemaglutanin, tetanus, and diptheria toxoid.
  • Other classes and examples of carriers suitable for use in accordance with the invention are known in the art. These, as well as later discovered or invented naturally occurring or synthetic carriers, can be adapted for application in accordance with the invention.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • Cerebrovascular therapy refers to therapy directed to the prevention and/or treatment of diseases and disorders associated with cerebral ischemia and/or hypoxia.
  • cerebral ischemia and/or hypoxia resulting from global ischemia resulting from a heart disease, including without limitation heart failure.
  • chemotherapeutic agent means anti-cancer and other anti-hyperproliferative agents.
  • chemotherapeutic agents are a subset of therapeutic agents in general.
  • Chemotherapeutic agents include, but are not limited to: DNA damaging agents and agents that inhibit DNA synthesis: anthracyclines (doxorubicin, donorubicin, epirubicin), alkylating agents (bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cyclophosphamide, dacarbazine, hexamethylmelamine, ifosphamide, lomustine, mechlorethamine, melphalan, mitotane, mytomycin, pipobroman, procarbazine, streptozocin, thiotepa, and triethylenemelamine), platinum derivatives (cisplatin, carboplatin, cis diammine-dichloroplatinum), and topoisomerase inhibitors (Camptos
  • Faslodex steroids such as dexamethasone
  • immuno-modulators cytokines such as IFN-beta and IL2
  • cytokines such as IFN-beta and IL2
  • inhibitors to integrins other adhesion proteins and matrix metalloproteinases
  • histone deacetylase inhibitors like suberoylanilide hydroxamic acid
  • inhibitors of signal transduction such as inhibitors of tyrosine kinases like imatinib (Gleevec); inhibitors of heat shock proteins like 17-N-allylamino-17-demethoxygeldanamycin
  • retinoids such as all trans retinoic acid
  • inhibitors of growth factor receptors or the growth factors themselves anti-mitotic compounds and/or tubulin- depolymerizing agents such as the taxoids (paclitaxel, docetaxel, taxotere, BAY 59-8862), navelbine, vinblastine, vincristine, vindesine and vin
  • chimeric antibody refers to a molecule comprising a heavy and/or light chain which is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly, et ai, infra; Morrison et ai, Proc. Natl. Acad. Sci. U.S.A., vol. 81 :6851 (1984)).
  • combination therapy refers to a therapeutic regimen that involves the provision of at least two distinct therapies to achieve an indicated therapeutic effect.
  • a combination therapy may involve the administration of two or more chemically distinct active ingredients, for example, a fast-acting chemotherapeutic agent and an anti-lipid antibody, or two different antibodies.
  • a combination therapy may involve the administration of an anti-lipid antibody together with the delivery of another treatment, such as radiation therapy and/or surgery.
  • a combination therapy may involve administration of an anti-lipid antibody together with one or more other biological agents (e.g., anti-VEGF, TGF , PDGF, or bFGF agent), chemotherapeutic agents and another treatment such as radiation and/or surgery.
  • the active ingredients may be administered as part of the same composition or as different compositions.
  • the compositions comprising the different active ingredients may be administered at the same or different times, by the same or different routes, using the same of different dosing regimens, all as the particular context requires and as determined by the attending physician.
  • one or more anti-lipid antibody species for example, an anti-LPA antibody
  • the drug(s) may be delivered before or after surgery or radiation treatment.
  • constant domain refers to the C-terminal region of an antibody heavy or light chain.
  • the constant domains are not directly involved in the binding properties of an antibody molecule to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • effector functions refer to the different physiological effects of antibodies (e.g., opsonization, cell lysis, mast cell, basophil and eosinophil degranulation, and other processes) mediated by the recruitment of immune cells by the molecular interaction between the Fc domain and proteins of the immune system.
  • the isotype of the heavy chain determines the functional properties of the antibody. Their distinctive functional properties are conferred by the carboxy-terminal portions of the heavy chains, where they are not associated with light chains.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • a “derivatized bioactive lipid” is a bioactive lipid, e.g., LPA, which has a polar head group and at least one hydrocarbon chain, wherein a carbon atom within the hydrocarbon chain is derivatized with a pendant reactive group [e.g., a sulfhydryl (thiol) group, a carboxylic acid group, a cyano group, an ester, a hydroxy group, an alkene, an alkyne, an acid chloride group or a halogen atom] that may or may not be protected.
  • This derivatization serves to activate the bioactive lipid for reaction with a molecule, e.g., for conjugation to a carrier.
  • A"derivatized bioactive lipid conjugate” refers to a derivatized bioactive lipid that is covalently conjugated to a carrier.
  • the carrier may be a protein molecule or may be a moiety such as polyethylene glycol, colloidal gold, adjuvants or silicone beads.
  • a derivatized bioactive lipid conjugate may be used as an immunogen for generating an antibody response according to the instant invention, and the same or a different bioactive lipid conjugate may be used as a detection reagent for detecting the antibody thus produced.
  • the derivatized bioactive lipid conjugate is attached to a solid support when used for detection.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH - VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH - VL polypeptide chain
  • Effective concentration refers to the absolute, relative, and/or available concentration and/or activity, for example of certain undesired bioactive lipids.
  • the effective concentration of a bioactive lipid is the amount of lipid available, and able, to perform its biological function.
  • an immune-derived moiety such as, for example, a monoclonal antibody directed to a bioactive lipid (such as, for example, C1 P) is able to reduce the effective concentration of the lipid by binding to the lipid and rendering it unable to perform its biological function.
  • the lipid itself is still present (it is not degraded by the antibody, in other words) but can no longer bind its receptor or other targets to cause a downstream effect, so "effective concentration" rather than absolute concentration is the appropriate measurement.
  • Methods and assays exist for directly and/or indirectly measuring the effective concentration of bioactive lipids.
  • epitope or “antigenic determinant” refers to that portion of an antigen that reacts with an antibody antigen-binding portion derived from an antibody.
  • expression cassette refers to a nucleotide molecule capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as an antibody of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide-coding sequence, and, optionally, with other sequences, e.g., transcription termination signals. Additional regulatory elements necessary or helpful in effecting expression may also be used, e.g., enhancers.
  • expression cassettes include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • a “fully human antibody” can refer to an antibody produced in a genetically engineered (i.e., transgenic) mouse (e.g. from Medarex) that, when presented with an immunogen, can produce a human antibody that does not necessarily require CDR grafting.
  • These antibodies are fully human (100% human protein sequences) from animals such as mice in which the non-human antibody genes are suppressed and replaced with human antibody gene expression. The applicants believe that antibodies could be generated against bioactive lipids when presented to these genetically engineered mice or other animals who might be able to produce human frameworks for the relevant CDRs.
  • a "hapten” is a substance that is non-immunogenic but can react with an antibody or antigen-binding portion derived from an antibody. In other words, haptens have the property of antigenicity but not immunogenicity.
  • a hapten is generally a small molecule that can, under most circumstances, elicit an immune response (i.e., act as an antigen) only when attached to a carrier, for example, a protein, polyethylene glycol (PEG), colloidal gold, silicone beads, or the like.
  • the carrier may be one that also does not elicit an immune response by itself.
  • a representative, non-limiting class of hapten molecules is proteins, examples of which include albumin, keyhole limpet hemocyanin, hemaglutanin, tetanus, and diphtheria toxoid.
  • Other classes and examples of hapten molecules are known in the art. These, as well as later discovered or invented naturally occurring or synthetic haptens, can be adapted for application in accordance with the invention.
  • heteroconjugate antibody can refer to two covalently joined antibodies. Such antibodies can be prepared using known methods in synthetic protein chemistry, including using crosslinking agents. As used herein, the term “conjugate” refers to molecules formed by the covalent attachment of one or more antibody fragment(s) or binding moieties to one or more polymer molecule(s).
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Or, looked at another way, a humanized antibody is a human antibody that also contains selected sequences from non-human (e.g., murine) antibodies in place of the human sequences.
  • a humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biologic activity.
  • Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary- determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties.
  • donor antibody such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • a humanized antibody will comprise all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin. See, e.g., Cabilly, et ai., U.S. Pat. No. 4,816,567; Cabilly, et ai., European Patent
  • hyperproliferative disorder refers to diseases and disorders associated with, the uncontrolled proliferation of cells, including but not limited to uncontrolled growth of organ and tissue cells resulting in cancers and benign tumors.
  • Hyperproliferative disorders associated with endothelial cells can result in diseases of angiogenesis such as angiomas, endometriosis, obesity, age-related macular degeneration and various retinopathies, as well as the proliferation of endothelial cells and smooth muscle cells that cause restenosis as a consequence of stenting in the treatment of atherosclerosis.
  • Hyperproliferative disorders involving fibroblasts include but are not limited to disorders of excessive scarring (i.e., fibrosis) such as age-related macular degeneration, cardiac remodeling and failure associated with myocardial infarction, excessive wound healing such as commonly occurs as a consequence of surgery or injury, keloids, and fibroid tumors and stenting.
  • an “immune-derived moiety” includes any antibody (Ab) or immunoglobulin (Ig), and refers to any form of a peptide, polypeptide derived from, modeled after or encoded by, an immunoglobulin gene, or a fragment of such peptide or polypeptide that is capable of binding an antigen or epitope (see, e.g., Immunobiology, 5th Edition, Janeway, Travers, Walport, Shlomchiked. (editors), Garland Publishing (2001)).
  • the antigen is a lipid molecule, such as a bioactive lipid molecule.
  • an "immunogen” is a molecule capable of inducing a specific immune response, particularly an antibody response in an animal to whom the immunogen has been administered.
  • the immunogen is a derivatized bioactive lipid conjugated to a carrier, i.e., a "derivatized bioactive lipid conjugate".
  • the derivatized bioactive lipid conjugate used as the immunogen may be used as capture material for detection of the antibody generated in response to the immunogen.
  • the immunogen may also be used as a detection reagent.
  • the derivatized bioactive lipid conjugate used as capture material may have a different linker and/or carrier moiety from that in the immunogen.
  • a treatment yielding “inhibition of tumorigenesis” may mean that tumors do not form at all, or that they form more slowly, or are fewer in number than in the untreated control.
  • an “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • label when used herein refers to a detectable compound or composition, such as one that is conjugated directly or indirectly to the antibody.
  • the label may itself be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • a "ligand” is a substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose.
  • an antigen may be described as a ligand of the antibody to which it binds.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant that is useful for delivery of a drug (such as the anti-sphingolipid antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature.
  • Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • a “liquid composition” refers to one that, in its filled and finished form as provided from a manufacturer to an end user (e.g., a doctor or nurse), is a liquid or solution, as opposed to a solid.
  • solid refers to compositions that are not liquids or solutions.
  • solids include dried compositions prepared by lyophilization, freeze-drying, precipitation, and similar procedures.
  • linear antibodies when used throughout this application refers to the antibodies described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1 -VH-CH1) that form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • metabolites refers to compounds from which LPAs are made, as well as those that result from the degradation of LPAs; that is, compounds that are involved in the lysophospholipid metabolic pathways.
  • metabolic precursors may be used to refer to compounds from which sphingolipids are made.
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • the individual antibodies comprising the population are essentially identical, except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.
  • the monoclonal antibodies herein specifically include chimeric antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)).
  • “Monotherapy” refers to a treatment regimen based on the delivery of one therapeutically effective compound, whether administered as a single dose or several doses over time.
  • multispecific antibody can refer to an antibody, or a monoclonal antibody, having binding properties for at least two different epitopes.
  • the epitopes are from the same antigen.
  • the epitopes are from two or more different antigens.
  • Methods for making multispecific antibodies are known in the art.
  • Multispecific antibodies include bispecific antibodies (having binding properties for two epitopes), trispecific antibodies (three epitopes) and so on.
  • multispecific antibodies can be produced recombinantly using the co-expression of two or more immunoglobulin heavy chain/light chain pairs.
  • multispecific antibodies can be prepared using chemical linkage.
  • One of skill can produce multispecific antibodies using these or other methods as may be known in the art.
  • Multispecific antibodies include multispecific antibody fragments.
  • a multispecific (in this case, bispecific) antibody comprehended by this invention is an antibody having binding properties for an S1 P epitope and a C1 P epitope, which thus is able to recognize and bind to both S1 P and C1 P.
  • Another example of of a bispecific antibody comprehended by this invention is an antibody having binding properties for an epitope from a bioactive lipid and an epitope from a cell surface antigen. Thus the antibody is able to recognize and bind the bioactive lipid and is able to recognize and bind to cells, e.g., for targeting purposes.
  • Neoplasia or “cancer'Yefers to abnormal and uncontrolled cell growth.
  • a “neoplasm”, or tumor or cancer is an abnormal, unregulated, and disorganized proliferation of cell growth, and is generally referred to as cancer.
  • a neoplasm may be benign or malignant.
  • a neoplasm is malignant, or cancerous, if it has properties of destructive growth, invasiveness, and metastasis.
  • Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically breaking through the basal laminas that define the boundaries of the tissues, thereby often entering the body's circulatory system.
  • Metastasis typically refers to the dissemination of tumor cells by lymphatics or blood vessels.
  • Metastasis also refers to the migration of tumor cells by direct extension through serous cavities, or subarachnoid or other spaces. Through the process of metastasis, tumor cell migration to other areas of the body establishes neoplasms in areas away from the site of initial appearance.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the "parent” antibody herein is one that is encoded by an amino acid sequence used for the preparation of the variant.
  • the parent antibody may be a native antibody or may already be a variant, e.g., a chimeric antibody.
  • the parent antibody may be a humanized or human antibody.
  • a "patentable" composition, process, machine, or article of manufacture according to the invention means that the subject matter satisfies all statutory requirements for patentability at the time the analysis is performed.
  • novelty, non-obviousness, or the like if later investigation reveals that one or more claims encompass one or more embodiments that would negate novelty, non-obviousness, eic, the claim(s), being limited by definition to "patentable” embodiments, specifically exclude the non-patentable embodiment(s).
  • the claims appended hereto are to be interpreted both to provide the broadest reasonable scope, as well as to preserve their validity.
  • pharmaceutically acceptable salt refers to a salt, such as used in formulation, which retains the biological effectiveness and properties of the agents and compounds of this invention and which are is biologically or otherwise undesirable.
  • the agents and compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of charged groups, for example, charged amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases.
  • a "plurality” means more than one.
  • promoter includes all sequences capable of driving transcription of a coding sequence in a cell.
  • promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
  • a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
  • Transcriptional regulatory regions suitable for use in the present invention include but are not limited to the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the E. coli lac or trp promoters, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • CMV human cytomegalovirus
  • recombinant DNA refers to nucleic acids and gene products expressed therefrom that have been engineered, created, or modified by man.
  • Recombinant polypeptides or proteins are polypeptides or proteins produced by recombinant DNA techniques, for example, from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
  • Synthetic polypeptides or proteins are those prepared by chemical synthesis.
  • sample-holding vessel The terms “separated”, “purified”, “isolated”, and the like mean that one or more components of a sample contained in a sample-holding vessel are or have been physically removed from, or diluted in the presence of, one or more other sample components present in the vessel.
  • Sample components that may be removed or diluted during a separating or purifying step include, chemical reaction products, non-reacted chemicals, proteins, carbohydrates, lipids, and unbound molecules.
  • solid phase is meant a non-aqueous matrix such as one to which the antibody of the present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • kits is used herein in various contexts, e.g., a particular species of chemotherapeutic agent. In each context, the term refers to a population of chemically indistinct molecules of the sort referred in the particular context.
  • the term "specific” or “specificity” in the context of antibody-antigen interactions refers to the selective, non-random interaction between an antibody and its target epitope.
  • the term "antigen” refers to a molecule that is recognized and bound by an antibody molecule or other immune-derived moiety.
  • the specific portion of an antigen that is bound by an antibody is termed the "epitope". This interaction depends on the presence of structural, hydrophobic/hydrophilic, and/or electrostatic features that allow appropriate chemical or molecular interactions between the molecules.
  • an antibody is commonly said to “bind” (or “specifically bind”) or be “reactive with” (or “specifically reactive with), or, equivalently, “reactive against” (or “specifically reactive against”) the epitope of its target antigen.
  • Antibodies are commonly described in the art as being “against” or “to” their antigens as shorthand for antibody binding to the antigen.
  • an “antibody that binds C1 P,” an “antibody reactive against C1 P,” an “antibody reactive with C1 P,” an “antibody to C1 P” and an “anti-C1 P antibody” all have the same meaning in the art.
  • Antibody molecules can be tested for specificity of binding by comparing binding to the desired antigen to binding to unrelated antigen or analogue antigen or antigen mixture under a given set of conditions.
  • an antibody according to the invention will lack significant binding to unrelated antigens, or even analogs of the target antigen.
  • “Specifically associate” and “specific association” and the like refer to a specific, non-random interaction between two molecules, which interaction depends on the presence of structural, hydrophobic/hydrophilic, and/or electrostatic features that allow appropriate chemical or molecular interactions between the molecules.
  • sphingolipid refers to the class of compounds in the art known as sphingolipids, including, but not limited to the following compounds (see http//www.lipidmaps.org for chemical formulas, structural information, etc. for the corresponding compounds):
  • the present invention relates to anti-lipid agents, including anti-sphingolipid antibodies, that are useful for treating or preventing hyperproliferative disorders such as cancer and cardiovascular or cerebrovascular diseases and disorders and various ocular disorders, as described in greater detail below.
  • the invention relates, among others, to antibodies to S1 P and its variants including but are not limited to sphingosine-1-phosphate [sphingene-1- phosphate; D-erythro-sphingosine-1 -phosphate; sphing-4-enine-1 -phosphate; (E,2S,3R)-2-amino-3-hydroxy- octadec-4-enoxy]phosphonic acid (AS 26993-30-6), DHS1 P is defined as dihydrosphingosine-1-phosphate
  • sphingolipid metabolite refers to a compound from which a sphingolipid is made, as well as a that results from the degradation of a particular sphingolipid.
  • a "sphingolipid metabolite” is a compound that is involved in the sphingolipid metabolic pathways. Metabolites include metabolic precursors and metabolic products.
  • metabolic precursors refers to compounds from which sphingolipids are made. Metabolic precursors of particular interest include but are not limited to SPC, sphingomyelin, dihydrosphingosine, dihydroceramide, and 3-ketosphinganine.
  • metabolic products refers to compounds that result from the degradation of sphingolipids, such as phosphorylcholine (e.g.,. phosphocholine, choline phosphate), fatty acids, including free fatty acids, and hexadecanal (e.g.,. palmitaldehyde).
  • phosphorylcholine e.g.,. phosphocholine, choline phosphate
  • fatty acids including free fatty acids
  • hexadecanal e.g.,. palmitaldehyde
  • stable refers to an interaction between two molecules (e.g., a peptide and a TLR molecule) that is sufficiently stable such that the molecules can be maintained for the desired purpose or manipulation.
  • a “stable” interaction between a peptide and a TLR molecule refers to one wherein the peptide becomes and remains associated with a TLR molecule for a period sufficient to achieve the desired effect.
  • a “subject” or “patient” refers to an animal in need of treatment that can be effected by molecules of the invention.
  • Animals that can be treated in accordance with the invention include vertebrates, with mammals such as bovine, canine, equine, feline, ovine, porcine, and primate (including humans and non-human primates) animals being particularly preferred examples.
  • a “surrogate marker” refers to laboratory measurement of biological activity within the body that indirectly indicates the effect of treatment on disease state.
  • surrogate markers for hyperproliferative and/or cardiovascular conditions include SPHK and/or S1 PRs.
  • a “therapeutic agent” refers to a drug or compound that is intended to provide a therapeutic effect including, but not limited to: anti-inflammatory drugs including COX inhibitors and other NSAIDS, anti-angiogenic drugs, chemotherapeutic drugs as defined above, cardiovascular agents, immunomodulatory agents, agents that are used to treat neurodegenerative disorders, opthalmic drugs, anti-fibrotics, etc.
  • a “therapeutically effective amount” refers to an amount of an active ingredient, e.g., an agent according to the invention, sufficient to effect treatment when administered to a subject in need of such treatment. Accordingly, what constitutes a therapeutically effective amount of a composition according to the invention may be readily determined by one of ordinary skill in the art. In the context of cancer therapy, a
  • terapéuticaally effective amount is one that produces an objectively measured change in one or more parameters associated with cancer cell survival or metabolism, including an increase or decrease in the expression of one or more genes correlated with the particular cancer, reduction in tumor burden, cancer cell lysis, the detection of one or more cancer cell death markers in a biological sample (e.g., a biopsy and an aliquot of a bodily fluid such as whole blood, plasma, serum, urine, eic), induction of induction apoptosis or other cell death pathways, etc.
  • a biological sample e.g., a biopsy and an aliquot of a bodily fluid such as whole blood, plasma, serum, urine, eic
  • induction of induction apoptosis or other cell death pathways etc.
  • the therapeutically effective amount will vary depending upon the particular subject and condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art. It will be appreciated that in the context of combination therapy, what constitutes a therapeutically effective amount of a particular active ingredient may differ from what constitutes a therapeutically effective amount of the active ingredient when administered as a monotherapy (; ' .e., a therapeutic regimen that employs only one chemical entity as the active ingredient).
  • compositions of the invention are used in methods of bioactive lipid-based therapy.
  • the terms “therapy” and “therapeutic” encompasses the full spectrum of prevention and/or treatments for a disease, disorder or physical trauma.
  • a “therapeutic” agent of the invention may act in a manner that is prophylactic or preventive, including those that incorporate procedures designed to target individuals that can be identified as being at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may act to slow the rate or extent of the progression of at least one symptom of a disease or disorder being treated; or may act to minimize the time required, the occurrence or extent of any discomfort or pain, or physical limitations associated with recuperation from a disease, disorder or physical trauma; or may be used as an adjuvant to other therapies and treatments.
  • treatment means any treatment of a disease or disorder, including preventing or protecting against the disease or disorder (that is, causing the clinical symptoms not to develop); inhibiting the disease or disorder (; ' .e., arresting, delaying or suppressing the development of clinical symptoms; and/or relieving the disease or disorder (; ' .e., causing the regression of clinical symptoms).
  • preventing and “suppressing” a disease or disorder because the ultimate inductive event or events may be unknown or latent.
  • Those "in need of treatment” include those already with the disorder as well as those in which the disorder is to be prevented. Accordingly, the term “prophylaxis” will be understood to constitute a type of “treatment” that encompasses both “preventing” and “suppressing”.
  • the term “protection” thus includes “prophylaxis”.
  • therapeutic regimen means any treatment of a disease or disorder using chemotherapeutic and cytotoxic agents, radiation therapy, surgery, gene therapy, DNA vaccines and therapy, siRNA therapy, anti- angiogenic therapy, immunotherapy, bone marrow transplants, aptamers and other biologies such as antibodies and antibody variants, receptor decoys and other protein-based therapeutics.
  • variable region of an antibody comprises framework and complementarity determining regions (CDRs, otherwise known as hypervariable regions).
  • CDRs complementarity determining regions
  • the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in six CDR segments, three in each of the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR).
  • the variable domains of native heavy and light chains each comprise four FRs (FR1 , FR2, FR3 and FR4, respectively), largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region” or "CDR" (for example residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • CDR complementarity determining region
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ), pages 647-669).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.
  • a “vector” or “plasmid” or “expression vector” refers to a nucleic acid that can be maintained transiently or stably in a cell to effect expression of one or more recombinant genes.
  • a vector can comprise nucleic acid, alone or complexed with other compounds.
  • a vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes.
  • Vectors include, but are not limited, to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • vectors include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA and include both the expression and non-expression plasmids.
  • Plasmids can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids as reported with published protocols.
  • the expression vectors may also contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the present invention provides patentable crystalline forms of an anti-lipid antibody or fragment thereof, which may further comprise a lipid ligand of said antibody and/or salts, metals, and/or co-factors. Methods for making such crystals are provided.
  • the lipid may be a bioactive lipid, such as a sphingolipid including S1 P. X-ray coordinates of one such crystal are provided, as are methods of using this information in antibody design or optimization.
  • Methods for designing a humanized antibody to a lipid are provided, which may be performed in silico. These methods may result in enhanced binding affinity of an antibody to its original target lipid, or may be intended to alter binding specificity.
  • Figure 1 Purification, crystallization, x-ray diffraction, and structure of the anti-S1 P Fab/S1 P complex.
  • Figure 1a shows the result of an SDS- PAGE analysis showing purity of the antibody Fab fragment and its separation from the Fc fragment contaminant.
  • Figure 1 b is a photograph of a hanging drop containing Fab/S1 P complex co-crystals viewed through the eyepiece of a stereomicroscope.
  • Figure 1 c is a one-degree oscillation image of x-rays diffracted by the Fab/S1 P crystals. Data were collected at 100K on an R-AxislV++ image plate detector at the SDSU MXCF.
  • Figure 1d is a ribbon diagram structure depicting the antibody Fab/S1 P complex crystal structure.
  • the heavy chain is depicted in dark orange while the light chain is represented in light orange.
  • S1 P is in a stick representation with cpk atom coloring.
  • the two grey spheres are Ca 2+ ions.
  • Figure 2 S1 P binding of LT1009 variants.
  • Figure 2a is a bar graph showing the calculated concentrations of LT1009 variants and WT that produce half-maximal S1 P binding using the direct-binding ELISA.
  • Figure 2b is a colored structure diagram showing the structure of the LT1009Fab/S1 P complex. Atoms in the light (green) and heavy (blue) chains are drawn as spheres. The atoms in the amino acid side chains substituted in the LT1009 variants are colored magenta. The carbon, oxygen and phosphorus atoms of the bound S1 P are colored grey, red, and yellow, respectively.
  • Figure 3 Effect of metal chelators and mutations on S1 P binding by LT1009.
  • Figure 3a is a ribbon model showing the interaction of S1 P (gray) with key amino acid residues in the anti-S1 P antibody. The calcium atoms are shown in purple.
  • Figure 3b is a line graph showing the negative effect of chelators EGTA and EDTA on LT1009-S1 P binding.
  • Figure 3c is a line graph showing the effect of mutation of certain amino acid residues on LT1009-S1 P binding. Numbering of amino acid residues is sequential.
  • FIG. 4 Conversion of antibody specificity. A single amino acid at position 50 of the light chain of LT1009 was mutated (GluL50 to GlnL50). The figure is a line graph showing that the resulting antibody variant has significantly higher affinity for LPA conjugate than for S1 P conjugate, as shown by direct ELISA.
  • Antibody molecules or immunoglobulins are large glycoprotein molecules with a molecular weight of approximately 150 kDa, usually composed of two different kinds of polypeptide chain.
  • the heavy chain (H) is approximately 50 kDa.
  • the light chain (L), is approximately 25 kDa.
  • Each immunoglobulin molecule usually consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds, the number of which varies between the heavy chains of different immunoglobulin isotypes. Each light chain is linked to a heavy chain by one covalent disulfide bond.
  • the two heavy chains and the two light chains are identical, harboring two identical antigen-binding sites, and are thus said to be divalent, i.e., having the capacity to bind simultaneously to two identical molecules.
  • the light chains of antibody molecules from any vertebrate species can be assigned to one of two clearly distinct types, kappa (k) and lambda (I), based on the amino acid sequences of their constant domains.
  • the ratio of the two types of light chain varies from species to species. As a way of example, the average k to I ratio is 20: 1 in mice, whereas in humans it is 2: 1 and in cattle it is 1 :20.
  • the heavy chains of antibody molecules from any vertebrate species can be assigned to one of five clearly distinct types, called isotypes, based on the amino acid sequences of their constant domains. Some isotypes have several subtypes.
  • the five major classes of immunoglobulin are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE).
  • IgG is the most abundant isotype and has several subclasses (IgG 1 , 2, 3, and 4 in humans).
  • the Fc fragment and hinge regions differ in antibodies of different isotypes, thus determining their functional properties. However, the overall organization of the domains is similar in all isotypes.
  • Antibodies may be raised in many species including mammalian species (for example, mouse, rat, camel, bovine, goat, horse, guinea pig, hamster, sheep and rabbit) and birds (duck, chicken). Antibodies raised may derive from a different species from the animal in which they are raised. For example, the XenoMouseTM (Abgenix, Inc., Fremont CA) produces fully human monoclonal antibodies.
  • native human antibodies such as autoantibodies to S1 P isolated from individuals who may show a titer of such S1 P autoantibody may be used.
  • a human antibody sequence library may be used to generate antibodies comprising a human sequence.
  • therapeutic agents that alter the activity or concentration of one or more undesired bioactive lipids, or precursors or metabolites thereof, are therapeutically useful. These agents, including antibodies, act by changing the effective concentration, i.e., the absolute, relative, effective and/or available concentration and/or activities, of certain undesired bioactive lipids. Lowering the effective concentration of the bioactive lipid may be said to "neutralize” the target lipid or its undesired effects, including downstream effects.
  • undesired refers to a bioactive lipid that is unwanted due to its involvement in a disease process, for example, as a signaling molecule, or to an unwanted amount of a bioactive lipid which contributes to disease when present in excess.
  • compositions and methods can be used to treat these diseases and disorders, particularly by decreasing the effective in vivo concentration of a particular target lipid, for example, S1 P or its variants.
  • compositions and methods of the invention are useful in treating diseases characterized, at least in part, by aberrant neovascularization, angiogenesis, fibrogenesis, fibrosis, scarring, inflammation, and immune response.
  • One way to control the amount of undesirable sphingolipids or other bioactive lipids in a patient is by providing a composition that comprises one or more humanized anti-sphingolipid antibodies to bind one or more sphingolipids, thereby acting as therapeutic "sponges” that reduce the level of free undesirable sphingolipids.
  • a compound is referred to as "free" the compound is not in any way restricted from reaching the site or sites where it exerts its undesirable effects.
  • a free compound is present in blood and tissue, which either is or contains the site(s) of action of the free compound, or from which a compound can freely migrate to its site(s) of action.
  • a free compound may also be available to be acted upon by any enzyme that converts the compound into an undesirable compound.
  • compositions and methods of the present disclosure may be applied to treat these diseases and disorders as well as cardiac and myocardial diseases and disorders.
  • sphingolipid-based therapy involves manipulating the metabolic pathways of sphingolipids in order to decrease the actual, relative and/or available in vivo concentrations of undesirable, toxic sphingolipids.
  • the invention provides compositions and methods for treating or preventing diseases, disorders or physical trauma, in which humanized anti-sphingolipid antibodies are administered to a patient to bind undesirable, toxic sphingolipids, or metabolites thereof.
  • Such humanized anti-sphingolipid antibodies may be formulated in a pharmaceutical composition and are useful for a variety of purposes, including the treatment of diseases, disorders or physical trauma.
  • Pharmaceutical compositions comprising one or more humanized anti-sphingolipid antibodies of the invention may be incorporated into kits and medical devices for such treatment.
  • Medical devices may be used to administer the pharmaceutical compositions of the invention to a patient in need thereof, and according to one embodiment of the invention, kits are provided that include such devices.
  • Such devices and kits may be designed for routine administration, including self-administration, of the pharmaceutical compositions of the invention.
  • Such devices and kits may also be designed for emergency use, for example, in ambulances or emergency rooms, or during surgery, or in activities where injury is possible but where full medical attention may not be immediately forthcoming (for example, hiking and camping, or combat situations).
  • Suitable pharmaceutically acceptable diluents, carriers, and excipients are well known in the art.
  • Suitable amounts to be administered for any particular treatment protocol can readily be determined. Suitable amounts might be expected to fall within the range of 10 g/dose to 10 g/dose, preferably within 10 mg/dose to 1 g/dose.
  • Drug substances may be administered by techniques known in the art, including but not limited to systemic, subcutaneous, intradermal, mucosal, including by inhalation, and topical administration.
  • the mucosa refers to the epithelial tissue that lines the internal cavities of the body.
  • the mucosa comprises the alimentary canal, including the mouth, esophagus, stomach, intestines, and anus; the respiratory tract, including the nasal passages, trachea, bronchi, and lungs; and the genitalia.
  • the mucosa also includes the external surface of the eye, i.e., the cornea and conjunctiva.
  • Local administration (as opposed to systemic administration) may be advantageous because this approach can limit potential systemic side effects, but still allow therapeutic effect.
  • compositions used in the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations used in the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).
  • Preferred carriers include those that are pharmaceutically acceptable, particularly when the composition is intended for therapeutic use in humans.
  • veterinarily acceptable carriers may be employed.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • compositions include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies, and liposomes.
  • an immune-derived moiety can be delivered to the eye via, for example, topical drops or ointment, periocular injection, intracamerally into the anterior chamber or vitreous, via an implanted depot, or systemically by injection or oral administration.
  • the quantity of antibody used can be readily determined by one skilled in the art.
  • Topical drops are convenient, but wash away primarily because of nasolacrimal drainage often delivering less than 5% of the applied drug into the anterior section of the eye and an even smaller fraction of that dose to the posterior segment of the globe.
  • sprays afford another mode for topical administration.
  • a third mode is ophthalmic ointments or emulsions can be used to prolong the contact time of the formulation with the ocular surface although blurring of vision and matting of the eyelids can be troublesome.
  • Such topical approaches are still preferable, since systemic administration of therapeutics to treat ocular disorders exposes the whole body to the potential toxicity of the drug.
  • Treatment of the posterior segment of the eye is medically important because age-related macular degeneration, diabetic retinopathy, posterior uveitis, and glaucoma are the leading causes of vision loss in the United States and other developed countries.
  • the most efficient mode of drug delivery to the posterior segment is intravitreal injection through the pars plana.
  • direct injections require a skilled medical practitioner to effect the delivery and can cause treatment-limiting anxiety in many patients.
  • Periocular injections an approach that includes subconjunctival, retrobulbar, peribulbar and posterior subtenon injections, are somewhat less invasive than intravitreal injections. Repeated and long-term intravitreal injections may cause complications, such as vitreous hemorrhage, retinal detachment, or
  • the anti-bioactive lipid antibody treatment might also be administered using one of the newer ocular delivery systems [Sultana, et al. (2006),Current Drug Delivery, vol 3: 207-217; and Ghate and Edelhauser (2006),
  • soluble, erodible, non-erodible or hydrogel-based corneal shields, eg, collagen-based bandage and contact lenses that provide controlled delivery of drug to the eye
  • in situ gelling systems that provide ease of administration as drops that get converted to gel form in the eye, thereby providing some sustained effect of drug in the eye
  • vesicular systems such as liposomes, niosomes/discomes, etc., that offers advantages of targeted delivery, bio- compatibility and freedom from blurring of vision
  • mucoadhesive systems that provide better retention in the eye
  • prodrugs f) penetration enhancers, (g) lyophilized carrier systems, (h) particulates, (i) submicron emulsions, (j) iontophoresis, (k) dendrimers,
  • transscleral iontophoresis (Eljarrat-Binstock and Domb (2006), Control Release, 110: 479-89] is an important advance and may offer an effective way to deliver antibodies to the posterior segment of the eye.
  • excipients might also be added to the formulated antibody to improve performance of the therapy, make the therapy more convenient or to clearly ensure that the formulated antibody is used only for its intended, approved purpose.
  • excipients include chemicals to control pH, antimicrobial agents, preservatives to prevent loss of antibody potency, dyes to identify the formulation for ocular use only, solubilizing agents to increase the concentration of antibody in the formulation, penetration enhancers and the use of agents to adjust isotonicity and/or viscosity.
  • Inhibitors of, e.g., proteases could be added to prolong the half life of the antibody.
  • the antibody is delivered to the eye by intravitreal injection in a solution comprising phosphate-buffered saline at a suitable pH for the eye.
  • the anti-S1 P agent e.g., a humanized antibody
  • the active form of the antibody is then released by action of an endogenous enzyme.
  • Possible ocular enzymes to be considered in this application are the various cytochrome p450s, aldehyde reductases, ketone reductases, esterases or N-acetyl- ⁇ - glucosamidases.
  • Other chemical modifications to the antibody could increase its molecular weight, and as a result, increase the residence time of the antibody in the eye.
  • Antibody affinities may be determined as described in the examples herein below.
  • Preferred humanized or variant antibodies are those which bind a sphingolipid with a Kd value of no more than about 1 x 10 7 M, preferably no more than about 1 x 10 8 M, and most preferably no more than about 5 x 10 9 M.
  • the antibody may be one that reduce angiogenesis and alter tumor progression.
  • the antibody has an effective concentration 50 (EC50) value of no more than about 10 ug/ml, preferably no more than about 1 ug/ml, and most preferably no more than about 0.1 ug/ml, as measured in a direct binding ELISA assay.
  • the antibody has an effective concentration value of no more than about 10 ug/ml, preferably no more than about 1 ug/ml, and most preferably no more than about 0.1 ug/ml, as measured in cell assays in presence of 1 uM of S1 P, for example, at these concentrations the antibody is able to inhibit sphingolipid-induced IL-8 release in vitro by at least 10%.
  • the antibody has an effective concentration value of no more than about 10 ug/ml, preferably no more than about 1 ug/ml, and most preferably no more than about 0.1 ug/ml, as measured in the CNV animal model after laser burn, for example, at these concentrations the antibody is able to inhibit sphingolipid-induced
  • neovascularization in vivo by at least 50%.
  • Assays for determining the activity of the anti-sphingolipid antibodies of the invention include ELISA assays as shown in the examples hereinbelow.
  • the humanized or variant antibody fails to elicit an immunogenic response upon administration of a therapeutically effective amount of the antibody to a human patient. If an immunogenic response is elicited, preferably the response will be such that the antibody still provides a therapeutic benefit to the patient treated therewith.
  • humanized anti-sphingolipid antibodies bind the "epitope" as herein defined.
  • an antibody of interest e.g., those that block binding of the antibody to sphingolipid
  • a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)
  • epitope mapping e.g., as described in Champe, et al. [J. Biol. Chem. 270: 1388-1394 (1995)] can be performed to determine whether the antibody binds an epitope of interest.
  • the antibodies of the invention have a heavy chain variable domain comprising an amino acid sequence represented by the formula: FR1 -CDRH1 -FR2-CDRH2-FR3-CDRH3-FR4, wherein "FR1-4" represents the four framework regions and "CDRH 1 -3" represents the three hypervariable regions of an anti-sphingolipid antibody variable heavy domain.
  • FR1 -4 may be derived from a "consensus sequence” (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) as in the examples below or may be derived from an individual human antibody framework region or from a combination of different framework region sequences.
  • variable heavy FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat, et al., above.
  • the human variable heavy FR sequence preferably has one or more substitutions therein, e.g., wherein the human FR residue is replaced by a corresponding nonhuman residue (by "corresponding nonhuman residue” is meant the nonhuman residue with the same Kabat positional numbering as the human residue of interest when the human and nonhuman sequences are aligned), but replacement with the nonhuman residue is not necessary.
  • a replacement FR residue other than the corresponding nonhuman residue can be selected by phage display.
  • variable heavy FR residues which may be substituted include any one or more of FR residue numbers: 37H, 49H, 67H, 69H, 71 H, 73H, 75H, 76H, 78H, and 94H (Kabat residue numbering employed here). Preferably at least two, or at least three, or at least four of these residues are substituted. A particularly preferred combination of FR substitutions is: 49H, 69H, 71 H, 73H, 76H, 78H, and 94H. With respect to the heavy chain hypervariable regions, these preferably have amino acid sequences listed in Table 2, below.
  • the antibodies of the preferred embodiment herein have a light chain variable domain comprising an amino acid sequence represented by the formula: FR1 -CDRL1-FR2-CDRL2-FR3-CDRL3-FR4, wherein "FR1 -4" represents the four framework regions and "CDRL1 -3" represents the three hypervariable regions of an anti-sphingolipid antibody variable heavy domain.
  • FR1-4 may be derived from a "consensus sequence” (for example, the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) as in the examples below or may be derived from an individual human antibody framework region or from a combination of different framework region sequences.
  • the variable light FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat, et al., above.
  • the human variable light FR sequence preferably has substitutions therein, e.g., wherein a human FR residue is replaced by a corresponding mouse residue, but replacement with the nonhuman residue is not necessary.
  • a replacement residue other than the corresponding nonhuman residue may be selected by phage display.
  • Exemplary variable light FR residues that may be substituted include any one or more of FR residue numbers, including, but not limited to, F4, Y36, Y49, G64, S67.
  • the sphingolipid antigen to be used for production of antibodies may be, e.g., intact sphingolipid or a portion of a sphingolipid (e.g., a sphingolipid fragment comprising an "epitope").
  • a sphingolipid antigen used to generate antibodies is described in the examples below.
  • the antigen is a derivatized form of the sphingolipid, and may be associated with a carrier protein.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyrog
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ug or 5 ug of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 0.1 to 0.2 times the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum may be suitably used to enhance the immune response.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler, et al., Nature, 256:495 (1975), or by other suitable methods, including by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro.
  • Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and M.C.-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 - 63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbant assay
  • the binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson, et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • amino acid sequence variants of these humanized antibodies may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly where these improve the binding affinity or other biological properties of the humanized antibody. Examples hereinbelow describe methodologies for generating amino acid sequence variants of an anti-sphingolipid antibody with enhanced affinity relative to the parent antibody.
  • Amino acid sequence variants of the anti-sphingolipid antibody are prepared by introducing appropriate nucleotide changes into the anti-sphingolipid antibody DNA, or by peptide synthesis.
  • Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-sphingolipid antibodies of the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the humanized or variant anti-sphingolipid antibody, such as changing the number or position of glycosylation sites.
  • a useful method for identification of certain residues or regions of the anti-sphingolipid antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244: 1081 -1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with sphingolipid antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an anti-sphingolipid antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag.
  • Other insertional variants of the anti-sphingolipid antibody molecule include the fusion to the N- or C-terminus of the anti-sphingolipid antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the anti-sphingolipid antibody molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary" substitutions listed below, or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide 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.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • cysteine residues not involved in maintaining the proper conformation of the humanized or variant anti- sphingolipid antibody also may be substituted, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene llll product of M13 packaged within each particle.
  • the phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • biological activity e.g., binding affinity
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Crystals (co-crystals) of the antigen - antibody complex include co-crystals of the antigen and the Fab or other fragment of the antibody, along with any salts, metals (including divalent metals), cofactors and the like.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X- serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • Nucleic acid molecules encoding amino acid sequence variants of the anti-sphingolipid antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide- mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-sphingolipid antibody.
  • human antibodies can be generated.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • JH antibody heavy-chain joining region
  • Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits, et al. Nature, 362:255-
  • Human antibodies can also be derived from phage-display libraries (Hoogenboom, et al, J. Mol. Biol,
  • human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275) or by other suitable methods.
  • the humanized or variant anti-sphingolipid antibody is an antibody fragment.
  • Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto, et al., Journal of Biochemical and Biophysical Methods 24: 107-117(1992); and Brennan, et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter, et al., Bio/Technology 10: 163-167 (1992)).
  • the F(ab')2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab')2 molecule.
  • Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • bispecific humanized or variant anti-sphingolipid antibodies having binding specificities for at least two different epitopes.
  • Exemplary bispecific antibodies may bind to two different epitopes of the sphingolipid.
  • an anti-sphingolipid arm may be combined with an arm which binds to a different molecule.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See, e.g., U.S. patent no. 5,731 ,168.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in, for example, U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary Vi_ and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary Vi_ and VH domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • the bispecific antibody may be a "linear antibody" produced as described in, fror example, Zapata, et al. Protein Eng. 8(10): 1057- 1062 (1995).
  • Antibodies with more than two valencies are also contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991 ).
  • an antibody (or polymer or polypeptide) of the invention comprising one or more binding sites per arm or fragment thereof will be referred to herein as "multivalent” antibody.
  • a "bivalent” antibody of the invention comprises two binding sites per Fab or fragment thereof whereas a “trivalent” polypeptide of the invention comprises three binding sites per Fab or fragment thereof.
  • the two or more binding sites per Fab may be binding to the same or different antigens.
  • the two or more binding sites in a multivalent polypeptide of the invention may be directed against the same antigen, for example against the same parts or epitopes of said antigen or against two or more same or different parts or epitopes of said antigen; and/or may be directed against different antigens; or a combination thereof.
  • a bivalent polypeptide of the invention for example may comprise two identical binding sites, may comprise a first binding sites directed against a first part or epitope of an antigen and a second binding site directed against the same part or epitope of said antigen or against another part or epitope of said antigen; or may comprise a first binding sites directed against a first part or epitope of an antigen and a second binding site directed against the a different antigen.
  • the invention is not limited thereto, in the sense that a multivalent polypeptide of the invention may comprise any number of binding sites directed against the same or different antigens.
  • An antibody (or polymer or polypeptide) of the invention that contains at least two binding sites per Fab or fragment thereof, in which at least one binding site is directed against a first antigen and a second binding site directed against a second antigen different from the first antigen, will also be referred to as "multispecific".
  • a "bispecific" polymer comprises at least one site directed against a first antigen and at least one a second site directed against a second antigen
  • a "trispecific” is a polymer that comprises at least one binding site directed against a first antigen, at least one further binding site directed against a second antigen, and at least one further binding site directed against a third antigen, etc.
  • a bispecific polypeptide of the invention is a bivalent polypeptide (per Fab) of the invention.
  • the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise any number of binding sites directed against two or more different antigens.
  • the invention also pertains to immunoconjugates comprising the antibody described herein conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate).
  • Conjugates are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyldi
  • anti-sphingolipid antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidyl choline, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin, et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. Another active ingredient is optionally contained within the liposome.
  • Enzymes or other polypeptides can be covalently bound to the anti-sphingolipid antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger, et al., Nature 312:604-608 (1984)).
  • an antibody fragment rather than an intact antibody, to increase penetration of target tissues and cells, for example.
  • Covalent modifications of the humanized or variant anti-sphingolipid antibody are also included within the scope of this invention. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Exemplary covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615, specifically incorporated herein by reference.
  • a preferred type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • the invention also provides isolated nucleic acid encoding the humanized or variant anti-sphingolipid antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.
  • the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • the antibody may be produced by homologous recombination, e.g., as described in U.S. Pat. No. 5,204,244.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, as described, for example, in U.S. Pat. No. 5,534,615.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram- negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
  • E. coli cloning host is E. coli 294 (ATCC 31 ,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31 ,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-sphingolipid antibody-encoding vectors.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe;
  • Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii
  • Suitable host cells for the expression of glycosylated anti-sphingolipid antibodies are derived from multicellularorganisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham, et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub, et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod.
  • SV40 monkey kidney CV1 line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham, et al., J. Gen Virol. 36:59 (1977)
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for anti-sphingolipid antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the anti-sphingolipid antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellular ⁇ , in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellular ⁇ , as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter, et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and
  • phenylmethylsulfonylfluoride over about 30 min.
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human heavy chains (Lindmark, et al., J. Immunol. Meth. 62: 1 -13 (1983)). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss, et al., EMBO J. 5: 15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • Therapeutic formulations of an antibody or immune-derived moiety of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly- (methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished for instance by filtration through sterile filtration membranes.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or polyvinyl alcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ -ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • Antibodies to bioactive lipids may be used as affinity purification agents.
  • the antibodies are immobilized on a solid phase such a Sephadex resin or filter paper, using methods well known in the art.
  • the immobilized antibody is contacted with a sample containing the sphingolipid to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the sphingolipid, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, for instance between pH 3 to pH 5.0, that will release the sphingolipid from the antibody.
  • Anti-lipid antibodies may also be useful in diagnostic assays for the target lipid, e.g., detecting its expression in specific cells, tissues (such as biopsy samples), or bodily fluids. Such diagnostic methods may be useful in diagnosis of a cardiovascular or cerebrovascular disease or disorder.
  • the antibody typically will be labeled with a detectable moiety.
  • a detectable moiety Numerous labels are available which can be generally grouped into the following categories:
  • Radioisotopes such as 35 S, 14 C, 125 l, 3 H, and 131 l.
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley- Interscience, New York, N.Y., Pubs. (1991 ), for example, and radioactivity can be measured using scintillation counting.
  • Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques.
  • the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically.
  • the enzyme may alter the fluorescence or
  • chemiluminescence of the substrate involves techniques for quantifying a change in fluorescence.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase beta-galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-
  • enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • alkaline phosphatase AP
  • para-Nitrophenyl phosphate as chromogenic substrate
  • ⁇ -D-Gal ⁇ - D- galactosidase
  • a chromogenic substrate e.g., p-nitrophenyl ⁇ -D-galactosidase
  • fluorogenic substrate 4-methylumbelliferyl- ⁇ -D-galactosidase
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten e.g., digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • the antibody need not be labeled, and the presence thereof can be detected using a labeled secondary antibody which binds to the anti-lipid antibody.
  • the antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the blood or tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
  • the antibodies may also be used for in vivo diagnostic assays.
  • the antibody is labeled with a radionuclide (such as 111 ln, "Tc, 14 C, 131 l, 125 l, 3 H, 32 P, or 35 S) so that the bound target molecule can be localized using immunoscintillography.
  • a radionuclide such as 111 ln, "Tc, 14 C, 131 l, 125 l, 3 H, 32 P, or 35 S
  • kits for example, a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides the detectable chromophore or fluorophore.
  • other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • antibodies to bioactive lipids are administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ug/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 g/kg to about 20 mg/kg or more, depending on the factors mentioned above.
  • the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging.
  • the effectiveness of the antibody in preventing or treating disease may be improved by administering the antibody serially or in combination with another agent that is effective for those purposes, such as chemotherapeutic anti-cancer drugs, for example.
  • another agent that is effective for those purposes, such as chemotherapeutic anti-cancer drugs, for example.
  • Such other agents may be present in the composition being administered or may be administered separately.
  • the antibody is suitably administered serially or in combination with the other agent.
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agent in the composition is the anti-sphingolipid antibody.
  • the label on, or associated with, the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • Lpath's proprietary Immune Y2TM technology allows the generation of monoclonal antibodies against bioactive lipids, including sphingolipids.
  • Lpath's mAbs Sonepcizumab and Lpathomab also referred to as LT1009 and LT3015, targeted to S1 P and LPA, respectively
  • LT1009 and LT3015 are first-in-class examples of antibody drugs against bioactive lipids.
  • Example 1 Murine Monoclonal Antibody to S1 P (SphinqomabTM ; LT1002)
  • One type of therapeutic antibody specifically binds undesirable sphingolipids to achieve beneficial effects such as, e.g., (1) lowering the effective concentration of undesirable, toxic sphingolipids (and/or the concentration of their metabolic precursors) that would promote an undesirable effect such as a cardiotoxic, tumorigenic, or angiogenic effect; (2) to inhibit the binding of an undesirable, toxic, tumorigenic, or angiogenic sphingolipids to a cellular receptor therefore, and/or to lower the concentration of a sphingolipid that is available for binding to such a receptor.
  • beneficial effects such as, e.g., (1) lowering the effective concentration of undesirable, toxic sphingolipids (and/or the concentration of their metabolic precursors) that would promote an undesirable effect such as a cardiotoxic, tumorigenic, or angiogenic effect; (2) to inhibit the binding of an undesirable, toxic, tumorigenic, or
  • therapeutic effects include, but are not limited to, the use of anti-S1 P antibodies to lower the effective in vivo serum concentration of available S1 P, thereby blocking or at least limiting S1 P's tumorigenic and angiogenic effects and its role in post-MI heart failure, cancer, or fibrongenic diseases.
  • Thiolated S1 P was synthesized to contain a reactive group capable of cross-linking the essential structural features of S1 P to a carrier molecule such as KLH. Prior to immunization, the thio-S1 P analog was conjugated via IOA or SMCC cross-linking to protein carriers (e.g., KLH) using standard protocols.
  • SMCC is a heterobifunctional crosslinker that reacts with primary amines and sulfhydryl groups, and represents a preferred crosslinker.
  • Fusions were subsequently carried out using the spleens of these mice and myeloma cells according to established procedures.
  • the resulting 1 ,500 hybridomas were then screened by direct ELISA, yielding 287 positive hybridomas.
  • 159 hybridomas were then expanded into 24-well plates.
  • the cell- conditioned media of the expanded hybridomas were then re-screened to identify stable hybridomas capable of secreting antibodies of interest.
  • Competitive ELISAs were performed on the 60 highest titer stable hybridomas.
  • mice were injected, producing a total of 125mL of ascites.
  • the antibodies were isotyped as lgG1 kappa, and were deemed >95% pure by HPLC.
  • the antibody was prepared in 20mM sodium phosphate with 150 mM sodium chloride (pH 7.2) and stored at -70°C. This antibody is designated LT1002 or SphingomabTM .
  • the positive hybridoma clone (designated as clone 306D326.26) was deposited with the ATCC (safety deposit storage number SD-5362), and represents the first murine mAb directed against S1 P.
  • the clone also contains the variable regions of the antibody heavy and light chains that could be used for the generation of a "humanized" antibody variant, as well as the sequence information needed to construct a chimeric antibody.
  • the thiolated-S1 P-BSA was incubated at 37°C for 1 hr. at 4°C overnight in the ELISA plate wells. The plates were then washed four times with PBS (137mM NaCI, 2.68mM KCI, 10.14mM Na 2 HP0 4 , 1.76mM KH 2 P0 4 ; pH 7.4) and blocked with PBST for 1 hr. at room temperature. For the primary incubation step, 75uL of the sample (containing the S1 P to be measured), was incubated with 25uL of 0.1 ug/mL anti-S1 P mAb diluted in PBST and added to a well of the ELISA plate. Each sample was performed in triplicate wells.
  • the ELISA plates were washed four times with PBS and incubated with 100ul per well of 0.1 ug/mL HRP goat anti-mouse secondary (Jackson Immunoresearch) for 1 hr. at room temperature. Plates were then washed four times with PBS and exposed to tetramethylbenzidine (Sigma) for 1 -10 minutes. The detection reaction was stopped by the addition of an equal volume of 1 M H2SO4. Optical density of the samples was determined by measurement at 450nm using an EL- X-800 ELISA plate reader (Bio-Tech).
  • a competitive ELISA was performed as described above, except for the following alterations.
  • the primary incubation consisted of the competitor (S1 P, SPH, LPA, etc.) and a biotin-conjugated anti- Si P mAb.
  • Biotinylation of the purified monoclonal antibody was performed using the EZ-Link Sulfo-NHS- Biotinylation kit (Pierce). Biotin incorporation was determined as per kit protocol and ranged from 7 to 1 1 biotin molecules per antibody.
  • the competitor was prepared as follows: lipid stocks were sonicated and dried under argon before reconstitution in DPBS/BSA [1 mg/ml fatty acid free BSA (Calbiochem) in DPBS (Invitrogen 14040-133)]. Purified anti-S1 P mAb was diluted as necessary in PBS/0.5% Triton X-100. Competitor and antibody solutions were mixed together so to generate 3 parts competitor to 1 part antibody. A HRP-conjugated streptavidin secondary antibody (Jackson Immunoresearch) was used to generate signal.
  • Another aspect of the competitive ELISA data is that it shows that the anti-S1 P mAb was unable to distinguish the thiolated-S1 P analog from the natural S1 P that was added in the competition experiment. It also demonstrates that the antibody does not recognize any oxidation products since the analog was constructed without any double bonds.
  • the anti-S1 P mAb was also tested against natural product containing the double bond that was allowed to sit at room temperature for 48 hours. Reverse phase HPLC of the natural S1 P was performed according to methods reported previously (Deutschman, et al. (July 2003), Am Heart J., vol. 146(1):62-8), and the results showed no difference in retention time.
  • the epitope recognized by the monoclonal antibody does not involve the hydrocarbon chain in the region of the double bond of natural S1 P.
  • the epitope recognized by the monoclonal antibody is the region containing the amino alcohol on the sphingosine base backbone plus the free phosphate. If the free phosphate is linked with a choline (as is the case with SPC), then the binding was somewhat reduced. If the amino group is esterfied to a fatty acid (as is the case with C1 P), no antibody binding was observed.
  • Binding kinetics The binding kinetics of S1 P to its receptor or other moieties has, traditionally, been problematic because of the nature of lipids. Many problems have been associated with the insolubility of lipids. For BIAcore measurements, these problems were overcome by directly immobilizing S1 P to a BIAcore chip. Antibody was then flowed over the surface of the chip and alterations in optical density were measured to determine the binding characteristics of the antibody to S1 P. To circumvent the bivalent binding nature of antibodies, S1 P was coated on the chip at low densities. Additionally, the chip was coated with various densities of S1 P (7, 20, and 1000 RU) and antibody binding data was globally fit to a 1 : 1 interaction model.
  • the affinity of the monoclonal antibody to S1 P was determined to be very high, in the range of approximately 88 picomolar (pM) to 99 nM, depending on whether a monovalent or bivalent binding model was used to analyze the binding data.
  • Microtiter ELISA plates (Costar, Cat No. 3361 ) were coated with rabbit anti-mouse IgG, F(ab')2 fragment specific antibody (Jackson, 315-005-047) diluted in1 M Carbonate Buffer (pH 9.5) at 37°C for 1 h. Plates were washed with PBS and blocked with PBS/BSA/Tween-20 for 1 hr at 37°C. For the primary incubation,
  • Microtiter ELISA plates (Costar, Cat No. 3361 ) were coated with LPA-BSA diluted in 1 M Carbonate Buffer
  • mAbs The specificity of mAbs was tested in ELISA assays.
  • Microtiter plates ELISA plates (Costar, Cat No. 3361 ) were coated with 18:0 LPA-BSA diluted in 1 M Carbonate Buffer (pH 9.5) at 37 °C for 1 h. Plates were washed with PBS (137 mM NaCI, 2.68 mM KCI, 10.1 mM Na 2 HP0 4 , 1.76 mM KH 2 P0 4 ; pH 7.4) and blocked with
  • a competitive ELISA demonstrates SPHINGOMAB's specificity for S1 P compared to other bioactive lipids.
  • SPHINGOMAB demonstrated no cross-reactivity to sphingosine (SPH), the immediate metabolic precursor of S1 P or lysophosphatidic acid (LPA), an important extracellular signaling molecule that is structurally and functionally similar to S1 P.
  • SPHINGOMAB did not recognize other structurally similar lipids and metabolites, including ceramide-1 -phosphate (C1 P), dihydrosphingosine (DH-SPH), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), or sphingomyelin (SM).
  • SPHINGOMAB did cross react with dihydrosphingosine-1-phosphate (DH-S1 P) and, to a lesser extent, sphingosylphorylcholine (SPC).
  • SPHINGOMAB has been shown to significantly reduce choroidal neovascularization (CNV) and scar formation in the eye in a murine model of CNV, and inhibits cardiac scar formation in mice as well.
  • Example 5 Cloning and Characterization of the variable domains of an S1 P murine monoclonal antibody (LT1002; Sphingomab)
  • LT1002 murine monoclonal antibody
  • Sphingomab This example reports the cloning of the murine mAb against S1 P.
  • the overall strategy consisted of cloning the murine variable domains of both the light chain (VL) and the heavy chain (VH).
  • the consensus sequence of 306D VH shows that the constant region fragment is consistent with a gamma 2b isotype.
  • the murine variable domains were cloned together with the constant domain of the light chain (CL) and with the constant domain of the heavy chain (CH1 , CH2, and CH3), resulting in a chimeric antibody construct.
  • the immunoglobulin heavy chain variable region (VH) cDNA was amplified by PCR using an MHV7 primer (MHV7: 5'-ATGGRATGGAGCKGGRTCTTTMTCTT-3' [SEQ ID NO: 1 ]) in combination with a lgG2b constant region primer MHCG1/2a/2b/3 mixture (MHCG1 : 5'-CAGTGGATAGACAGATGGGGG-3' [SEQ ID NO: 2]; MHCG2a: 5'- CAGTGGATAGACCGATGGGGC-3 [SEQ ID NO: 3]; MHCG2b: 5'- CAGTGGATAGACTGATGGGGG -3' [SEQ ID NO: 4]; MHCG3: 5'- CAAGGGATAGACAGATGGGGC -3' [SEQ ID NO: 5]).
  • the product of the reaction was ligated into the pCR2.1®-TOPO® vector (Invitrogen) using the TOPO-TA cloning® kit and sequence.
  • the variable domain of the heavy chain was then amplified by PCR from this vector and inserted as a Hind III and Apa I fragment and ligated into the expression vector pG1 D200 (see U.S. patent no. 7,060,808) or pG4D200 (id.) containing the HCMVi promoter, a leader sequence, and the gamma-1 constant region to generate the plasmid pG1 D200306DVH.
  • the consensus sequence of 306D VH ( shown below) showed that the constant region fragment was consistent with a gamma 2b isotype.
  • VK immunoglobulin kappa chain variable region
  • MKV 20 primer 5'- GTCTCTGATTCTAGGGCA-3' [SEQ ID NO: 6]
  • MKC 5'- ACTGGATGGTGGGAAGATGG-3' [SEQ ID NO: 7]
  • the product of this reaction was ligated into the pCR2.1®- TOPO® vector using the TOPO-TA cloning® kit and sequence.
  • the variable domain of the light chain was then amplified by PCR and then inserted as a Bam HI and Hind III fragment into the expression vector pKN 100 (see U.S. patent no. 7,060,808) containing the HCMV promoter, a leader sequence, and the human kappa constant domain, generating plasmid pKN100306DVK.
  • the heavy and light chain plasmids pG1 D200306DVH plus pKN 100306DVK were transformed into DH4a bacteria and stocked in glycerol. Large-scale plasmid DNA was prepared as described by the manufacturer
  • EndoFree Plasmid Mega/Maxi Kit were sequenced using an ABI 3730x1 automated sequencer, which also translates the fluorescent signals into their corresponding nucleobase sequence. Primers were designed at the 5' and 3' ends so that the sequence obtained would overlap. The length of the primers was 18-24 bases, and preferably they contained 50% GC content and no predicted dimers or secondary structure.
  • the amino acid sequences for the mouse VH and VL domains from SphingomabTM are SEQ ID NOS: 8 and 9, respectively (Table 2).
  • the CDR residues are underlined in Table 2, and are shown separately below in Table 3.
  • the amino acid sequences of several chimeric antibody variable (VH and VL> domains are compared in Table 4. These variants were cloned into expression vectors behind germ line leader sequences.
  • the germ line leader sequences are underlined in Table 4 on the pATH200 (first 19 amino acids) and pATH300 sequences (first 22 amino acids).
  • the CDRs are shown in bold.
  • Amino acids that follow the C-terminus of each of the heavy and light chain sequences in Table 4 are shown in italics. These are the first few amino acids of the constant domain and not part of the variable domain.
  • pATH200 and pATH300 series numbers usually refer to a vector containing a particular variable domain variant sequence, for convenience this nomenclature may be used herein to refer to and distinguish the variant variable domains per se.
  • Table 4 Amino acid sequences of the humanized VH (pATH200 series)and VL (pATH300 series) domains from the humanized anti-S1 P antibody variants. Leaders underlined, CDRs are in bold.
  • the heavy and light chain plasmids of both pG1 D200306DVH plus pKN 100306DVK were transformed into DH4a bacteria and stocked in glycerol.
  • Large scale plasmid DNA was prepared as described by the manufacturer (Qiagen, endotoxin-free MAXIPREPTM kit Cat. No.12362).
  • plasmids were transfected into the African green monkey kidney fibroblast cell line COS 7 by electroporation (0.7ml at 10 7 cells/ml) using 10 ug of each plasmid. Transfected cells were plated in 8 ml of growth medium for 4 days. The chimeric 306DH1 x 306DVK-2 antibody was expressed at 1 ⁇ g/ml in transiently co-transfected COS cell conditioned medium. The binding of this antibody to S1 P was measured using the S1 P ELISA.
  • the expression level of the chimeric antibody was determined in a quantitative ELISA as follows. Microtiter plates (Nunc MaxiSorp immunoplate, Invitrogen) were coated with 100 ⁇ aliquots of 0.4 ⁇ / ⁇ goat anti-human IgG antibody (Sigma, St. Louis, MO) diluted in PBS and incubate overnight at 4°C. The plates were then washed three times with 200 ⁇ /well of washing buffer (1 x PBS, 0.1 % TWEEN). Aliquots of 200 ⁇ of each diluted serum sample or fusion supernatant were transferred to the toxin-coated plates and incubated for 37°C for 1 hr.
  • the goat anti-human kappa light chain peroxidase conjugate (Jackson Immuno Research) was added to each well at a 1 :5000 dilution. The reaction was carried out for 1 hr at room temperature, plates were washed 6 times with the washing buffer, and 150 ⁇ of the K-BLUE substrate (Sigma) was added to each well, incubated in the dark at room temperature for 10 min. The reaction was stopped by adding 50 ⁇ of RED STOP solution (SkyBio Ltd.) and the absorption was determined at 655 nm using a Microplater Reader 3550 (Bio- Rad Laboratories Ltd.).
  • the heavy and light chain plasmids were transformed into Top 10 E. coli (One Shot Top 10 chemically competent E.coli cells (Invitrogen, C4040-10)) and stocked in glycerol. Large scale plasmid DNA was prepared as described by the manufacturer (Qiagen, endotoxin-free MAXIPREPTM kit CatNo12362).
  • plasmids were transfected into the human embryonic kidney cell line 293F (Invitrogen) using 293fectin (Invitrogen) and using 293F-FreeStyle Media (Invitrogen) for culture.
  • Light and heavy chain plasmids were both transfected at 0.5 g/mL.
  • Transfections were performed at a cell density of 10 6 cells/mL.
  • Supernatants were collected by centrifugation at 1 100 rpm for 5 minutes at 25°C 3 days after transfection. Expression levels were quantified by quantitative ELISA (see previous examples) and varied from -0.25-0.5 g/mL for the chimeric antibody.
  • Monoclonal antibodies were purified from culture supernatants by passing culture supernatants over protein A/G columns (Pierce, Cat.No 53133) at 0,.5 mL/min.
  • Mobile phases consisted of 1X Pierce IgG binding Buffer (Cat.No 21001 ) and 0.1 M glycine pH 2.7 (Pierce, Elution Buffer, Cat.No 21004).
  • Antibody collections in 0.1 M glycine were diluted 10 % (v/v) with 1 M Phosphate Buffer, pH 8.0, to neutralize the pH.
  • IgGi collections were pooled and dialyzed exhaustively against 1X PBS (Pierce Slide-A-Lyzer Cassette, 3,500 MWCO, Cat.No 66382).
  • Eluates were concentrated using Centricon YM-3(10,000 MWCO Amicon Cat.No 4203) by centrifugation for 1 h at 2,500 rcf.
  • the antibody concentration was determined by quantitative ELISA as described above using a commercial myeloma IgGi stock solution as a standard.
  • Heavy chain types of mAbs were determined by ELISA using
  • Table 5 shows a comparative analysis of mutants with the chimeric antibody.
  • bound antibody was detected by a second antibody, specific for the mouse or human IgG, conjugated with HRP.
  • the chromogenic reaction was measured and reported as optical density (OD).
  • the concentration of the panel of antibodies was 0.1 ug/ml. No interaction of the second antibody with S1 P-coated matrix alone was detected.
  • Table 5 Comparative binding to S1 P on variants of the chimeric anti-S1 P antibody.
  • S1 P was diluted into the HBS running buffer to a concentration of 0.1 mM and injected for different lengths of time producing 2 different density S1 P surfaces (305 and 470 RU).
  • binding data for the mAb was collected using a 3-fold dilution series starting with 16.7 nM, 50.0nM, 50.0nM, 16.7 nM, and 16.7 nM for the mouse, 201308, 201309, and 207308 antibodies respectively.
  • chimeric antibody refers to a molecule comprising a heavy and/or light chain which is identical with or homologous to corresponding seguences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly, et al., supra; Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81 :6851 (1984)).
  • a chimeric antibody to S1 P was generated using the variable regions (Fv) containing the active S1 P binding regions of the murine antibody from a particular hybridoma (ATCC safety deposit storage number SD-5362) with the Fc region of a human lgG1 immunoglobulin.
  • the Fc regions contained the CL, ChL, and Ch3 domains of the human antibody.
  • chimeric antibodies could also have been generated from Fc regions of human lgG1 , lgG2, lgG3, lgG4, IgA, or IgM.
  • "humanized" antibodies can been generated by grafting the complementarity determining regions (CDRs, e.g. CDR1 -3) of the murine anti-S1 P mAb with a human antibody framework regions (e.g., Fr1 , Fr4, etc.) such as the framework regions of an IgG 1.
  • the chimeric antibody to S1 P had similar binding characteristics to the fully murine monoclonal antibody.
  • ELISAs were performed in 96-well high-binding ELISA plates (Costar) coated with 0.1 ug of chemically-synthesized, thiolated S1 P conjugated to BSA in binding buffer (33.6mM Na2C03, 100mM NaHCOs; pH 9.5). The thiolated S1 P-BSA was incubated at 37°C for 1 hr. or at 4°C overnight in the ELISA plate.
  • the preferred method of measuring either antibody titer in the serum of an immunized animal or in cell-conditioned media (for example, supernatant) of an antibody-producing cell such as a hybridoma involves coating the ELISA plate with a target ligand (e.g., a thiolated analog of S1 P, LPA, etc.) that has been covalently linked to a protein carrier such as BSA.
  • a target ligand e.g., a thiolated analog of S1 P, LPA, etc.
  • chimeric antibodies could be generated against other lipid targets such as LPA, PAF, ceramides, sulfatides, cerebrosides, cardiolipins, phosphotidylserines, phosphotidylinositols, phosphatidic acids, phosphotidylcholines, phosphatidylethanolamines, eicosinoids, and other leukotrienes, etc. Further, many of these lipids could also be glycosylated and/or acetylated, if desired.
  • Example 7 Generation and characterization of humanized anti-S1 P monoclonal antibody LT1009 (Sonepcizumab)
  • LT1002 The murine anti-S1 P monoclonal antibody 306D (LT1002; SphingomabTM), which specifically binds S1 P, has been shown to potently suppress angiogenesis and tumor growth in various animal models.
  • LT1002 was humanized using sequence identity and homology searches for human frameworks into which to graft the murine CDRs and a computer-generated model to guide some framework backmutations.
  • the humanized variants were humanized using sequence identity and homology searches for human frameworks into which to graft the murine CDRs and a computer-generated model to guide some framework backmutations.
  • Two variants, HuMAbHCLCs (LT1004) (
  • HuMAbHCcysAiaLC 3 (LT1007) and HuMAbHCc y sAiaLC 5 (LT1009) in which a free-cysteine residue in HCDR2 was replaced with alanine exhibited a binding affinity in the picomolar range.
  • All humanized variants inhibited angiogenesis in the choroid neovascularization (CNV) model of age-related macular degeneration (AMD), with HuMAbHCcysAiaLCs (LT1009) exhibiting superior stability and in vivo efficacy compared to the parent murine antibody.
  • the variant huMAbHCcysalaLCs (LT1009) was designated SonepcizumabTM.
  • variable domains of murine mAb LT1002 were humanized via CDR grafting (Winter U.S. Pat. No. 5,225,539).
  • the CDR residues were identified based on sequence hypervariability as described by Kabat et al. 1991.
  • acceptor structures were selected based on a homology search of human antibodies in the IMGT and Kabat databases using a structural alignment program (SR v7.6).
  • the initial step was to query these human heavy variable (VH) and light variable (VL) sequence databases with LT1002 VH and VL protein sequences respectively, to identify human frameworks (FR) with high sequence identity in the FR, at Vernier (Foote, J. & Winter.G. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol. Biol.
  • CDR loop structures are dependent not only on the CDR loop sequence itself, but also on the underlying framework residues (canonical residues). Therefore a human framework with matching canonical CDR structures and/or CDR lengths is likely to hold the grafted mouse CDRs in the most appropriate orientation to maintain antigen binding affinity. This was achieved for all CDRs except CDR H3, by the choice of human framework sequences. Additionally, frameworks with unusual cysteine or proline residues were excluded where possible. These calculations were performed separately for the heavy and light chain sequences. Finally, individual sequence differences, throughout the framework region, in the best matching sequences were compared.
  • the antibodies AY050707 and AJ002773 were selected as the most appropriate human framework provider for the light chain and the heavy chain respectively.
  • the AY050707 framework was described by van den Brink, et al. (Blood, 15 April 2002, Vol. 99, No. 8, pp 2828-2834) and its sequence is available via Genbank (accession no. AY050707; Homo sapiens clone WR3VL immunoglobulin light chain variable region mRNA, partial cds.; submitted Nov 13, 2001 , last revision April 8, 2002).
  • IMGT/LIGM immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences from human and other vertebrate species. This database was created in 1989 by Marie-Paule Lefranc, LIGM, adjoin, France, and has been available online since July 1995.
  • the second step was to generate a molecular model of the variable regions of LT1002 and to identify FR residues which might affect antigen binding but were not included in the group of Vernier, Canonical and Interface residues. Many structural features of the graft donor and acceptor variable domains were examined in order to better understand how various FR residues influence the conformation of the CDR loops and vice versa. Non- conserved FR residues in LT1002 that were likely to impact the CDRs were identified from the Vernier and Canonical definitions (see above) and thus several residues of the human FR were restored to the original murine amino acids (backmutated).
  • the initial denaturation was carried out at 95°C for 30 s, followed by 16 cycles of amplification: 95°C for 30 s, 55°C for60 s and 68°C for 8 min. Following temperature cycling, the final reaction was then digested with Dpnl digest at 37°C for 1 h to remove methylated parental DNA. The resultant mutant was transformed into competentXL1 -Blue E.coli and plated on LB-agar containing 50 ⁇ g/ml Ampicillin. The colonies were then checked by sequencing. Each of the mutants were then cultured in 1 liter shake flasks and purified using the EndoFree Plasmid Purification Kit from Qiagen, catalog #12362.
  • a mouse-human chimeric antibody (chMAb S1 P) was constructed by cloning the variable domains of LT1002 into a vector that contained the human constant regions of the kappa and heavy chains to allow expression of the full length antibody into mammalian cells.
  • the generation of the humanized heavy chain was the result of the graft of the Kabat CDRs 1 , 2 and 3 from LT1002 VH into the acceptor framework of AJ002773.
  • the nearest germ line gene to AJ002773 was VH5-51 , whose leader sequence was incorporated, as a leader sequence, into the humanized heavy chain variant.
  • the protein sequence of pATH200, the first humanized version of LT1002 VH, with the VH5-51 leader sequence, is shown in Table 4.
  • the generation of the humanized light chain was the result of the graft of the Kabat CDRs 1 , 2 and 3 from LT1002 VL into the acceptor framework of AY050707.
  • the nearest germ line gene to AY050707 was L11 , whose leader sequence was incorporated into the humanized light chain construct.
  • the protein sequence of pATH300 (LT1002 light chain) is shown in Table 4. Germline leader sequences are indicated by underlining in Table 4. In the case of VL, four non-conserved Vernier positions 4, 36, 49, 64 were selected for backmutation to murine residues as they are involved in supporting the structure of the CDR loops.
  • variable regions of the basic grafted versions (pATH 200 and pATH 300) and all the variants containing backmutations were cloned into expression vectors containing the human VH or VL constant regions. All the humanized variants were produced in mammalian cells under the same conditions as the chimeric (chMAb) antibody and were tested for binding to S1 P by ELISA. The yield was approximately 10-20 mg /I for the humanized variants and 0.3-0.5 mg/l for chMAb S1 P. SDS-PAGE under reducing conditions revealed two bands at 25 kDa and 50 kDa with high purity (>98%), consistent with the expected masses of the light and heavy chains. A single band was observed under non-reducing conditions with the expected mass of ⁇ 150k. chMAb was used as a standard in the humanized antibody binding assays because it contained the same variable regions as the parent mouse antibody and bore the same constant regions as the humanized antibodies and therefore could be detected using the same ELISA protocol.
  • the initial humanized antibody in which the six murine CDRs were grafted into unmutated human frameworks, did not show any detectable binding to S1 P.
  • the kappa light chain containing the 4 backmutations (Y49S, Y36F, F4V and G64S), in association with chimeric heavy chain, exhibited suboptimal binding to S1 P as measured by ELISA.
  • the incorporation of an additional mutation at position Y67 significantly improved the binding.
  • Version pATH308 which contained backmutations Y49S, Y36F, F4V, G64S and S67Y and version pATH309 which contained the backmutations Y49S, G64S and S67Y, in association with chimeric heavy chain, both generated antibodies which bound S1 P similarly to the chimeric antibody as determined by ELISA.
  • the 2 mutations Y36F and F4V were not considered necessary backmutations from the viewpoint of S1 P binding.
  • the engineering of 3 to 5 backmutations in the VL framework was required to restore activity.
  • humanization of the LT1002 VH domain required only one amino acid from the murine framework sequence whereas the murine VL framework domain, three or five murine residues had to be retained to achieve binding equivalent to the murine parent LT1002.
  • the murine anti-S1 P antibody contains a free cysteine residue in CDR2 (Cys50) of the heavy chain that could potentially cause some instability of the antibody molecule.
  • Cys50 CDR2
  • variants of pATH201 were created with substitution of the cysteine residue with alanine (huMAbHCcysalaLCs) (pATH207), glycine (huMAbHCcysalaLCs), serine (huMAbHCcysserLCs), and phenylalanine (huMAbHCcyspheLCs).
  • the variants were expressed in mammalian cells and then characterized in a panel of in vitro assays. Importantly, the expression rate of the humanized variants was significantly higher than for chMAb SI P.
  • the humanized variants were tested for specificity in a competitive ELISA assay against S1 P and several other biolipids. This assay has the added benefit to allow for epitope mapping.
  • the humanized antibody LT1009 demonstrated no cross-reactivity to sphingosine (SPH), the immediate metabolic precursor of S1 P, or LPA (lysophosphatidic acid), an important extracellular signaling molecule that is structurally and functionally similar to S1 P.
  • SPH sphingosine
  • LPA lysophosphatidic acid
  • rhuMAb S1 P did not recognize other structurally similar lipids and metabolites, including ceramide (CER), ceramide-1-phosphate (C1 P).
  • LT1009 did cross react with sphingosyl phosphocholine (SPC), a lipid in which the free phosphate group of S1 P is tied up with a choline residue.
  • SPC sphingosyl phosphocholine
  • all the humanized variants exhibited a specificity profile comparable to the mouse antibody.
  • Binding affinity Biacore measurements of IgG binding to a S1 P coated chip showed that the variants LT1004 or LT1006 exhibited binding affinity in the low nanomolar range similar to chMAb S1 P.
  • the humanized variants LT1007 and LT1009 in which the cysteine residue was replaced with alanine exhibited a binding affinity in the picomolar range similar to the murine parent LT1002 (SphingomabTM).
  • SphingomabTM murine parent LT1002
  • the humanized variants were tested for stability after challenge at high temperature. The approximate midpoints of the thermal unfolding transitions (7M) were determined for every humanized variant by subjecting the supernatants to temperatures ranging from 60 to 74°C. These temperatures were chosen based on the denaturation profile observed for the murine antibody molecule after thermochallenging between a broad range of temperatures between 50 and 80°C. The binding properties of each variant were determined before and after thermochallenge. The murine antibody exhibited a 7 of 65°C.
  • the variant huMAbHCcysalaLCs (LT1009) exhibited superior 7M compared to all other variants. Table 6 shows the lead humanized candidates and their characteristics.
  • LT1009 includes three complementarity determining regions (each a "CDR") in each of the two light chain polypeptides and each of the two heavy chain polypeptides that comprise each antibody molecule.
  • CDR complementarity determining regions
  • the amino acid sequences for each of these six CDRs is provided immediately below ("VI” designates the variable region of the immunoglobulin light chain, whereas "VH” designates the variable region of the immunoglobulin heavy chain):
  • CDR2 VL EGNILRP [SEQ ID NO: 11 ]
  • CDR2 VH AISPRHDITKYNEMFRG [SEQ ID NO: 18]
  • LT1009 a recombinant humanized monoclonal antibody that binds with high affinity to the bioactive lipid sphingosine-1 -phosphate (S1 P).
  • S1 P bioactive lipid sphingosine-1 -phosphate
  • LT1009 is a full-length IgGl k isotype antibody composed of two identical light chains and two identical heavy chains with a total molecular weight of approximately 150kDa. The heavy chain contains an N-linked glycosylation site.
  • the nature of the oligosaccharide structure has not yet been determined but is anticipated to be a complex biantennary structure with a core fucose. The nature of the glycoform that will be predominant is not known at this stage.
  • LT1009 was originally derived from a murine monoclonal antibody (LT1002; SphingomabTM) that was produced using hybridomas generated from mice immunized with S1 P.
  • the humanization of the murine antibody involved the insertion of the six murine CDRs in place of those of a human antibody framework selected for its structure similarity to the murine parent antibody.
  • a series of substitutions were made in the framework to engineer the humanized antibody. These substitutions are called back mutations and replace human with murine residues that are play a significant role in the interaction of the antibody with the antigen.
  • the final humanized version contains one murine back mutation in the human framework of variable domain of the heavy chain and five murine back mutations in the human framework of the variable domain of the light chain.
  • one residue present in the CDR #2 of the heavy chain was substituted to an alanine residue. This substitution was shown to increase stability and potency of the antibody molecule.
  • the humanized variable domains (both heavy and light chain) were cloned into the Lonza's GS gene expression system to generate the plasmid pATH1009.
  • the Lonza GS expression system consists of an expression vector carrying the constant domains of the antibody genes and the selectable marker glutamine synthetase (GS).
  • GS is the enzyme responsible for the biosynthesis of glutamine from glutamate and ammonia.
  • the vector carrying both the antibody genes and the selectable marker is transfected into a proprietary Chinese hamster ovary host cell line (CHOK1 SV) adapted for growth in serum-free medium and provides sufficient glutamine for the cell to survive without exogenous glutamine.
  • CHOK1 SV Chinese hamster ovary host cell line
  • GS inhibitor methionine sulphoximine (MSX)
  • MSX methionine sulphoximine
  • the latter leader sequences can be seen as 19 amino acids beginning "mewswv,” at the N- terminus of the LT1009 heavy chain (SEQ ID NO: 19 and 24), and the LC leader is 20 amino acids beginning "msvpt" (as shown at the N- terminus of SEQ ID NO: 20 and 26).
  • LH 1 275 is the name given to the lead clone of the LH1 CHO cell line containing the pATH1009 vector selected for the creation of a Master Cell Bank (MCB) for production of all lots of LT1009 antibody product. Material for toxicology studies and clinical development were then produced for tox and clinical development.
  • ATCC deposits E. coli StB12 containing the pATH 1009 plasmid has been deposited with the American Type Culture Collection (deposit number PTA-8421 ).
  • CHO cell line LH1 275, which contains the pATH1009 vector has also been deposited with the American Type Culture Collection (deposit number PTA-8422).
  • nucleotide and amino acid sequences for the heavy and light chain polypeptides of LT1009 are listed immediately below. Leader sequences (from Lonza GS expression vector) are underlined; CDRs are in bold.
  • nucleotide sequences encoding the heavy and light chain variable domains are listed immediately below.
  • Leader sequences from Lonza GS expression vector
  • sequences preceding the leader are Hindll I cut site (aagctt) and Kozak consensus sequence (gccgccacc), which plays a major role in the initiation of translation process;
  • CDRs are in bold:
  • cDNA full length HC nucleotide sequence [SEQ ID NO: 23] with CDRs in bold and leader region underlined; hinge region is in italics. Sequences preceding the leader are Hindi 11 cut site (aagctt) and Kozak sequence (gccgccacc):
  • LT1009 LC full length nucleotide sequence [SEQ ID NO: 25] with leader underlined and CDRs in bold; sequences preceding the leader are Hindi 11 cut site (aagctt) and Kozak sequence (gccgccacc):
  • LT1009 full length heavy chain amino acid sequence without leader (and without preceding nuclease cleavage site and Kozak sequence) and including hinge (underlined) (SEQ ID NO: 31 ) :
  • nucleotide sequences (without leaders or preceding nuclease or Kozak sites) are below. It will be understood that due to the degeneracy of the genetic code, alternative nucleotide sequences also may encode virtually any given amino acid sequence.
  • LT1009 full length heavy chain nucleotide (cDNA) sequence [SEQ ID NO: 33]:
  • the C-terminal lysine on the LT1009 heavy chain may not always be present on the mature heavy chain protein. While the nucleotide and amino acid sequences for LT1009 heavy chain reveal a lysine as the last (most C- terminal) amino acid residue of the protein, LT1009 when expressed, for example, in CHO cell clone LH1 275, does not contain the C-terminal lysine. This is shown by peptide mapping and, while not wishing to be bound by theory, is believed to result from posttranslational modification of the protein in mammalian systems. Again not wishing to be bound by theory, it is believed that in other expression systems, particularly nonmammalian systems, the C-terminal lysine is present on the mature LT1009 heavy chain.
  • LT1009 heavy chain amino acid sequence as expressed in CHO cells i.e., without leaders and without the C-terminal lysine
  • CDRs are in bold, hinge in italics
  • SEQ ID NO: 36 An example of a nucleotide sequence that could encode this amino acid sequence is shown below as SEQ ID NO: 36. It will be understood that, due to the degeneracy of the genetic code, multiple nucleotide sequences may encode the same amino acid sequence, and for this reason, these and other nucleotide sequences shown herein as encoding amino acid sequences are recognized to be for purposes of exemplification. CDRs are shown in bold and the hinge region is in italics:
  • Example 9 In vivo efficacy of murine mAb (Sphingomab) vs, humanized mAb (Sonepcizumab)
  • Sphingomab (LT1002) and Sonepcizumab (LT1009) were compared in an assortment of animal and in vitro models as disclosed in US patent application serial no. 1 1/924,890 (attorney docket no. LPT-3010-UT), filed on October 26, 2007, entitled “Compositions and Methods for Binding Sphingosine-1-Phosphate,” which is incorporated herein in its entirety.
  • the humanized antibody variants and the murine antibody were compared for their ability to inhibit neovascularization in the CNV animal model of AMD.
  • Three of the humanized variants inhibited angiogenesis essentially equivalently to the murine antibody as assessed by measurement of CNV area.
  • Both the murine mAb LT1002 (SphingomabTM) and the humanized mAb LT1009 (SonepcizumabTM) significantly decreased lesion size in this mouse model of CNV. All mAbs tested showed approximately 80-98% reduction of lesion size, which was significant (p ⁇ 0.001 vs. saline) in all cases.
  • LT1007 and LT1009 also showed significant inhibition (p ⁇ 0.05) compared to non-specific antibody control.
  • LT1009 Percent inhibition of lesion size was approximately 80% for LT1002 (murine), 82% for LT1004 (humanized), 81 % for LT1006 and 99% for LT1009. Thus, LT1009 was most active in this in vivo model of neovascularization.
  • LT1009 was also effective in reducing the development of retinal neovascularization in murine model of retinopathy of prematurity [US patent application serial no. 11/924,890 (attorney docket no. LPT-3010-UT), filed on October 26, 2007, entitled “Compositions and Methods for Binding Sphingosine-1-Phosphate,” which is incorporated herein in its entirety].
  • Intravitreal administration of LT1009 (5.0 resulted in a nearly 4-fold reduction in retinal neovascularization compared to saline control.
  • LT1009 also blocked nearly 80% of VEGF-induced Angiogenesis in a Matrigel plug assay. This reduction is significant (p ⁇ 0.05 compared to VEGF alone) and confirms the potent anti-angiogenic activity of LT1009 and strongly suggest that LT1009 is capable of significantly inhibiting VEGF induced angiogenesis. This finding is consistent with data from Lpath's oncology program whereby that S1 P antibody reduced serum levels of several angiogenic factors, including VEGF, in a murine orthotopic breast cancer model.
  • LT1009 also significantly reduces choroidal neovascularization and vascular leakage following laser rupture of Bruch's membrane.
  • the area of choroidal neovascularization (stained by PECAM-1 ) was approximately 0.015mm 2 for animals treated with LT1009 and approximately 0.03 mm 2 for saline-treated control animals. This is a 50% reduction in neovascularization (p-0.018).
  • the area of leakage from choroidal neovascularization was approximately 0.125 mm 2 for animals treated with LT1009 and approximately 0.2 mm 2 for saline- treated control animals. This is approximately a 38% reduction (p-0.017) in blood vessel leakage.
  • Example 10 Anti-S1 P antibodies LT1002 and LT1009 decrease lymphocyte counts when administered to c57/b!6 mice or cynomoloqous monkeys, respectively
  • the purpose of this study was to determine the toxicity and toxicokinetic profile of the murine anti-S1 P monoclonal antibody, LT1002, following daily administration to C57/BL6 mice.
  • the study was conducted by an independent contract laboratory organization, LAB Research, Inc.
  • the LT1002 dosing solutions were administered for 28 consecutive days to animals in each group by bolus intravenous injection via the tail vein (Days 1 -14) and then by bolus intraperitoneal injection (Days 15-28), over a period of approximately 0.5-1.0 minute.
  • lymphocyte counts were significantly (p ⁇ 0.001) reduced in all LT1002-treated dosing groups with a weak dose-response effect.
  • LT1002 caused substantial reductions in lymphocyte counts correlated with reductions in axonal degeneration, demyelination and infiltration of inflammatory cells.
  • LT1009 was administered by 30-minute intravenous infusion every third day for 28 days (10 doses).
  • blood samples were collected from all animals at several timepoints on Days 1 , 16 and 28.
  • blood was collected from recovery animals 48, 72, 144 and 240 hours following the end of the last dose.
  • Parameters monitored during this study included mortality, clinical signs, body weight, qualitative evaluation of the food consumption, ophthalmology, electrocardiography, and clinical pathology (hematology, clinical chemistry, coagulation and urinalysis).
  • Blood samples were also collected for immunophenotyping assessments, at pre- treatment, on the last day of treatment, and on days 35, 42 and at the end of the recovery period. At termination, a macroscopic examination was performed and selected organs were weighed. Histological evaluation of tissues was conducted on all animals.
  • NOTEL No Observed Toxic Effect Level
  • lymphocyte counts are consistent with the scientific literature suggesting that S1 P is involved in lymphocyte trafficking and egress from primary and secondary lymphoid tissue into the peripheral circulation. Consequently in humans, it is possible that changes in lymphocyte counts could be a pharmocodynamic marker that could indicate in vivo biological activity of the humanized LT1009 drug candidate formulated for systemic administration. Further, it is possible that systemic administration of LT1009 could be used to alter lymphocyte trafficking with resulting lymphopenia necessary for the treatment of multiple sclerosis or other disorders which might benefit from reduced peripheral blood lymphocyte counts.
  • S1 P anti-sphingosine-1 -phosphate
  • LT1009 e.g., from the transfected CHO cell line LH 1 275 (ATCC Accession No. PTA-8422)
  • intracellular pools of S1 P can be released into the media as a result of normal cellular signaling and/or as a consequence of cell rupture after cell death.
  • the LT1009 antibody expressed in the cell-conditioned medium (supernatant) is able to bind to this S1 P.
  • more S1 P may be released and accumulate in the supernatant as a complex with LT1009.
  • LT1009 antibody preparations may contain in excess of 0.5 moles (50 mole percent, mol%) of S1 P per mole of antibody.
  • steps must be taken in both upstream and downstream processing to minimize the amount of S1 P in the crude harvest and to promote removal of that S1 P during purification.
  • the S1 P concentrations in various preparations of the LT1009 antibody were measured at WindRose Analytica by RP-HPLC-MS-MS method. Mass spectrometry is rapid and sensitive and, if applied properly, can quantify picogram amounts of analyte.
  • the approach taken in this analytical method is to introduce the S1 P into an electrospray mass spectrometer source by reversed phase liquid chromatography (RPC).
  • RPC reversed phase liquid chromatography
  • the RPC step separates the S1 P from protein, salts and other contaminants.
  • the results verify sample identity in three dimensions of analysis: RPC retention time, parent ion m/z of 380, and daughter ion m/z of 264. It is unlikely that any other compound would satisfy all three of these criteria.
  • the MS-MS step maximizes signal-to-noise and therefore increases sensitivity significantly. Since there is no extraction step required there is no need for an internal standard. Additionally, the direct injection of sample into the HPLC-MS increases recovery and sensitivity and decreases complexity and analysis time.
  • the concentration of S1 P in extracts of selected antibody preparations was determined using a S1 P-quantification ELISA.
  • a 4-fold excess of 1 :2 chloroform:methanol was added to 1 mg/ml antibody samples to extract the S1 P.
  • the aqueous/organic solution was extensively vortexed and sonicated to disrupt antibody-lipid complexes and incubated on ice. After centrifugation, the soluble fraction was evaporated using a speed-vac, and the dried S1 P was resuspened in delipidated human serum.
  • the S1 P concentration in the resuspended sample was determined by a competitive ELISA using an anti-S1 P antibody and a S1 P-coating conjugate.
  • the coating conjugate a covalently linked S1 P-BSA
  • S1 P-BSA was prepared by coupling a chemically synthesized thiolated S1 P with maleimide-activated BSA.
  • mono-layer S1 P was solubilized in 1 % BSA in PBS (137 mM NaCI, 2.68 mM KCI, 10.1 mM Na2HP04, 1.76 mM KH2P04; pH 7.4) by sonication to obtain 10 uM S1 P (S1 P-BSA complex).
  • the S1 P-BSA complex solution was further diluted with delipidated human serum to appropriate concentrations (up to 2 uM).
  • Microtiter ELISA plates (Costar, high-binding plate) were coated with S1 P- coating material diluted in 0.1 M sodium carbonate buffer (pH 9.5) at 37 °C for 1 hour. Plates were washed with PBS and blocked with PBS/1 % BSA/0.1 % Tween-20 for 1 hr at room temperature. For the primary incubation, 0.4 ug/mL biotin-labeled anti-S1 P antibody, designated amounts of S1 P-BSA complex and samples to be tested were added to wells of the ELISA plates.
  • culturing the CHO cells in serum-free medium is essential because serum contains contaminating S1 P that could add to that produced by the CHO cells themselves.
  • serum-free medium In addition to use of serum-free medium, harvesting the antibody from the bioreactor prior to extensive cell death will prevent intracellular pools of S1 P to be released into the medium.
  • initiating the downstream processing immediately after harvest minimizes the time the LT1009 spends in the presence of S1 P and the amount of lipid carried over to the final preparation.
  • significant S1 P often remains in the crude harvest which typically ranges between 0.1 - 0.2 molar ratio (10-20 mol%) of bound S1 P per mol of antibody .
  • Lpath developed downstream methods to remove lipids from antibody preparations in order to generate LT1009 material with very low S1 P carry-over levels. These methods (described immediately below) were developed by Lpath and transferred to Laureate Pharma, Inc. to incorporate into their processing methods. As a result, the final drug product produced by Laureate has very low levels of bound S1 P ( ⁇ 0.4 mol % measured by HPLC-MS-MS).
  • Lpath developed a method that involved premixing of two volumes of crude LT1009 antibody harvest, produced from CHO cells bioreactor campaign, with one volume of Protein A IgG binding buffer ("Pierce binding buffer,” Pierce Protein Research Products, Thermo Fisher Scientific, Rockford IL), containing 50 mM Potassium Phosphate, 1 M NaCI, 2 mM EDTA and 5% glycerol, pH 8.0.
  • Protein A column was equilibrated with Pierce binding buffer, loaded with premixed crude harvest and washed with 10 column volumes of the same binding buffer.
  • the resulting purified LT1009 contained 2-fold less mole percent of S1 P as judged by the S1 P-quantification ELISA.
  • a metal chelator e.g., EDTA
  • EDTA metal chelator
  • titration of LT1009 with EDTA which chelates divalent metal cations, abrogates S1 P binding.
  • the ability of EDTA to dissociate S1 P from LT1009 is believed to facilitate removal of S1 P during purification of LT1009.
  • Addition of 2 mM EDTA in the binding and washing buffers effectively lowered the S1 P carryover twofold in the eluted antibody fractions.
  • S1 P levels in this study are relatively low initially, and including EDTA should produce greater reduction in lipid carryover in samples with higher initial S1 P levels.
  • other metal chelators such as EGTA, histidine, malate and phytochelatin may be useful in dissociating S1 P from the antibody.
  • EGTA and EDTA are presently preferred divalent metal chelators for separating S1 P from anti-S1 P antibodies. Based on these results, a new high salt buffer was developed by Lpath that was comparable in pH and conductivity to the Pierce buffer, and the new premixing step was incorporated in the LT1009 manufacturing process.
  • the dialyzed solution is passed through a material, such as C8 silica resin (e.g., SepPak cartridges, Waters, Cat no WAT036775), that binds the lipid and facilitates separation of the protein free of lipid.
  • C8 silica resin e.g., SepPak cartridges, Waters, Cat no WAT036775
  • the free lipid irreversibly binds the hydrophobic resin (in the case of C8 silica resin) while the antibody flows through without significant loss (-90% recovery).
  • Most of the lipid can be removed with one pass through the cartridge, but modest gains in lipid removal can be achieved with an additional pass (Table 7, below).
  • EDTA metal chelation and pHiL methods described above can easily be incorporated into a single purification procedure.
  • EDTA is compatible with most buffers and does not adversely affect antibody stability, solubility or protein-A binding.
  • washing the bound IgG with copious amount of EDTA-containing buffer will remove a portion of the S1 P from the S1 P-LT1009 complex as well as potentially dissociate other metal-dependant antigens-antibody complexes. If the EDTA wash does not sufficiently remove the lipid, the eluate from the protein-A column can be treated using the pHiL method. Elution of bound IgG from protein-A is typically achieved using low pH buffers (pH ⁇ 3.0).
  • the sample can simply be applied to the C8 silica resin to remove the lipid. If necessary, the pH can be easily adjusted prior to applying it to the resin.
  • Example 12 Formulations containing the humanized monoclonal antibody LT1009
  • LT1009 is an engineered full-length IgGl k isotype antibody that contains two identical light chains and two identical heavy chains, and has a total molecular weight of about 150 kDa.
  • the complementarity determining regions (CDRs) of the light and heavy chains were derived from a murine monoclonal antibody generated against S1 P, and further include a Cys to Ala substitution in one of the CDRs.
  • human framework regions contribute approximately 95% of the total amino acid sequences in the antibody, which binds S1 P with high affinity and specificity.
  • the purpose of the testing described in this example was to develop one or more preferred formulations suitable for systemic administration that are capable of maintaining stability and bioactivity of LT1009 over time.
  • maintenance of molecular conformation, and hence stability is dependent at least in part on the molecular environment of the protein and on storage conditions.
  • Preferred formulations should not only stabilize the antibody, but also be tolerated by patients when injected.
  • the various formulations tested included either 1 1 mg/mL or 42 mg/mL of LT1009, as well as different pH, salt, and nonionic surfactant concentrations. Additionally, three different storage temperatures (5°C, 25°C, and 40°C) were also examined (representing actual, accelerated, and temperature stress conditions, respectively).
  • Stability was assessed using representative samples taken from the various formulations at five different time points: at study initiation and after two weeks, 1 month, 2 months, and 3 months. At each time point, testing involved visual inspection, syringeability (by pulling through a 30- gauge needle), and size exclusion high performance liquid chromatography (SE-HPLC). Circular dichroism (CD) spectroscopy was also used to assess protein stability since above a certain temperature, proteins undergo denaturation, followed by some degree of aggregate formation. The observed transition is referred to as an apparent denaturation or "melting" temperature (T m ) and indicate the relative stability of a protein.
  • T m apparent denaturation or "melting" temperature
  • the formulation samples (-0.6 mL each) were generated from an aqueous stock solution containing 42 mg/mL LT1009 in 24 mM sodium phosphate, 148 mM NaCI, pH 6.5.
  • Samples containing 1 1 mg/mL LT1009 were prepared by diluting a volume of aqueous stock solution to the desired concentration using a 24 mM sodium phosphate, 148 mM NaCI, pH 6.5, solution.
  • the pH of each concentration of LT1009 (11 mg/mL and 42 mg/mL) was adjusted to 6.0 or 7.0 with 0.1 M HCI or O.1 M NaOH, respectively, from the original 6.5 value.
  • the vials Prior to placement into stability chambers, the vials were briefly stored at 2-8°C; thereafter, they were placed upright in a stability chamber adjusted to one of three specified storage conditions: 40°C( ⁇ 2°C)/75%( ⁇ 5%) relative humidity (RH); 25°C( ⁇ 2°C)/60%( ⁇ 5%) RH; or 5°C( ⁇ 3°C)/ambient RH.
  • RH relative humidity
  • 5°C( ⁇ 3°C)/ambient RH A summary of the formulation variables tested appears in Table 8, below.
  • Circular dichroism spectroscopy was performed separately from the formulation studies.
  • An Aviv 202 CD spectrophotometer was used to perform these analyses.
  • Near UV CD spectra were collected from 400 nm to 250 nm. In this region, the disulfides and aromatic side chains contribute to the CD signals. In the far UV wavelength region (250-190 nm), the spectra are dominated by the peptide backbone.
  • Thermal denaturation curves were generated by monitoring at 205 nm, a wavelength commonly used for b-sheet proteins. Data was collected using 0.1 mg/ml samples with heating from 25°C to 85°C. Data were collected in 1 °C increments. The total time for such a denaturation scan was between 70 and 90 minutes. The averaging time was 2 seconds. 3. Results and Discussion
  • LT1009 adopts a well-defined tertiary structure in aqueous solution, with well-ordered environments around both Tyr and Trp residues. It also appeared that at least some of the disulfides in antibody molecules experience some degree of bond strain, although this is not uncommon when both intra- and inter-chain disulfides are present.
  • the secondary structure of LT1009 was found to be unremarkable, and exhibited a far UV CD spectrum consistent with ⁇ -sheet structure. The observed transition is referred to as an apparent denaturation or "melting" temperature (T m ). Upon heating, LT1009 displayed an apparent Tm of approximately 73°C at pH 7.2.
  • a preferred aqueous LT1009 formulation is one having 24 mM phosphate, 450 mM NaCI, 200 ppm polysorbate-80, pH 6.1.
  • the relatively high tonicity of this formulation should not pose a problem for systemic applications since the drug product will likely be diluted by injection into iv-bags containing a larger volume of PBS prior to administration to a patient.
  • Example 13 Production and purification of anti-S1 P and anti-LPA antibodies
  • a stable CHO cell line that produces >0.5 mg/L of anti-S1 P antibody is used. While maintaining a viability of >95%, cells are seeded at a density of 0.4 x 10 6 cells/ml into 1 liter shaker flasks with 500 ml of CD-CHO medium (Invitrogen, San Diego, cat. No. 10743-029) containing 25 ⁇ L-methionine sulphoximine (Sigma, St. Louis MO, Cat. No. M5379). Cells are grown in an atmosphere of 7.5% CO 2 for ten days or until the viability dropped to 45-50%.
  • CD-CHO medium Invitrogen, San Diego, cat. No. 10743-029
  • 25 ⁇ L-methionine sulphoximine Sigma, St. Louis MO, Cat. No. M5379
  • Supernatants are then harvested by centrifugation at 1500 rpm for 10 minutes and sterile-filtered through a 0.22 micron filter system (Corning, Lowell MA, cat no. 431098).
  • the clarified supernatants are concentrated tenfold using a Labscale Tangential Flow Filtration system installed with a Pellicon XL Biomax 50 cartridge (Millipore, Billerica MA, Cat. no PXB050A50) according to manufacturer's protocol assuring that all tubing and vessels were cleaned prior to use with 0.5% NaOH and thoroughly rinsed with DNase and RNase-free distilled water (Invitrogen, San Diego CA, cat no. 10977-015).
  • Fractions with a absorption at 280 nm (A280) of greater than 0.1 were pooled and concentrated using an Amicon stirred cell equipped with a 50 kDa molecular weight cut off (MWCO) filter (Millipore, Cat No PBQK07610).
  • the concentrated antibody was extensively dialyzed against 1X PBS (Cellgro, Manassas VA, Cat No 21 -040), filtered through a 0.22 uM syringe-driven filter unit (Millipore, Cat No SLGP033RS) and stored at 4°C.
  • Anti-LPA antibody is produced and purified in substantially the same manner as the S1 P antibody.
  • Example 14 Isolation of Fab Fragments from Anti-S1 P and Anti-LPA Monoclonal Antibodies.
  • Fab fragment consisting of both variable domains and the Ck and Ch1 constant domains from the Fc domain, which contains a pair of Ch2 and Ch3 domains.
  • the Fab fragment retains one entire variable region and, therefore, serves as a useful tool for biochemical characterization of a 1 : 1 interaction between the antibody and epitope.
  • the Fab fragment is generally an excellent platform for structure studies via single crystal x-ray diffraction.
  • Purified, intact anti-S1 P IgG was digested with activated papain (incubated 10 mg/ml papain in 5.5 mM cysteine-HCL, 1 mM EDTA, 70 ⁇ 2-mercaptoethanol for 0.5 hours at 37 °C) in digestion buffer (100: 1
  • the anti-LPA Fab fragment is prepared similarly.
  • the concentration of the isolated Fab fragment was calculated from the A280 value using an extinction coefficient of 1.4 ml/mg.
  • a 5-fold molar excess of 1 mM S1 P (Avanti, Cat No 860429P) suspended in methanol was dried in 13x100 mm borosilicate glass tubes by holding in a low vacuum for three hours.
  • the lipids were resuspended in 500 ⁇ of purifed anti-S1 P Fab by pipetting and filtered through a 0.22 ⁇ Costar Spin-X centrifugal cellulose acetate filter (Corning, Cat No 8160).
  • the complex is concentrated to approximately 12 mg/ml using the centriprep-10 centrifugal concentrator (Millipore).
  • the concentrated Fab/lipid complexes were stored at 4 °C.
  • Fab/LPA complexes are prepared using LPA (Avanti, Cat No 857120X) and isolated LPA Fab.
  • Example 16 Crystallization of the Fab/lipid complexes.
  • initial crystallization conditions were determined by the use of a sparse matrix screen (Hampton Research, Aliso Viejo CA) and the hanging drop vapor diffusion method.
  • the Fab/S1 P complex single crystals suitable for diffraction studies were grown at room temperature. 1 microliter of 12 mg/ml Fab/S1 P complex was mixed with 1 microliter of reservoir solution containing 22% (w/v) polyethylene glycol 3350, 100 mM MgS04, 100 mM sodium citrate (pH 6.0) and 10% (v/v) ethylene glycol and sealed over 1 milliliter of reservoir solution. Crystals grew to a final size of 0.2 x 0.2 x 0.2 mm in two days. The crystals were harvested from the crystallization drop with nylon loops and flash cooled directly in liquid nitrogen.
  • X-ray crystallography is a powerful tool that enables researchers to visualize the mechanisms of molecular recognition at the atomic level. This information is extremely valuable to understand the mode of action for therapeutic antibodies as well as engineer antibodies for enhanced binding characteristics or novel antigen specificities.
  • a combination of x-ray crystallography with innovative biochemical methods is used herein to study two monoclonal antibodies that specifically recognize two bioactive lipids. In addition, these techniques will be used to engineer antibodies with novel specificities for other lipids. This technology grants researchers new tools for studying lipid pathways, metabolism and signaling and hopefully arms clinicians with powerful new weapons against lipid- based pathologies. As lipidomics emerges as an important field in medicine and as more bioactive lipids become implicated in human disease, antibodies that recognize lipids and other non-proteinaceous targets will likely play a significant role in biomedical research.
  • X-rays were produced by an RU-H3R rotating anode x-ray generator functioning at 100 mA and 50 kV with Osmic Blue confocal optics (Rigaku). Data indexing and scaling were carried out using HKL2000. Otwinowski.Z. and W. Minor (1997) Methods Enzymol. 276:307-326. Cryo-cooled crystals were tested on the San Diego State University Macromolecular X-ray Crystallography Facility and were observed to diffract x-rays to beyond 2.7 A resolution ( Figure 1 c). The data coordinates for this crystal are provided in U.S. Application Serial No. 12/631 ,784, filed 4 December 2009, the contents of which are hereby incorporated by reference in their entirety.
  • the LT1009 Fab fragment structure exhibits the standard immunoglobulin domain folds.
  • the S1 P ligand In its complex-bound state the S1 P ligand adopts a slightly curled conformation as it perfectly fits the refined electron density with near ideal stereochemistry, bond lengths, and angles.
  • the most striking feature of the S1 P:LT1009Fab complex structure is the extent (approx. 70%) to which the ligand is almost completely engulfed by its antibody.
  • the exposed portions include most of the phosphate head group and the terminal carbon atom of the hydrocarbon tail, which was the point of attachment when the derivatized S1 P hapten was prepared for immunization.
  • the LT1009 Fab intimately contacts nearly all of the S1 P atoms.
  • the metal atoms are coordinated by the side chains of four aspartic acid residues, including two bivalent interactions from aspartic acids D30 and D32 of the CDR L1.
  • Figure 3a when either unbound whole LT1009 IgG or Fab fragments were analyzed by ICP, the amount of detected Ca 2+ corresponded to less than one metal ion per binding site.
  • the coordination sphere is made up of both amino acid side chains from the LT1009 light chain and the phosphate group of S1 P.
  • Both Ca 2+ are octahedrally coordinated through one terminal syn ⁇ 1 bond from either aspartic acid D31 in the antibody light chain to one calcium (designated Ca1 ) and from aspartic acid D92 in the antibody light chain to the other calcium (designated Ca2).
  • Two bridging interactions with the side chains of aspartic acids at positions 30 and 32 in the light chain provide another pair of bonds to each metal ion.
  • Two separate pairs of water molecules occupy symmetrically similar positions about the ions providing the fourth and fifth ligands.
  • an oxygen atom from the phosphate head group of S1 P completes the coordination of both ions via a bridge. This ligand arrangement allows the two Ca 2+ to come within 3.81 A of each other without any linking atoms directly between them.
  • the remaining contacts between S1 P and the LT1009 Fab are hydrophobic in nature. These include amino acid residues leucine L94 and phenylalanine F96 from the light chain and threonine T33, histidine H35, alanine A50, serine S52, histidine H54, isoleucine I56, lysine K58, phenylalanine F97, tyrosine Y98, threonine T100A and tryptophan W100C from the antibody heavy chain (Kabat numbering). Although some of these contain polar or charged side chains, each contributes to create a network of closely packed carbon atoms and create a hydrophobic channel that surrounds the lipid aliphatic tail.
  • the CDR-H3 loop of the heavy chain appears to fold over the top of the lipid upon S1 P binding to the antibody, with tyrosine Y98 thought to function as a "gate” or "latch” that passes over the bound S1 P molecule and fastens to the side chains of leucine at position 94 of the light chain and lysine at position 58 of the heavy chain through van der Waals forces.
  • LT1009 Fab binding to an immobilized S1 P derivative was measured using Surface Plasmon Resonance (SPR) in the presence and absence of calcium.
  • SPR Surface Plasmon Resonance
  • LT1009 Fab was passed over C18 thiolated S1 P (S1 P-SH) coated on a ProteOn GLM sensor chip using sulfo-MBS coupling.
  • KD equilibrium dissociation binding constant
  • Lpath's Immune Y2 technology provides a powerful, sensitive and robust method for rapidly analyzing the lipid-binding characteristics of many antibody variants. This platform is disclosed in Lpath's patent applications US20070281320 (attorney docket no. LPT-3100-UT1 ), US20080138334 (attorney docket no. LPT-3100-UT2) and US20080090303A1 (attorney docket no. LPT-3100-UT3), all of which are herein incorporated in their entirety for all purposes.
  • the Immune Y2 platform relies upon a derivatized bioactive lipid for immunogen preparation and for detection and characterization methods.
  • the highly reactive sulfhydryl group covalently attached to the terminal carbon of the aliphatic lipid chain enables the thiolated S1 P and LPA (including C12 and C18 isoforms) to be directly coupled to a surface plasma resonance (SPR) chip or conjugated with a protein (e.g., albumin) to serve as the coating material for enzyme-linked immunosorbent assays (ELISA).
  • SPR surface plasma resonance
  • a protein e.g., albumin
  • ELISA enzyme-linked immunosorbent assays
  • This competition ELISA measures the crossreactivity of either wild type (WT) or mutant antibodies to a variety of structurally related lipids. ELISAS are described in Examples 1 and 2, and below. The ELISA results confirm that the anti-S1 P and anti-LPA antibodies LT1009 and LT3015 are highly specific for their lipid targets. The direct-binding ELISA, competition ELISA and SPR methods are used to determine the effect of mutating amino acids in the variable domains of the anti-S1 P and anti-LPA antibodies on the ability of those variants to recognize and bind lipids.
  • the resultant mutants are transformed into competent XL1 -Blue E.coli and plated on LB-agar containing 50 ⁇ g/ml ampicillin. The colonies are screened by DNA sequencing to confirm the presence of the mutation. Each mutant is cultured in 1 liter shake flasks and purified using the EndoFree Plasmid Purification Kit from Qiagen, Valencia CA (Cat. No 12362).
  • b. Expression and Production of Mutant Antibodies in Mammalian Cells Purified plasmids containing the mutations are transfected into the human embryonic kidney cell line 293F using 293fectin and using 293F-FreeStyle Media (Invitrogen) for culture.
  • Light and heavy chain plasmids are both transfected at 0.5 ⁇ g/mL following manufacturer's instructions. The purity and structurally integrity is judged using SDS-PAGE. Under reducing conditions, the expected masses of the heavy and light chains are 25 kDa and 50 kDa, while a single band is observed under non-reducing conditions with the expected mass of ⁇ 150 kDa.
  • Quantitative ELISA Goat-anti human IgG-Fc antibody (Bethyl, Montgomery TX.Cat no.
  • A80-104A 1 mg/ml is diluted 1 :100 in carbonate buffer (100 mM NaHC0 3 , 33.6 mM Na 2 C0 3 , pH 9.5). Plates are coated with 100 ul/well of coating solution and incubated at 37°C for 1 hour. The plates are then washed 4X with TBS-T (50 mM Tris, 0.14 M NaCI, 0.05% Tween-20, pH 8.0) and blocked with 200 ⁇ /well TBS/BSA (50mM Tris, 0.14 M NaCI, + 1 % BSA, pH 8.0) for 1 hour at 37°C . Samples and standards are prepared on non-binding plates with enough volume to run in duplicate.
  • the standard is prepared by diluting human reference serum (Bethyl RS10-1 10; 4 mg/ml) in TBS-T/BSA (50 mM Tris, 0.14 NaCI, 1 % BSA, 0.05 % Tween-20, pH 8.0) to the following dilutions: 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml, 31.25 ng/ml, 15.625 ng/ml, 7.8125 ng/ml, and 0.0 ng/ml.
  • human reference serum Bethyl RS10-1 10; 4 mg/ml
  • TBS-T/BSA 50 mM Tris, 0.14 NaCI, 1 % BSA, 0.05 % Tween-20, pH 8.0
  • the samples are prepared by making appropriate dilutions in TBS-T/BSA so that the samples OD fall within the range of this standard curve, the most linear range being from 125 ng/ml to15.625 ng/ml.
  • 100 ⁇ of the standard/samples preparation is added to each well and incubated at 37°C for 1 hour.
  • the plates are washed 4 times with TBS-T and then incubated for 1 hour at 37 °C with 100 ul/well of HRP-goat anti-human IgG antibody (Bethyl A80-104P, 1 mg/ml) diluted 1 :150,000 in TBS-T/BSA.
  • the plates are washed 4 additional times with TBS-T and developed using 100 ⁇ /well TMB substrate at 4 °C. After 7 minutes, the reaction is stopped by adding 100 ⁇ /well of 1 M H2SO4. The OD is measured at 450 nm. Data is analyzed using Graphpad Prizm software.
  • Direct-Binding ELISA Microtiter ELISA plates (Costar, Corning Inc., Lowell MA, Cat No. 3361 ) are coated overnight with either S1 P or LPA conjugated to delipidated BSA diluted in 0.1 M Carbonate Buffer (pH 9.5) at 37 °C for 1 h.
  • the thiolated lipid is diluted into the HBS running buffer to a concentration of 10, 1 and 0.1 ⁇ and injected for 7 minutes producing different lipid density surfaces (-100, -300 and -1400 RU).
  • binding data for the WT and mutant antibodies is collected using a 3-fold dilution series starting with 25 nM as the highest concentration. Surfaces are regenerated with a 10 second pulse of 100 mM HCI. All data is collected at 25 °C. Controls are processed using a reference surface as well as blank injections. In order to extract binding parameters, the data is globally fit using 1 - site and 2-site models.
  • mutations in the anti-S1 P and anti-LPA antibodies are designed to test the x-ray structures with biochemical technigues.
  • Amino acids in the variable domains that directly contact the lipids in the complex are substituted with amino acids designed to reduce binding in the SPR and direct-binding ELISA.
  • the importance of the electrostatic charge, polarity and hydrophobicity of the amino acids are thus investigated. Based on preliminary data, it is presently believed that amino acids recognize the S1 P head group using electrostatic and hydrogen bonding interactions, whereas hydrophobic residues stabilize the aliphatic carbon chain of S1 P. Therefore, it is believed that mutating residues that contact the lipid head groups to alanine or a residue with opposite charge will abrogate lipid binding.
  • select residues that form the hydrophobic pocket are substituted with charged, polar residues (such as glutamic acid) designed to dramatically alter the electrostatic surface of the variable domain and seguester water into the hydrophobic binding pocket and dramatically reduce stability of the complex.
  • An interesting feature detected in the LT1009Fab/S1 P structure is the position of Y102 in the CDR H3.
  • the side chain of this tyrosine residue appears to fold over the hydrocarbon tail of S1 P, clamping down on the lipid.
  • the lipid is unable to freely dissociate from the antibody.
  • a conformational change in the CDR H3 or the Y102 side chain rotomer position is believed to take place which allows the lipid to dissociate. While not wishing to be bound by theory, this is believed to play an important role in the lifetime of the LT1009-S1 P complex.
  • the Vh framework may present a favorable, universal binding pocket for lysolipids.
  • the LT1009 and LT3015 Vh sequences are 93% identical outside the CDRs (as expected, the CDRs have lower identity, in this case 46%).
  • the Vk sequences are 59% identical outside the CDRs (19% identity within the CDRs).
  • the less conserved Vk domain exclusively contacts the head group of S1 P, which is dissimilar to LPA, whereas the highly conserved Vh domain primarily contacts the hydrocarbon chain, which is chemically conserved between S1 P and LPA.
  • cocrystals e.g., humanized anti-LPA antibody and LPA
  • information from multiple co-crystals particularly bioactive lipid-antibody co-crystals, may be used together in the design of new anti-lipid antibodies.
  • S1 P sphingosine-1 - phosphate
  • the ammonium group forms a single, electrostatic interaction with side chain of glutamic acid at position 50, which protrudes from the antibody light chain.
  • this glutamic acid was mutated to glutamine (GlnL50) and binding of these antibodies to either S1 P or LPA was assayed using a direct ELISA.
  • the wild-type (WT) LT1009 shows high affinity for the S1 P-BSA coating material, while no binding activity was observed for C12 LPA-BSA coated on the plate.
  • the LT1009 GlnL50 mutant antibody shows significantly higher affinity for the C12 LPA-BSA conjugate compared to S1 P-BSA ( Figure 4 ), suggesting that this amino acid plays a significant role in S1 P specificity and changes at this position alters target specificity.
  • Example 20 Additional variants designed based on antibody:lipid co-crystal structure:
  • a number of amino acid residues in both the light chain and the heavy chain of LT1009 have been identified as likely to play a role in antibody binding to S1 P because these residues are shown to be in contact with the lipid, based on the structure of the antibody-lipid complex.
  • positions include aspartic acid residue at position 28 (D28), glycine residue G51 , leucine residue L94 and phenylalanine residue F96 of the light chain, and histidine residue H35, serine residue S52, proline residue P52A, histidine residue H54, isoleucine residue I56, lysine residue K58, phenylalanine residue F97, tyrosine residue Y98, glycine residue G99, serine residue 100, threonine residue T100A, tryptophan residue W100C, arginine residue R54, arginine residue R65 and tyrosine residue Y102 of the heavy chain.
  • aspartic acid D28 was replaced by asparagine (D28N); glycine G51 was replaced by glutamic acid (G51 E) or by tyrosine (G51 Y), and phenylalanine residue F96 was replaced by alanine (F96A).
  • histidine residue H35 was replace by alanine (H35A), glutamine (H35Q) or glutamic acid (H35E), serine residue S52 was replaced by glycine (S52G) or alanine (S52A), proline residue P52A was replaced by glycine (P52AG), histidine residue H54 was replaced with phenylalanine (H54F) or with tryptophan (H54W), isoleucine residue I56 was replaced with tryptophan (I56W) or with lysine (I56K), lysine residue K58 was replaced with glutamine (K58Q), phenylalanine residue F97 was replaced with tyrosine (F97Y), tyrosine residue Y98 was replaced with alanine (Y98A) or with tryptophan (Y98W), glycine residue G99 was replaced with lysine (G99K), threonine residue T100A was replaced with glutamic acid (T
  • Certain amino acid residues in the light chain and heavy chain of LT1009 have been identified as likely to play a role in specificity of the antibody for S1 P. These positions include the glutamic acid at position 50 (E50), asparagine residue at position 93 (N93), and tyrosine at position 105A (T105A) of the light chain and glycine at position 99 (G99) and serine at position 100 (S100) of the heavy chain.
  • aspartic acid residues D30, D31 , D32 and D92 of the light chain were found to coordinate the metal atoms that bridge the antibody and its epitope.
  • changing the aspartic acid to alanine at positions D30 and D32 eliminated S1 P binding by the antibody. Additional substitution mutations at these sites have been made to gain further information about the role of each amino acid residue in metal binding (and thus, antibody binding to S1 P).
  • Aspartic acid D30 was replaced either by lysine (D30K), threonine (D30T), arginine (D30R) or tyrosine (D30Y).
  • Aspartic acid D31 was replaced by alanine (D31A) or arginine (D31 R).
  • Aspartic acid D32 was replaced by asparagine (D32N) or by arginine (D32R).
  • Aspartic acid D92 was replaced either by alanine (D92A), arginine (D92R) or by glutamine (D92Q).
  • *%WT refers to the ELISA signal for each variant relative to WT (LT1009) at a particular concentration (6.4 ug/ml), which is useful for purposes of comparison.
  • AAG values are theoretical, calculated using computational methods to predict lipid binding.
  • Binding is a qualitative comparison of S1 P binding (+++ high affinity, ++ moderate affinity, + low affinity, - no binding), based on evaluation of EC50, Bmax, and % WT.
  • Example 21 Purification and production of anti-LPA antibodies
  • LT3015 a mammalian cell line (CHO CK1 sv) that expresses >0.5 mg/ml of the humanized, anti-LPA mAb, LT3015.
  • This stable cell line was utilized in a 50 liter bioreactor campaign to produce large quantities of non-GMP material. Purification of LT3015 from the bioreactor supernatant resulted in >10 grams of antibody material.
  • LT3015 was formulated at 18 mg/ml in 24 mM PBS, 148 mM NaCI, pH 6.5, and this preparation meets strict specifications for purity, aggregation and LPA-binding properties. Therefore, suitable material is available for papain digestion, isolation of the Fab fragment, complex formation with LPA, and crystallization of the LT3015Fab/LPA complex.
  • Example 22 Information gained from comparison of anti-S1 P and anti-LPA humanized antibodies
  • LT1009 and LT3015 are compared.
  • the relatively minor differences in the amino acid sequences of the antibody hypervariable regions function to discriminate between LPA and S1 P, two bioactive lipids with such high structural and chemical identity.
  • the anti-LPA and anti-S1 P VH sequences are 93% identical outside the CDRs (as expected, the CDRs have lower identity, in this case 46%).
  • the Vk sequences are 59% identical outside the CDRs (19% identity within the CDRs).
  • compositions and methods described and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit and scope of the invention as defined by the appended claims.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Theoretical Computer Science (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Evolutionary Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Medical Informatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des procédés de conception d'anticorps optimisés ciblant des lipides bioactifs. Ces procédés peuvent être mis en œuvre in silico et peuvent avoir pour but de renforcer l'affinité de liaison d'un anticorps avec son lipide cible initial et/ou de modifier ladite affinité de liaison. L'invention concerne également des variants d'anticorps inédits obtenus par ces procédés, ainsi que leurs procédés de fabrication et d'utilisation.
PCT/US2011/039039 2010-06-04 2011-06-03 Variants d'anticorps anti-s1p inédits de lt1009 WO2011153416A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35182610P 2010-06-04 2010-06-04
US61/351,826 2010-06-04

Publications (2)

Publication Number Publication Date
WO2011153416A2 true WO2011153416A2 (fr) 2011-12-08
WO2011153416A3 WO2011153416A3 (fr) 2013-04-11

Family

ID=45067309

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/039039 WO2011153416A2 (fr) 2010-06-04 2011-06-03 Variants d'anticorps anti-s1p inédits de lt1009

Country Status (1)

Country Link
WO (1) WO2011153416A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100098700A1 (en) * 2008-04-05 2010-04-22 Sabbadini Roger A Pharmaceutical compositions for binding sphingosine-1-phosphate
WO2010065921A2 (fr) * 2008-12-05 2010-06-10 Lpath, Inc. Conception d'anticorps à l'aide de structures cristallines d'anticorps anti-lipidiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100098700A1 (en) * 2008-04-05 2010-04-22 Sabbadini Roger A Pharmaceutical compositions for binding sphingosine-1-phosphate
WO2010065921A2 (fr) * 2008-12-05 2010-06-10 Lpath, Inc. Conception d'anticorps à l'aide de structures cristallines d'anticorps anti-lipidiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
0' BRIEN, N. ET AL.: 'Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies' JOURNAL OF LIPID RESEARCH vol. 50, 09 June 2009, pages 2245 - 2257 *
WOJCIAK, J. M. ET AL.: 'The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen' PROC. NATL. ACAD. SCI. USA vol. 106, no. 42, 07 October 2009, pages 17717 - 17722 *

Also Published As

Publication number Publication date
WO2011153416A3 (fr) 2013-04-11

Similar Documents

Publication Publication Date Title
US20100292443A1 (en) Humanized platelet activating factor antibody design using anti-lipid antibody templates
US8026342B2 (en) Compositions and methods for binding sphingosine-1-phosphate
US20110044990A1 (en) Antibody design using anti-lipid antibody crystal structures
US9163091B2 (en) Compositions and methods for binding lysophosphatidic acid
US8614103B2 (en) Compositions and methods for treating sphingosine-1-phosphate (S1P) related ocular diseases and conditions
US8604172B2 (en) Humanized antibody compositions and methods for binding lysophosphatidic acid
US20110212088A1 (en) Anti-paf antibodies
US20130261287A1 (en) Antibody design using anti-lipid antibody crystal structures
US8361465B2 (en) Use of anti-sphingosine-1-phosphate antibodies in combination with chemotherapeutic agents
US20140186339A1 (en) Compositions and methods for treating ocular diseases and conditions
WO2011153416A2 (fr) Variants d'anticorps anti-s1p inédits de lt1009
US20120034631A1 (en) Anti-lysophospholipid antibody design using antibody structures
AU2013273727A1 (en) Compositions and methods for treating ocular diseases and conditions
AU2016204486A1 (en) Composition and Methods for Treating Ocular Diseases and Conditions

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC DATED 19.03.13

122 Ep: pct application non-entry in european phase

Ref document number: 11790452

Country of ref document: EP

Kind code of ref document: A2