+

WO2005086568A2 - Anticorps anti-icam 1 humains et utilisations de ceux-ci - Google Patents

Anticorps anti-icam 1 humains et utilisations de ceux-ci Download PDF

Info

Publication number
WO2005086568A2
WO2005086568A2 PCT/IB2005/002041 IB2005002041W WO2005086568A2 WO 2005086568 A2 WO2005086568 A2 WO 2005086568A2 IB 2005002041 W IB2005002041 W IB 2005002041W WO 2005086568 A2 WO2005086568 A2 WO 2005086568A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
icam
seq
functional fragment
antigen
Prior art date
Application number
PCT/IB2005/002041
Other languages
English (en)
Other versions
WO2005086568A3 (fr
Inventor
Tobias Litzenburger
Margit Urban
Original Assignee
Morphosys Ag
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 Morphosys Ag filed Critical Morphosys Ag
Publication of WO2005086568A2 publication Critical patent/WO2005086568A2/fr
Publication of WO2005086568A3 publication Critical patent/WO2005086568A3/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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • 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
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/75—Agonist effect on antigen
    • 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

  • intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the immunoglobulin superfamily and has five extracellular Ig-like domains (designated DI to D5). See Staunton et al., (1988) Cell 52(6): 925-33. Under non-inflammatory conditions, ICAM-1 is expressed at low levels in a variety of tissues, including endothelial cells, epithelial cells and leukocytes (Dustin et al., 1986). Also, ICAM-1 is strongly induced on endothelial cells by inflammatory
  • cytokines such as interferon-gamma (IFN- ⁇ ), interleukin-1 (I -1) and tumour
  • necrosis factor alpha (TNF- ⁇ ) (Pober et al., 1986).
  • ICAM-1 ⁇ 2-integrins, mainly LFA-1 (CDl la/CD18) and Mac-1 (CDl lb/CD18) on activated leukocytes (Marlin and Springer, 1987; Diamond et al., 1990; Smith et al., 1989), ICAM-1 also plays a role in inflammatory reactions that mediate leukocyte extravasation.
  • the interaction of integrins with ICAM-1 is tightly regulated, such that activation of leukocytes leads to conformational changes in the integrin and results in strongly enhanced binding to ICAM-1 (reviewed by Binnerts and van Kooyk, 1999).
  • the affinities of ICAM-1 for its major counter-receptor LFA-1 have been determined in various assays utilizing purified and recombinant proteins. These studies revealed dissociation constants (Kd) in the range of 60-170 nM and 4-60 nM for monomeric and dimeric ICAM-1 preparations, respectively (Woska, Jr. et al., 1996; Woska, Jr. et al., 1998; Jun et al, 2001).
  • ICAM-1 functions as an accessory molecule on antigen-presenting cells such as monocytes, macrophages and dendritic cells and delivers co-stimulatory signals to T cells through interaction with LFA-1, which is required for a functional T cell response (Nan Seventer et al., 1990; Dougherty et al, 1988). It is known that ICAM-1 that is expressed on the T lymphocytes also is involved in co-stimulation transmitting anti-apoptotic signals and expanding the number of memory T cells (Chirathaworn et al., 2002; Kohlmeier et al., 2003). ICAM-1 can be a targeted molecule for anti-inflammation therapy, due to its role in inflammation.
  • disorders or conditions associated with inflammation include psoriasis, rheumatoid arthritis ("RA"), burn injury, inflammatory bowel disease or multiple sclerosis.
  • RA rheumatoid arthritis
  • ICAM-1 is believed to play an important role in leukocyte migration into the synovial joint (Smith et al., 2001; Tanaka, 2001).
  • An anti-ICAM-1 antibody is, thus, expected to reduce infiltration of inflammatory leukocytes into synovial tissue.
  • an anti-ICAM-1 antibody may inhibit T cell or monocyte effector functions such as cytokine secretion, which contributes to synovial and cartilage tissue damage.
  • Psoriasis is a complex disorder of the skin, characterized by keratinocyte hyperproliferation, altered epidermal differentiation, and infiltration of activated T cells and other leukocytes into the skin (reviewed by Leitz, 2003). Although the precise cellular mechanism responsible for psoriasis is not fully understood, it is postulated to be of autoimmune origin induced by autoreactive T cells. Inflammatory cytokines, in particular IF ⁇ -gamma and T ⁇ F-alpha, induce strong expression of ICAM-1 on keratinocytes, dermal infiltrates and vascular endothelial cells in psoriatic skin lesions (Griffiths et al., 1989; Terajima et al., 1998).
  • An antibody against ICAM-1 could act at several levels in treating psoriasis by inhibiting the extravasation of inflammatory leukocytes and suppressing the pathogenic T cell attack by disrupting the costimulatory signal mediated by antigen presenting cells such as keratinocytes.
  • Animal model studies and clinical trials alike have demonstrated that blocking of the ICAM-1 /LFA-1 interaction is of considerable value in the treatment of psoriasis (Gott Kunststoff et al., 2002; Mehta et al., 2000).
  • blocking of ICAM-1 demonstrated efficacy in acute inflammatory disorders such as deep dermal burn (Mileski et al., 2003).
  • Anti-ICAM-1 mAb therapy which was well tolerated, resulted in transient alteration of T lymphocyte recirculation and effected disease improvement in more than 50% of RA patients.
  • the induction and persistence of T cell hyporesponsiveness reflected by decreased IL-2 production, correlated with sustained therapeutic benefit in treated patients (Davis et al, 1995).
  • persistently decreased IL-6 levels in peripheral blood mononuclear cells induced by anti-ICAM-1 mAb is thought to be associated with the long-term beneficial effect of this therapy (Schulze-Koops et al., 1996).
  • BIRR-1 BIRR-1 phenotype induces HAMA reactions in humans, which renders this antibody unsafe for chronic applications;
  • mouse IgG2a isotype of BIRR-1 was shown to activate neutrophils in a complement-dependent manner, which tends to explain the adverse side effects associated with BIRR-1 therapy (Nuorte et al., 1999).
  • Another murine anti-ICAM-1 antibody has been humanized (WO 03/035696).
  • the resulting chimeric human/mouse antibodies still comprise murine CDR sequences, so that about 30% of the NH amino acid residues and about 24% of the VL amino acid residues are still of non-human origin.
  • the invention provides an isolated human antibody or functional antibody fragment that contains an antigen-binding region that is specific for an epitope of ICAM-1.
  • An antibody of the invention may be an IgG (e.g. , IgG ), while an antibody fragment may be a Fab or scFv, for example.
  • epitope describes the set of critical binding residues as identified by epitope mapping studies using sets of overlapping peptides corresponding to the sequence of a protein (Reineke, 2004).
  • epitope may be linear (i.e. the critical residues identified in epitope mapping studies may be adjacent to each other in the protein sequence), whereas for others it may be discontinuous (i.e. the critical residues identified in epitope mapping studies may be found in two or more different parts of the protein sequence).
  • An antibody or functional fragment thereof having one or more of these properties may contain an antigen-binding region that contains an H-CDR3 region depicted in SEQ ID NO: 5, 6, 7 or 8; the antigen-binding region may further include an H-CDR2 region depicted in SEQ ID NO: 5, 6, 7 or 8; and the antigen-binding region also may contain an H-CDR1 region depicted in SEQ ID NO: 5, 6, 7 or 8.
  • An antibody of the invention may contain an antigen-binding region that contains an L-CDR3 region depicted in SEQ ID NO: 13, 14, 15 or 16; the antigen-binding region may further include an L-CDR1 region depicted in SEQ ID NO: 13, 14, 15 or 16; and the antigen-binding region also may contain an L-CDR2 region depicted in SEQ ID NO: 13, 14, 15 or 16.
  • Certain human antibodies (and functional fragments thereof) of the invention are cross-reactive with rabbit and/or at least one rodent species selected from the group consisting of mouse, rat and hamster, as determined by immunohistochemistry.
  • the rodent is mouse.
  • Such an antibody additionally may be cross-reactive with one or more species selected from the group of cynomolgus, marmoset, rhesus and baboon.
  • An antibody or functional fragment thereof having these properties may contain an antigen-binding region that contains an H-CDR3 region depicted in SEQ ID NO: 7; the antigen-binding region may further comprise an H-CDR2 region depicted in SEQ ID NO: 7; and the antigen-binding region also may contain an H-CDR1 region depicted in SEQ ID NO: 7.
  • a species cross-reactive antibody of the invention may contain an antigen-binding region that contains an L-CDR3 region depicted in SEQ ID NO: 15; the antigen-binding region may further comprise an L-CDR1 region depicted in SEQ ID NO: 15; and the antigen-binding region also may contain an L-CDR2 region depicted in SEQ ID NO: 15.
  • Peptide variants of the sequences disclosed herein also are embraced by the present invention.
  • the invention includes human anti-ICAM-1 antibodies having a heavy chain amino acid sequence with: at least 60 percent sequence identity in the CDR regions with the CDR regions depicted in SEQ ID NO: 5, 6, 7 or 8; and/or at least 80 percent sequence homology in the CDR regions with the CDR regions depicted in SEQ ID NO: 5, 6, 7 or 8. Further included are human anti-ICAM-1 antibodies having a light chain amino acid sequence with: at least 60 percent sequence identity in the CDR regions with the CDR regions depicted in SEQ ID NO: 13, 14, 15 or 16; and/or at least 80 percent sequence homology in the CDR regions with the CDR regions depicted in SEQ ID NO: 13, 14, 15 or 16.
  • the invention also is related to isolated nucleic acid sequences, each of which can encode an antigen-binding region of a human antibody or functional fragment thereof that is specific for an epitope of ICAM-1.
  • a nucleic acid sequence may encode a variable heavy chain of an antibody and include a sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3 and 4, or a nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of SEQ ID NO: 1, 2, 3 or 4.
  • the nucleic acid might encode a variable light chain of an isolated antibody or functional fragment thereof, and may contain a sequence selected from the group consisting of SEQ ID NOS: 9, 10, 11 and 12, or a nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of SEQ ID NO: 9, 10, 11 or 12.
  • Nucleic acids of the invention are suitable for recombinant production.
  • the invention also relates to vectors and host cells containing a nucleic acid sequence of the invention.
  • Compositions of the invention may be used for therapeutic or prophylactic applications.
  • the invention therefore, includes a pharmaceutical composition containing an antibody of the invention (or functional antibody fragment) and a pharmaceutically acceptable carrier or excipient therefor.
  • the invention provides a method for treating a disorder or condition associated with the undesired presence of ICAM-1.
  • Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an antibody of the invention as described or contemplated herein.
  • BRIEF DESCRIPTION OF THE FIGURES Figure la provides nucleic acid sequences of various novel antibody variable heavy regions.
  • Figure lb provides amino acid sequences of various novel antibody variable heavy regions.
  • CDR regions HCDRl, HCDR2 and HCDR3 are designated from N- to C-terminus in boldface.
  • Figure 2a provides nucleic acid sequences of various novel antibody variable light regions.
  • Figure 2b provides amino acid sequences of various novel antibody variable light regions.
  • CDR regions LCDR1, LCDR2 and LCDR3 are designated from N- to C-terminus in boldface.
  • Figure 3 provides amino acid sequences of variable heavy regions of various consensus antibody sequences.
  • CDR regions HCDRl, HCDR2 and HCDR3 are designated from N- to C-terminus in boldface.
  • Figure 4 provides amino acid sequences of variable light regions of various consensus antibody sequences.
  • CDR regions LCDRl, LCDR2 and LCDR3 are designated from N- to C-terminus in boldface.
  • Figure 5 provides the amino acid sequence for the variable light and heavy chains for the R6.5 antibody (BIRR-1).
  • Figure 6 provides the amino acid sequence of mature ICAM-1.
  • the lg domain 1 (DI) region is from amino acids 1 to 88; the D2 region is from amino acids 89 to 185; the D3 region is from amino acids 186 to 284; the D4 region is from amino acids 285 to 385; and the D5 region is from amino acids 386 to 453.
  • DI lg domain 1
  • the present invention is based on the discovery of novel human antibodies that are specific to or have a high affinity for ICAM-1, including human ICAM-1, and can deliver a therapeutic benefit to a subject.
  • the antibodies of the invention can be used in many contexts, which are more fully described herein.
  • a “human” antibody or functional antibody fragment is hereby defined as one that is not chimeric (e.g., not “humanized”) and not from (either in whole or in part) a non-human species.
  • a human antibody or functional antibody fragment can be derived directly from a human or can be derived at least partially in silico from synthetic sequences that are based on the analysis of known human antibody sequences.
  • an antibody “binds specifically to,” is “specific to/for” or “specifically recognizes” an antigen (here, ICAM-1) if such antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property.
  • binding is referring to the ability of the antibody to discriminate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods.
  • Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
  • a standard ELISA assay can be carried out.
  • the scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogenperoxide).
  • the reaction in certain wells is scored by the optical density, for example, at 450 nm.
  • determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.
  • “specific binding” also may refer to the ability of an antibody to discriminate between the target antigen and one or more closely related antigen(s), which are used as reference points, e.g. between ICAM-1 and ICAM-2 or -3. Additionally, “specific binding” may relate to the ability of an antibody to discriminate between different parts of its target antigen, e.g.
  • an antibody is said to have a "high affinity" for an antigen (e.g. a receptor) if the affinity measurement is at least 100 nM.
  • Certain antibodies according to the invention have an affinity for ICAM-1 of less than 30 nM, preferably less than 10 nM, and more preferably less than 3 nM.
  • an "immunoglobulin" (lg) hereby is defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), and includes all conventionally known antibodies and functional fragments thereof.
  • a “functional fragment” of an antibody/immunoglobulin hereby is defined as a fragment of an antibody/immunoglobulin (e.g., a variable region of an IgG) that retains the antigen-binding region.
  • An "antigen-binding region" of an antibody typically is found in one or more hypervariable region(s) of an -antibody, i.e., the CDR-1, -2, and/or -3 regions; however, the variable "framework" regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs.
  • the "antigen-binding region” comprises at least amino acid residues 4 to 103 of the variable light (NL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111 of NH, and particularly preferred are the complete NL and VH chains (amino acid positions 1 to 109 of NL and 1 to 113 of NH; numbering according to WO 97/08320).
  • a preferred class of immunoglobulins for use in the present invention is IgG.
  • "Functional fragments" of the invention include the domain of a F(ab') 2 fragment, a Fab fragment and scFv.
  • the F(ab') 2 or Fab may be engineered to minimize or completely remove the intermolecular disulphide interactions that occur between the CHI and C domains.
  • An antibody of the invention may be derived from a recombinant antibody library that is based on amino acid sequences that have been designed in silico and encoded by nucleic acids that are synthetically created. In silico design of an antibody sequence is achieved, for example, by analyzing a database of human sequences and devising a polypeptide sequence utilizing the data obtained therefrom. Methods for designing and obtaining in ⁇ ///co-created sequences are described, for example, in Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al, J.
  • LAC 1555 represents an antibody having a variable heavy region corresponding to SEQ ID NO: 5 and a variable light region corresponding to SEQ ID NO: 13.
  • LAC 3040 represents an antibody having a variable heavy region corresponding to SEQ ID NO: 6 and a variable light region corresponding to SEQ ID NO: 14.
  • LAC 3041 represents an antibody having a variable heavy region corresponding to SEQ ID NO: 7 and a variable light region corresponding to SEQ ID NO: 15.
  • LAC 3043 represents an antibody having a variable heavy region corresponding to SEQ ID NO: 8 and a variable light region corresponding to SEQ ID NO: 16.
  • Antibody R 6.5 represents the BIRR-1 antibody, which has a variable heavy region corresponding to SEQ ID NO: 23 and a variable light region corresponding to SEQ ID NO: 24.
  • the invention provides antibodies having an antigen-binding region that can bind specifically to or has a high affinity for one or more regions of ICAM-1, whose amino acid sequence is depicted by SEQ ID NO: 25.
  • An inventive antibody or antigen-binding region preferably can specifically bind to ICAM-1 with an affinity of about less than 100 nM, more preferably less than about 75 nM, and still more preferably less than about 30 nM. Further preferred are antibodies that specifically bind to ICAM-1 with an affinity of less than about 10 nM, and more preferably less than 3 about nM.
  • the affinity of LACs 1555, 3040, 3041 and 3043 was measured by surface plasmon resonance (Biacore) on immobilized recombinant ICAM-1 and by a flow cytometry procedure utilizing the ICAM-1 - expressing human ECN304 cell line.
  • the Biacore studies were performed on either directly immobilized antigen (ICAM-1 -Fc fusion protein) or on ICAM-1 -Fc captured via anti-Fc antibodies.
  • Kd dissociation constants
  • the Kd values ranged from approximately 3 to 31 nM.
  • the functional affinities (avidities) of HuCAL ® IgG4 and BIRR-1 mIgG2a were determined in the same cell-based system as for the Fabs. All IgG4 binders in Table 1 are characterized by similar binding strength in the low nanomolar range comparable to BIRR-1 mIgG2a (Table 1). In the IgG format, 3040 and 3041 antibodies showed the strongest binding (both 0.7 nM), similar to BIRR-1 (1.0 nM) and slightly stronger than 1555 (1.7 nM) and 3043 (2.5 nM).
  • clones 3040 and 3041 exhibited a strong improvement of up to 45-fold in the bivalent format as compared to monovalent Fabs.
  • Another feature of preferred antibodies of the invention is their specificity for an area within the DI region (the most N-terminal domain) of ICAM-1.
  • the DI region is made up of amino acids 28 to 115 of ICAM-1 (amino acids 1 to 88 of mature ICAM-1, see Fig. 6).
  • the major counter-receptor of ICAM-1, the ⁇ 2 integrin LFA-1 binds to the DI region of ICAM-1 (Staunton et al., 1990).
  • an antibody that is specific for the DI region is expected to block the interaction of ICAM-1 and LFA-1 more effectively than antibodies specific for another region.
  • Representative antibodies of the invention that can bind specifically to the DI region of ICAM-1 include LACs 1555, 3040, 3041 and 3043.
  • Antibodies of the invention advantageously can block the interaction between ICAM-1 and LFA-1.
  • ICAM-1 specific mAb RR1.1 an antibody that blocks the interaction of ICAM-1 with LFA-1, but not Mac-1, and showed therapeutic benefit in models of inflammation and ischemia (Zhao et al., 1997; Norman et al., 1994).
  • An antibody of the invention that blocks or hinders the interaction between ICAM-1 and LFA-1 is, thus, particularly suitable for anti- inflammation therapy.
  • An antibody of the invention may have a specificity for an epitope that differs from the epitope to which known anti-ICAM-1 antibodies bind. Competition assays can be employed to confirm such different specificity, wherein a lack of competition is evidence that binding to a different epitope occurs.
  • the present inventors have shown that the BIRR-1 antibody (R6.5, ATCC deposit no. HB-9580) does not compete with certain antibodies of the invention (LACS 1555, 3041 and 3043). It is known that BIRR-1 recognizes an epitope at the top of domain 2 (Staunton et al, 1990; Ockenhouse et al., 1992). A competition assay (i.e., the co-incubation of an inventive antibody (e.g., LAC 1555, 3041 or 3043) and a known anti-ICAM-1 antibody such as BIRR-1) can be used to obtain these data.
  • an inventive antibody e.g., LAC 1555, 3041 or 3043
  • a known anti-ICAM-1 antibody such as BIRR-1
  • an antibody of the invention binds to a different epitope.
  • An antibody of the invention additionally may be specific to, or have a heightened affinity for, a region of ICAM-1 other than DI, such as the D2 region.
  • Antibody no. 3040 is one particular antibody of the invention that may bind specifically to residues of D 1 and D2 of IC AM- 1.
  • the type of epitope to which an antibody of the invention binds may be linear (i.e. one consecutive stretch of amino acids) or discontinuous (i.e. multiple stretches of amino acids).
  • the skilled worker can analyze the binding of antibodies to overlapping peptides (e.g., 13-mer peptides with an overlap of 11 amino acids) covering different domains of ICAM-1 (e.g., domains 1 and 2). Using this analysis, the inventors have discovered that LACS 1555, 3040 and 3041 recognize similar, discontinuous epitopes in domain 1 of ICAM-1, whereas the epitope of 3043 can be described as linear. All the determined epitopes contain amino acid residue K39 of ICAM-1 (DI region), which was previously described to be involved in binding to LFA-1 (Fisher et al., 1997).
  • DI region amino acid residue
  • An antibody of the invention preferably is species cross-reactive with humans and at least one other species, which may be rabbit, a rodent species or a non-human primate.
  • the non-human primate can be rhesus, baboon and/or cynomolgus.
  • the rodent species can be mouse, rat and/or hamster.
  • Table 2 provides species cross-reactivity immunohistochemistry (IHC) data for representative antibodies of the invention LACS-1555, -3040, -3041 and -3043, and compares the species cross-reactivity of the variable regions of these antibodies (in bivalent Fab-dHLX format) with that of the BIRR-1 murine antibody.
  • Table 2 provides species cross-reactivity immunohistochemistry (IHC) data for representative antibodies of the invention LACS-1555, -3040, -3041 and -3043, and compares the species cross-reactivity of the variable regions of these antibodies (in bivalent Fab-dHLX format) with that of the BIRR-1 murine
  • inventive antibodies listed in Table 2 has a distinct species cross-reactivity profile, all of which are distinct from that of BIRR-1.
  • inventive antibodies therefore, provide much greater flexibility and benefits over known anti-ICAM-1 antibodies, for purposes of conducting in vivo studies in multiple species with the same antibody. Indeed, the present invention holds great promise for relatively rapid development of a therapeutic and/or diagnostic product.
  • an antibody of the invention does not evoke a substantial neutrophil-activating response in a subject; more preferably, there is no neutrophil-activating response observed in a subject that has been administered an inventive antibody.
  • the inventive antibody is in a format, which is known not to elicit complement activation, e.g., by not having complement-fixing capacity, such as in an Fab antibody fragment format or as a human IgG4 antibody. Less preferably, the inventive antibody is a human IgGl antibody.
  • the skilled worker can perform in vitro assays, e.g., by measuring i) intracellular DCFH oxidation and (ii) cell surface CDl lb expression (Nuorte et al., 1999).
  • An antibody of the invention that does not evoke a substantial neutrophil response, or any neutrophil response at all, is more suitable for in vivo administration than known antibodies.
  • murine antibody R6.5 when administered at concentrations achieved in clinical trials, activated peripheral blood neutrophils in whole blood in vitro, as indicated by a significant increase in expression of CD lib/CD 18 adhesion molecule and an enhancement of the intracellular oxidative burst. This effect was mediated by prior activation of complement, and it can well explain, at least in part, the observed adverse effects associated with R6.5 therapy.
  • Peptide Variants Antibodies of the invention are not limited to the specific peptide sequences provided herein.
  • blocking activity means a functional characteristic ascribed to an anti-ICAM-1 antibody of the invention. Blocking activity, thus, includes the ability to hinder or inhibit the interaction between ICAM-1 and a ligand, e.g., LFA-1.
  • a variant can include, for example, an antibody that has at least one altered complementarity determining region (CDR) (hyper-variable) and/or framework (FR) (variable) domain position, vis-a-vis a peptide sequence disclosed herein.
  • CDR complementarity determining region
  • FR framework
  • variable domain position vis-a-vis a peptide sequence disclosed herein.
  • An antibody is composed of two peptide chains, each containing one (light chain) or three (heavy chain) constant domains and a variable region (NL, NH), the latter of which is in each case made up of four FR regions and three interspaced CDRs.
  • the antigen-binding site is formed by one or more CDRs, yet the FR regions provide the structural framework for the CDRs and, hence, play an important role in antigen binding.
  • Tables 3a and 3b delineate the CDR and FR regions for certain antibodies of the invention and compare amino acids at a given position to each other and to corresponding consensus or "master gene" sequences (as described in U.S. Patent No. 6,300,064):
  • VH Framework 3 CDR3 Framework 4 9 10 11 Position 9 0 1 2 3 4 5 6 7 0 1 2 a b c 3 4 5 0 1 2 3 4 5 6 7 8 9 0 a b c d e f g h l j 1 2 3 4 5 6 7 B 9 0 1 2 3 BssH ⁇ Styl Blpl
  • variants are constructed by changing amino acids within one or more CDR regions; a variant might also have one or more altered framework regions.
  • candidate residues that can be changed include residues 28, 51, and/or 94 of the variable light chains of LACs 3040 and 3043, since these are positions of variance vis-a-vis each other. Alterations also may be made in the framework regions. For example, a peptide FR domain might be altered where there is a deviation in a residue compared to a germline sequence.
  • candidate residues that can be changed include residues 30, 34, 90 and/or 94 of the variable light chain of LAC 3041.
  • the skilled worker could make the same analysis by comparing the amino acid sequences disclosed herein to known sequences of the same class of such antibodies, using, for example, the procedure described by Knappik et al. (2000), and U.S. Patent No. 6,300,064 issued to Knappik et al.
  • variants may be obtained by using one LAC as starting point for optimization by diversifying one or more amino acid residues in the LAC, preferably amino acid residues in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties.
  • Particularly preferred is diversification of one or more amino acid residues in CDR-3 of NL, CDR-3 of NH, CDR-1 of NL and/or CDR-2 of NH.
  • Diversification can be done by synthesizing a collection of D ⁇ A molecules using trinucleotide mutagenesis (TRIM) technology (Nirnekas et al. , 1994).
  • TAM trinucleotide mutagenesis
  • Polypeptide variants may be made that conserve the overall molecular structure of an antibody peptide sequence described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine;
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d).
  • glycine and proline may be substituted for one another based on their ability to disrupt ⁇ -helices.
  • certain amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in ⁇ -helices
  • valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in ⁇ -pleated sheets.
  • Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns.
  • substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, N, L and I.
  • S and T amino acid position 3 in SEQ ID ⁇ OS: 5, 6, 7 and/or 8 can be substituted from a Q to an E.
  • sequence identity indicates the percentage of amino acids that are identical between the sequences.
  • sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Preferred polypeptide sequences of the invention have a sequence identity in the CDR regions of at least 60%, more preferably, at least 70% or 80%, still more preferably at least 90% and most preferably at least 95%.
  • Preferred antibodies also have a sequence similarity in the CDR regions of at least 80%, more preferably 90% and most preferably 95%.
  • DNA molecules of the invention The present invention also relates to the DNA molecules that encode an antibody of the invention. These sequences include, but are not limited to, those DNA molecules set forth in Figures la and 2a.
  • DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the invention may be described by reference to their physical properties in hybridization.
  • DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homolog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, for example, Sambrook et al, 1989 and Ausubel et al, 1999. Structural similarity between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another. As used herein, the term "stringency" refers to the extent that the conditions disfavor hybridization.
  • T m is the melting temperature of a nucleic acid duplex
  • T m 69.3 + 0.41(G+C)%
  • the T m of a duplex DNA decreases by 1 °C with every increase of 1% in the number of mismatched base pairs.
  • Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the "binding" phase and the “washing” phase. First, in the binding phase, the probe is bound to the target under conditions favoring hybridization. Stringency is usually controlled at this stage by altering the temperature. For high stringency, the temperature is usually between 65 °C and 70°C, unless short ( ⁇ 20 nt) oligonucleotide probes are used.
  • a representative hybridization solution comprises 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100 ⁇ g of nonspecific carrier DNA. See Ausubel et al, (1999). Of course, many different, yet functionally equivalent, buffer conditions are known. Where the degree of relatedness is lower, a lower temperature may be chosen. Low stringency binding temperatures are between about 25°C and 40°C. Medium stringency is between at least about 40°C to less than about 65°C. High stringency is at least about 65°C. Second, the excess probe is removed by washing. It is at this stage that more stringent conditions usually are applied. Hence, it is this "washing" stage that is most important in determining relatedness via hybridization. Washing solutions typically contain lower salt concentrations.
  • One exemplary medium stringency solution contains 2X SSC and 0.1% SDS.
  • a high stringency wash solution contains the equivalent (in ionic strength) of less than about 0.2X SSC, with a preferred stringent solution containing about 0.1X SSC.
  • the temperatures associated with various stringencies are the same as discussed above for "binding.”
  • the washing solution also typically is replaced a number of times during washing. For example, typical high stringency washing conditions comprise washing twice for 30 minutes at 55° C. and three times for 15 minutes at 60° C.
  • the present invention includes nucleic acid molecules that hybridize to the molecules of set forth in Figures la and 2a under high stringency binding and washing conditions, where such nucleic molecules encode an antibody or functional fragment thereof having properties as described herein.
  • Preferred molecules are those that have at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein.
  • nucleic acid position 7 in SEQ ID NOS: 1, 2, 3 and/or 4 can be substituted from a C to an G, thereby changing the codon from CAA to GAA.
  • Functionally Equivalent Variants Yet another class of DNA variants within the scope of the invention may be described with reference to the product they encode (see the peptides listed in figures lb and 2b).
  • SEQ ID NOS: 9 and 26 are an example of functionally equivalent variants, as their nucleic acid sequences are different, yet they encode the same polypeptide — SEQ ID NO: 13. It is recognized that variants of DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as completely synthetic DNAs. Methods of efficiently synthesizing ohgonucleotides in the range of 20 to about 150 nucleotides are widely available. See Ausubel et al. (1999).
  • Overlapping ohgonucleotides may be synthesized and assembled in a fashion first reported by Khorana et al (1971); see also Ausubel et al. (1999).
  • Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector. As indicated, a method of generating variants is to start with one of the
  • DNAs disclosed herein and then to conduct site-directed mutagenesis See Ausubel et al. (1999).
  • a target DNA is cloned into a single-stranded DNA bacteriophage vehicle.
  • Single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alterations).
  • the complementary strand is synthesized and the double stranded phage is introduced into a host.
  • Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing.
  • various methods are available that increase the probability that the progeny phage will contain the desired mutant. These methods are well known to those in the field and kits are commercially available for generating such mutants.
  • the present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention.
  • the recombinant constructs of the present invention are used in connection with a vector, such as a plasmid or viral vector, into which a DNA molecule encoding an antibody of the invention is inserted.
  • a vector such as a plasmid or viral vector
  • the encoded gene may be produced by techniques described in Sambrook et al, 1989, and Ausubel et al, 1999.
  • the DNA sequences may be chemically synthesized using, for example, synthesizers.
  • Recombinant constructs of the invention may be contained within expression vectors that are capable of expressing the RNA and/or protein products of the encoded DNA(s).
  • the vector may further comprise regulatory sequences, including a promoter operably linked to the open reading frame (ORF).
  • the vector may further comprise a selectable marker sequence. Specific initiation and bacterial secretory signals also may be required for efficient translation of inserted target gene coding sequences.
  • the present invention further provides host cells containing at least one of the DNAs of the present invention. The host cell can be virtually any cell for which expression vectors are available.
  • eukaryotic host cell such as a mammalian cell
  • a lower eukaryotic host cell such as a yeast cell
  • a prokaryotic cell such as a bacterial cell.
  • Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation or phage infection.
  • Bacterial Expression Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and bacterial origin of replication derived from commercially available plasmids typically containing elements of the well known cloning vector pBR322 (ATCC 37017).
  • the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced for, say, the generation of antibodies or to screen peptide libraries, then vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of an antibody contemplated by the invention.
  • a "therapeutically effective" amount hereby is defined as the amount of an antibody that is of sufficient quantity to effectively block the interaction between ICAM-1 and ligand(s) thereto in the treated area — either as a single dose or according to a multiple dose regimen, alone or in combination with other agents, which leads to the alleviation of an adverse condition, yet is toxicologically tolerable.
  • the subject may be a human or non-human animal (e.g-, rabbit, rat, mouse, monkey or other lower-order primate). Some methods contemplate combination therapy with known medicaments.
  • Antibodies according to the invention can be used as a therapeutic or a diagnostic tool in a variety of situations where ICAM-1 and/or a ligand to ICAM- 1 (e.g., LFA-1) is/are undesirably expressed or found.
  • Preferred disorders and conditions for treatment with an antibody of the invention are rheumatoid arthritis, psoriasis, deep dermal burn, ocular inflammation such as diabetic retinopathy and age-related macular degeneration, and cancer.
  • an anti-ICAM-1 antibody of the invention can be used to treat tissue necrosis, which is distinct from tissue inflammation.
  • T cell inflammatory responses such as inflammatory skin diseases in addition to psoriasis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); adult respiratory distress syndrome; dermatitis; encephalitis; meningitis; certain allergic conditions (e.g., eczema and asthma); atherosclerosis; stroke; uveitic; autoimmune diseases such as systemic lupus erythematosus (SLE), Reynaud's syndrome, diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, autoimmune thyroiditis, juvenile onset diabetes, Sjorgen's syndrome, and immune responses associated with delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, polymyositis, sarcoidosis, vasculitis and granulomatosis; diseases involving leukocyte diapedesis; per
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • An antibody of the invention can be administered by any suitable means, which can vary depending on the type of disorder being treated. Possible administration routes include parenteral (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous), intrapulmonary, intraocular, intravitreal and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration.
  • an antibody of the invention might be administered by pulse infusion, with, e.g., declining doses of the antibody.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, and whether other drugs are administered.
  • the skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated. Determining a therapeutically effective amount of the novel polypeptide, according to this invention, largely will depend on particular patient characteristics, route of administration, and the nature of the disorder being treated.
  • determining a therapeutically effective amount will depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples. As indicated, an antibody of the invention can be used to treat asthma.
  • the agents of the present invention intranasally, e.g., by nasal spray or swab. It is especially preferred to administer such agents by oral inhalation, or via an oral spray or oral aerosol.
  • the administration may be by continuous infusion, or by single or multiple boluses.
  • An anti-ICAM-1 antibody or antibody fragment of the invention may be administered either alone or in combination with one or more additional anti-asthma agents (such as methylxanthines (such as thiophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins, and ephedrine), glucocorticoids (such as hydrocortisone), chromones (such as cromolyn sodium) and anticholinergics (such as atropine).
  • An antibody of the invention can be used to treat various eye disorders.
  • ICAM-1 is expressed on inflammatory cells belonging to ocular tissue.
  • ICAM-1 may be caused from trauma, disease such as age- related macular degeneration or diabetic retinopathy, post-surgical inflammation or post-surgical corneal graft rejection, for example.
  • trauma disease
  • disease such as age- related macular degeneration or diabetic retinopathy, post-surgical inflammation or post-surgical corneal graft rejection
  • the binding of cell adhesion molecules e.g., ICAM-1 to their corresponding counter-receptors expressed on ocular tissues fosters the development of inflammation in the eye.
  • an antibody of the invention to block ICAM-1 can be inhibited.
  • the present invention relates to a method of treating a subject suffering from a disease or condition selected from the group consisting of ocular inflammation, trauma, disease, post-surgical inflammation and post-surgical corneal graft rejection, which method comprises the steps of administering to the subject a therapeutically effective amount of an antibody contemplated herein, along with a suitable pharmaceutically acceptable diluent, carrier, or excipient.
  • a suitable pharmaceutically acceptable diluent, carrier, and excipients are well known in the art. The skilled artisan will appreciate that the 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.
  • These substances may be administered by techniques known in the art (e.g., systemically, via periocular injection, or topically to the eye as eye drops or ophthalmic ointment). Local administration can limit potential systemic side effects, but still allow control of ocular inflammation.
  • an antibody of the invention might be administered intracamerally into the anterior chamber or vitreous of the eye, via a depot attached to the intraocular lens implant inserted during surgery, or via a depot placed in the eye sutured in the anterior chamber or vitreous.
  • an anti-ICAM- 1 antibody of the invention may be employed in order to image or visualize a site of possible infection and/or inflammation in a patient.
  • an antibody can be detectably labeled, through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc. Procedures for accomplishing such labeling are well known to the art. Clinical application of antibodies in diagnostic imaging are reviewed by Grossman, H. B., Urol. Clin. North Amer. 13:465-474 (1986)), Unger, E. C. et al., Invest. Radiol. 20:693-700 (1985)), and Khaw, B.
  • the detection of foci of such detectably labeled antibodies is indicative of a site of inflammation or tumor development.
  • this examination for inflammation is done by removing samples of tissue or blood and incubating such samples in the presence of the detectably labeled antibodies.
  • this technique is done in a non-invasive manner through the use of magnetic imaging, fluorography, etc.
  • Such a diagnostic test may be employed in monitoring organ transplant recipients for early signs of potential tissue rejection.
  • Such assays may also be conducted in efforts to determine an individual's predilection to, e.g., rheumatoid arthritis or other chronic inflammatory or LFA-1 -mediated diseases described herein.
  • An antibody of the invention also can be used to take advantage of the observation made in U.S. Patent No. 5,472,849 issued to Rothlein et al. (which is hereby incorporated by reference), namely, the discovery of cICAM-1 in bodily fluids and the observation that higher than normal levels of cICAM-1 in the bodily fluids of a patient are indicative of the presence of inflammation, i.e., the reactions of the specific defense system, and the reactions of the non-specific defense system. Accordingly, an antibody of the invention can be utilized in the assays described in the '849 patent.
  • compositions can be formulated according to known methods to prepare pharmaceutically useful compositions, wherein an antibody of the invention (including any functional fragment thereof) is combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • a pharmaceutically acceptable carrier vehicle Suitable vehicles and their formulation are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990).
  • REMINGTON'S PHARMACEUTICAL SCIENCES 18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990.
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of one or more of the antibodies of the present invention, together with a suitable amount of carrier vehicle. Preparations may be suitably formulated to give controlled-release of the active compound.
  • Controlled-release preparations may be achieved through the use of polymers to complex or absorb anti-ICAM-1 antibody.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinyl-acetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate anti-ICAM-1 antibody into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine- microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the invention further is understood by reference to the following examples, which are intended to illustrate and, hence, not limit the invention. EXAMPLES
  • Phages were PEG-precipitated from the supernatant, resuspended in PBS / 20 % glycerol and stored at -80°C. Phage amplification between two panning rounds was conducted as follows: mid- log phase TGI cells were infected with eluted phages and plated onto LB -agar supplemented with 1 % of glucose and 34 ⁇ g/ml of chloramphenicol (LB-CG). After overnight incubation at 30°C, colonies were scraped off, adjusted to an OD 6 oo of 0.5 and helper phage added as described above.
  • LB-CG chloramphenicol
  • HuCAL GOLD ® antibody-phages were divided into three pools comprising different NH master genes. These pools were individually subjected to a solid phase panning on ICAM-1-Fc fusion protein (R&D Systems, Minneapolis, M ⁇ ) directly coated on Maxisorp ® wells ( ⁇ alge ⁇ unc, Rochester, ⁇ Y) as solid support (1 st and 3 rd round) and to an intermediate panning-step on ICAM-1 expressing JY lymphoma cells (2 nd round).
  • ICAM-1-Fc fusion protein R&D Systems, Minneapolis, M ⁇
  • Maxisorp ® wells ⁇ alge ⁇ unc, Rochester, ⁇ Y
  • the antigen was diluted to a concentration of 10 ⁇ g/ml in PBS and coated, 0.3 ml per well, overnight at 4°C. After one washing step with PBS / 1 % Tween20 (Sigma, St. Louis, MO), the wells were incubated with blocking buffer containing 2 % BSA (Sigma) and human gamma-globulin at 10 ⁇ g/ml in PBS for 2 h at room temperature (RT). Prior to the selections, the phages were pre-adsorbed in blocking buffer for 2 h at RT. Four different pannings were performed: A-D.
  • pannings A and D no specific blocking reagents were added.
  • B recombinant ICAM-1 domains DI and D3 were included in the blocking buffer at concentrations of 50 and 35 ⁇ g/ml, respectively.
  • ICAM-1 domain DI specific Fabs 3090, 1555 and 1557 were added to the blocking buffer, each at a concentration of 10 ⁇ g/ml.
  • 0.3 ml of the pre-adsorbed phages were added to the wells and incubated for 1 h at RT; This incubation was followed by 5 wash cycles with both PBS / 1 % Tween20 and PBS.
  • Bound phages were eluted by competitive elution with BIRR-1 mAb at 25 ⁇ g/ml for panning A and by standard acid elution for pannings B, C and D.
  • 20 mM DTT, 10 mM Tris pH 8.0 was added for 10 min at RT in order to remove residual, strongly binding phages.
  • the eluted phages from both steps were pooled and 10 ⁇ l thereof were subjected to titration.
  • the remaining phage eluate was mixed with a 20 ml culture of E. coli TGI grown to an OD o 0nm of 0.6-0.8 and incubated for 45 min at 37°C.
  • the bacterial pellet was resuspended in 2 x TY medium, plated on 2 x TY-CG agar plates and incubated overnight at 30°C. The selected clones were then scraped from the plates, rescued and amplified as described above.
  • the second round of selections was performed on JY lymphoma cells with 2.6 x 10 7 cells per panning. The same panning parameters were applied as in the solid phase panning, except that BSA was replaced by 5% fetal calf serum (FCS) in the blocking buffer. Cells were incubated with pre-blocked phages for 2 h at 4°C on an overhead shaker, then washed three times with PBS, 5% FCS and once with PBS.
  • FCS fetal calf serum
  • the Fab encoding inserts of the selected HuCAL GOLD ® phages were subcloned into the expression vector pMORPHx9_Fab_FS (Rauchenberger et al, 2003) to facilitate rapid expression of soluble Fab.
  • the DNA of the selected clones was digested with Xb ⁇ i and EcoR ⁇ , thereby cutting out the Fab encoding insert (ompA-VLCL and phoA-Fd), and cloned into the Xbal / EcoRI cut vector pMORPHx9_Fab_FS.
  • Fabs expressed in this vector carry two C-terminal tags
  • the scFv-encoding inserts of the selected clones were subcloned into the expression vector pMORPHx7_FS via Xbal I EcoRI as described in Krebs et al., 2001.
  • the scFv 1555 selected from these pannings was converted into the Fab format by the following steps: the scFv-derived VH region was inserted via Mfel and Blbl sites into expression vector pMORPHx9_Fab_FS.
  • the N-lambda insert was amplified by PCR from the corresponding scFv expression vector, followed by digestion with enzymes EcoRV and Dralll.
  • Periplasmic extracts of cell pellets were prepared and Fab fragments isolated by Strep-tactin ® chromatography (IBA GmbH). The apparent molecular weights were determined by SDS-PAGE and size exclusion chromatography (SEC). Concentrations were determined by UN- spectrophotometry.
  • SEC size exclusion chromatography
  • variable domain fragments of heavy (NH) and light chains (NL) were subcloned from Fab expression vectors into appropriate pMORPH_hIg vectors. Restriction enzymes EcoRI, Mfel, Blpl were used for subcloning of the VH domain fragment into pMORPH_hIgG4-l; EcoRN; Bs ⁇ Wi, for subcloning of the NL domain fragment into pMORPH_hIg ⁇ _l or pMORPH_h_ Ig ⁇ _l vectors.
  • IgG constructs were expressed in H ⁇ K293 cells (ATCC CRL-1573) by transient transfection using standard calcium phosphate-D ⁇ A-coprecipitation technique .
  • 1555 IgG4 the complete heavy and light chains including constant regions were transferred into the pEE vector series (Lonza Biologies, Slough, UK) resulting in a pEE double gene vector encoding for heavy and light chains of 1555 IgG4 ⁇ .
  • the construct was transfected into the appropriate NSO cell line obtained from Lonza (ATCC CRL-1827). Stable clones were selected for glutamine synthetase (GS) activity.
  • GS glutamine synthetase
  • GS positive clones were screened for IgG production; a master clone (8E7) was selected for IgG production and expanded to growth in T175 flasks. Production of more than 100 mg IgG was performed in CELLine 1000 production units. IgGs were purified from cell culture supernatants by affinity chromatography via a Protein A Sepharose column. Further down stream processing included a buffer exchange by gel filtration and sterile filtration of purified IgG. Quality control revealed a purity of >90 % by reducing SDS-PAGE and >90 % monomeric IgG as determined by analytical size exclusion chromatography. The endotoxin content of the material was determined by a kinetic LAL-based assay (Cambrex European Endotoxin Testing Service, Belgium).
  • Morphosys' Fab expression vector pMORPHx9_Fab_FS Expression and purification of the mouse-human chimeric BIRR-1 Fab was performed as described above for HuCAL ® Fabs. For the production of research grade material, the cleared cell lysate of six 10-liter fermentations was purified over a Streptactin ® column (IB A GmbH, G ⁇ ttingen, Gemany), followed by a concentration step, gel filtration (desalting and polishing step) and endotoxin removal.
  • ICAM-1 Domains Various domains of ICAM-1 were expressed using MorphoSys' proprietary ESTTM technology (Frisch et al., 2003). Five fragments comprising domain 1 (DI, aa 1-88), domain 2 (D2; aa 89-185), domain 3 (D3; aa 186-284), all three N-terminal Ig-like domains (D1-D3; aa 1-284) and domains 4-5 (D4-D5; aa 285-453) were amplified by PCR from ICAM-1 cDNA. These DNA fragments were ligated to the NI -domain of g3p of filamentous phage Ml 3. Expression in E. coli and purification were done as described (Frisch et al., 2003).
  • EXAMPLE 3 Cell Culture and Cell Adhesion Assays A. Cells and cell culture All cells were cultured under standardized conditions at 37°C and 5% CO 2 in a humidified incubator.
  • the B cell lymphoma line JY was a kind gift from Boehringer Ingelheim (Vienna, Austria).
  • SKW-3 and ECV304 cell lines were purchased from Cell Lines Service (Heidelberg, Germany), the mouse endothelial cell line bEnd.3 and Chinese hamster ovary (CHO) cells were obtained from ATCC (Manassas, VA). JY and SKW-3 cells were grown in RPMI 1640 (PAN biotech GmbH, Aidenbach, Germany).
  • ECV304 cells were cultured in Medium 199 (Invitrogen, Carlsbad, CA). For bEnd.3 and CHO cells, DMEM (Invitrogen) was used. All media were supplemented with 10 % FCS (PAN biotech GmbH), 50 U/ml penicillin, 50 ⁇ -g/ml streptomycin and 2 mM glutamine (Invitrogen).
  • JY cell-cell adhesion assay The semi-quantitative JY cell-cell adhesion assay was described previously (Rothlein et al., 1986). Briefly, 2 x 10 5 cells in 100 ⁇ l RPMI 1640 medium with 10% FCS were added to 80 ⁇ l of test compound (e.g. antibody) diluted in PBS (Invitrogen) in a flat-bottomed 96-well plate (Nunc). After incubation for 10 min at RT, 20 ⁇ l of phorbol 12-myristate 13 -acetate (PMA, Sigma), at a concentration of 1 ⁇ g/ml in PBS [final: 100 ng/ml], was added resulting in a final volume of 200 ⁇ l.
  • test compound e.g. antibody
  • PBS Invitrogen
  • PMA phorbol 12-myristate 13 -acetate
  • the cells were then incubated at 37°C between 10 and 60 min until evaluation with an inverted microscope.
  • the degree of aggregation i.e. percentage of cells within aggregates
  • the degree of aggregation was scored by two independent observers applying the following scale: 0: no aggregation; 0.5: ⁇ 10% 1: - 20%; 1.5: - 30%; 2: - 40%; 2.5: ⁇ 50%; 3: ⁇ 60%; 3.5: - 70%; 4: > 80% aggregation.
  • the degree of aggregation was normalized to values obtained in the absence of antibodies.
  • the percentage of inhibition of cell adhesion was then calculated as: 100% - % of aggregation.
  • the chamber was inverted for 7 min at RT to allow unbound cells to fall out of the wells. After this washing procedure, the fluorescence corresponding to the adherent cells was again measured and the percentage of adherent cells was calculated as [fluorescence after wash / fluorescence before wash] x 100 %.
  • a standard analysis software PRISM ® , Graph Pad Software
  • Fluorescence labeling of SKW-3 cells SKW-3 cells were resuspended in pre-warmed culture medium (RPMI 1640 with 10 % FCS) at 2 x 10 6 cells/ml and Calcein AM (Molecular Probes, Eugene, OR) was added at a final concentration of 1 ⁇ g/ml.
  • EXAMPLE 4 Preparation of Single Cell Suspensions from Peripheral Blood and Spleen
  • PBMC Peripheral blood mononuclear cells
  • Histopaque ® -1077 Single cell suspensions prepared from rabbit spleen were obtained from Aurigon (Tutzing, Germany).
  • Red blood cells were depleted from these cell suspensions by incubation in ACK Lysis Buffer (0.15 MNH 4 C1, 10 mM KHCO 3 , 0.1 M EDTA) for 5 min at RT. Cells were washed twice with PBS and then further processed for immune fluorescence staining and flow cytometry.
  • EXAMPLE 5 Immune Fluorescence and Flow Cytometry Techniques All stainings were performed in round bottom 96-well culture plates (Nalge Nunc) with 2 x 10 5 cells per well. Cells were incubated with Fabs or IgG antibodies at the indicated concentrations in 50 ⁇ l FACS buffer (PBS, 3% FCS, 0.02% NaN 3 ) for 40 min at 4°C. Cells were washed twice and then incubated with R-Phycoerythrin (PE) conjugated goat-anti-human or goat-anti-mouse IgG (H+L) F(ab') 2 (Jackson Immuno Research), diluted 1:200 in FACS buffer, for 30 min at 4°C.
  • PE R-Phycoerythrin
  • EXAMPLE 6 Enzyme Linked ⁇ mmunosorbent Assay (ELISA) Techniques Antigens such as ICAM-Fc recombinant proteins (R&D Systems) or protein domains expressed via MorphoSys' EST technology (WO 01/02588) were coated at 10 ⁇ g/ml (5 ⁇ g/ml for ESTs) in PBS onto Maxisorp ® microtiter plates overnight at 4°C. Alternatively, goat anti-human IgG (Fc ⁇ fragment specific) antibodies coated at 10 ⁇ g/ml in PBS at 4°C overnight, were used for capturing of ICAM-1-Fc fusion proteins applied at 1 ⁇ g/ml in PBS for 2 h at RT.
  • ELISA Enzyme Linked ⁇ mmunosorbent Assay
  • the following secondary antibodies were applied: peroxidase (PO)-conjugated anti-His6 mAb (Roche, Basel, Switzerland) for detection of Fab-dHLX_MH antibodies; alkaline phospatase (AP)-conjugated AffiniPure F(ab') 2 fragment, goat anti-human IgG (Jackson Immuno Research) or alternatively (in case ICAM-1-Fc was captured) a mixture of AP-conjugated goat anti-human kappa light chain (1:10.000) and goat anti-human lambda light chain (1:5000) antibodies (Sigma) for detection of HuCAL ® Fab and IgG.
  • PO peroxidase
  • AP alkaline phospatase
  • AffiniPure F(ab') 2 fragment goat anti-human IgG
  • ICAM-1-Fc goat anti-human IgG
  • ICAM-1-Fc For each capturing step, ICAM-1-Fc at a concentration of 5 ⁇ g/ml was injected for 3 min at a flow rate of 5 ⁇ l/min. Kinetic measurements were done in PBS (136 mM NaCl, 2.7 mM KCl, lOmM Na 2 HPO 4 , 1,76 mM KH 2 PO 4 pH 7.4) at a flow rate of 20 ⁇ l/min using Fab concentration range from 1.5-500 nM. Injection time for each concentration was 1 min, followed by 3 min dissociation phase. For regeneration 5 ⁇ l lOmM HCl was used. All sensograms were fitted globally using BIA evaluation software 3.1 (Biacore).
  • EXAMPLE 8 Selection of ICAM-1 Specific Antibodies from HuCAL ® Libraries For the generation of therapeutic antibodies against ICAM-1, selections with the MorphoSys HuCAL ® -scFv 3 and HuCAL GOLD ® phage display libraries were carried out.
  • HuCAL ® -scFv 3 is a single chain Fv library with diversified H- and L-chain CDR3 regions
  • HuCAL GOLD ® is a Fab library, in which all six CDRs are diversified.
  • Both phagemid libraries are based on the HuCAL ® concept (Knappik et al., 2000) and employ the CysDisplayTM technology for linking Fab or scFv to the phage surface (L ⁇ hning, 2001).
  • ICAM-1 expressing cell lines JY and ECV304 revealed 40 unique cell binders out of 1104 screened clones. These 40 Fab clones were purified on a small scale and further screened in a cell-based assay for their potential to inhibit homotypic adhesion of JY lymphoma cells, which is dependent on the interaction of ICAM-1 and LFA-1 (Rothlein et al., 1986). Four Fabs that demonstrated significant inhibition of JY cell-cell adhesion were analyzed in a second functional assay, where the adhesion of LFA-1 expressing human T cells (leukemia T cell line SKW-3) on recombinant ICAM-1 was monitored.
  • LFA-1 expressing human T cells leukemia T cell line SKW-3
  • This assay solely focuses on the interaction between ICAM-1 and LFA-1, not being influenced by other receptor- ligand interactions.
  • two binders designated 3041 and 3043, which showed the strongest inhibitory activity, were selected as lead antibody candidates (LACs) for further studies.
  • LACs lead antibody candidates
  • a third clone, 3040 was selected due to its particular antigen binding characteristics. Clones 3040 and 3041 were isolated from conventional pannings without specific selection parameters, while clone 3043 is derived from a panning that included Fabs against D 1 as blocking reagents.
  • EXAMPLE 9 Characterization of Antibodies
  • the four ICAM-1 specific HuCAL ® antibodies 1555, 3040, 3041 and 3043 were characterized with respect to specificity, affinity/avidity and in vitro efficacy.
  • the four LACs were generated in monovalent Fab format and in bivalent antibody formats such as human IgG4 and Fab-dHLX, the latter was used only in immunohistochemistry studies.
  • Boehringer Ingelheim's ICAM-1 specific mouse IgG2a monoclonal antibody BIRR-1 served as reference compound, against which the HuCAL ® antibodies were compared in various assays.
  • the mouse lg variable regions were cloned and genetically fused to human CHl/CL regions.
  • the resulting "chimeric" BIRR- 1 Fab construct was expressed in E. coli, in the same system as MorphoSys' HuCAL ® Fabs (for details, see example 2C).
  • the in vitro efficacy of the LACs was characterized in two types of cell adhesion assays already applied during antibody selection.
  • the cell adhesion assays mimic cell-cell interactions, which take place in vivo in inflammatory immune responses and thus facilitate the evaluation of the therapeutic potential of the lead candidates.
  • the inhibition of cell adhesion was assessed for the monovalent Fab, as well as for the bivalent IgG4 format of all LACs.
  • the antibodies were tested for inhibition of the homotypic adhesion of human lymphoblastoid JY cells, which is dependent on the interaction between ICAM-1 and LFA-1 (Rothlein et al., 1986).
  • This assay is semi-quantitative and was evaluated by scoring the percentage of cell aggregation and the inhibition of aggregation by LACs as described in example 3B. All four LACs were capable of inhibiting ICAM-1 /LFA-1 mediated JY cell-cell adhesion in a concentration dependent manner.
  • All HuCAL ® clones showed similar inhibitory activities, which were slightly weaker than that of the murine IgG2a (BIRR-1) antibody.
  • BIRR-1 murine IgG2a
  • Fabs clear differences in the inhibitory activity are evident, with Fab 1555 being the most efficient one and even stronger than BIRR-1 Fab.
  • Fabs 3041 and 3043 were both slightly weaker than BIRR-1 Fab followed by Fab 3040.
  • the LACs were evaluated in a cell-protein adhesion assay as described in example 3C.
  • the binding of LFA-1 expressing leukocytes on immobilized ICAM-1 and thus the inhibitory potency of the LACs were determined in a quantitative way.
  • Two out of four HuCAL ® IgG4s, 1555 and 3041 showed very similar inhibitory activities corresponding to IC50 values of 2-3 nM, while the other two IgG4 antibodies, 3040 and 3043, are slightly weaker (IC50 values of 11 and 7 nM, respectively).
  • ICAM-2 and ICAM-3 which display a sequence identity with ICAM-1 of 36 % and 52 %, respectively (de Fougerolles et al., 1993; Gahmberg et al., 1997).
  • ICAM-2 shows a similar tissue distribution as ICAM-1, but is expressed constitutively and not induced by cytokines.
  • ICAM-3 is a costimulatory molecule found on leukocytes but absent from endothelial cells (for review see Gahmberg et al., 1997). Binding of antibodies to recombinant ICAM-2 and ICAM-3 proteins was analyzed in ELISA.
  • Binding of 1555 and 3041 is neither inhibited by BIRR-1 nor by RRl.l.
  • LAC 3040 may recognize a determinant consisting of residues in DI and D2 as it competes with RRl.l as well as BIRR-1. This finding was further supported by Biacore experiments showing inhibition of binding of 3040 to immobilized recombinant ICAM- 1 by BIRR- 1 and vice versa.
  • the Kd values ranging from 3 to 31 nM.
  • Both the indirect Biacore and the FACS based Scatchard analysis resulted in similar affinity rankings although the FACS based assays yielded 2-3 fold lower Kd values for two of the binders, 3040 and 3041.
  • the different affinities reflect conformational differences in the epitopes of the immobilized recombinant ICAM-1 and the native protein on the cell surface recognized by these two LACs.
  • the affinity ranking of Fabs nicely correlated with the ranking of functional activities in in vitro adhesion assays.
  • Fab 1555 which displayed the strongest affinity, is the most efficient inhibitor of cell adhesion, while Fab 3040 with the weakest affinity showed the lowest activity in inhibition of cell adhesion.
  • the functional affinities (avidities) of HuCAL ® IgG4 and BIRR-1 mIgG2a were determined in the same cell based system as for the Fabs. All IgG4 binders are characterized by similar binding strength in the low nanomolar range comparable to BIRR-1 mIgG2a. In the IgG format, 3040 and 3041 antibodies showed the strongest binding (both 0.7 nM), similar to BIRR-1 (1.0 nM) and slightly stronger than 1555 (1.7 nM) and 3043 (2.5 nM). In particular, clones
  • ICAM-1 is expressed at moderate levels on vascular endothelial cells, on activated lymphocytes and on monocytes. In addition, ICAM-1 is detected in germinal center cells as well as in fibroblast-like and epithelial cells of various organs (Dustin et al., 1986). The low expression of ICAM-1 on endothelial and epithelial cells is strongly induced by inflammatory cytokines such as TNF-alpha, IL-1 and INF-gamma (Pober et al., 1986; Dustin et al, 1986).
  • LAC 1555 showed immunoreactivity with Rhesus, Baboon and faintly with Marmoset tissue, but was negative on Cynomolgus.
  • antibody 3043 demonstrated specific staining of all four studied NHP species, while 3040 reacted with Rhesus and Baboon, and 3041 recognized Baboon only.
  • the mouse mAb BIRR-1 was negative on Baboon, but positive on the other three NHP species, Cynomolgus, Marmoset and Rhesus. Since rabbits are used as experimental animals in burn injury models
  • EXAMPLE 10 Production and stability testing of antibody research grade material For preclinical in vivo studies, psoriasis and rabbit burn animal models (described elsewhere), research grade material of the antibodies 1555 IgG4, chimeric BIRR-1 Fab and BIRR-1 mouse IgG2a was generated in amounts of 100 mg up to 625 mg.
  • BIRR-1 mIgG2a was produced from hybridoma cells, human 1555 IgG4 was expressed in Lonza's GS System in NS0 cells, and chimeric BIRR-1 Fab was produced in E. coli in a fermenter-based process.
  • the IgG compounds showed a purity of more than 90 % and an endotoxin content below 1 EU/mg, the chimeric BIRR-1 Fab demonstrated a purity of at least 86 % in the native state and contained approximately.4 EU endotoxin /mg.
  • the binding to the target antigen was confirmed in Biacore and ELISA for IgG and Fab, respectively. All three compounds showed bioactivity, which was assessed in the JY cell-cell adhesion assay described above. Stability of the material was tested after one and three months at storage temperatures of -80°C, 4°C and ambient temperature.
  • EXAMPLE 11 The use of antibodies to treat Psoriasis
  • LAC 1555 in the IgG 4 format was used in conjunction with a psoriasis SCID mouse model.
  • mice were anaesthetized by intraperitoneal injection of lOOmg/kg ketamine and 5mg/kg xylazine.
  • Spindle-shaped pieces of split-skin measuring 1cm in diameter were grafted onto corresponding excisional full-thickness defects of the shaved central dorsum of the mice and fixed by 6-0 atraumatic monofilament sutures.
  • the grafts were protected from injury by suturing a skin pouch over the transplanted area using the adjacent lateral skin. The sutures and over-tied pouches were left in place until they resolved spontaneously after 2-3 weeks.
  • mice were allowed 3 weeks for acceptance and healing onto the mice. During the following 4 weeks (days 22 to 50 after transplantation), the mice underwent one of the following protocols: daily intragastric applications of 200 ⁇ l PBS served as negative control; and the positive controls received daily intragastric applications of dexamathasone 0.2 mg/kg body weight in 200 ⁇ l PBS.
  • LAC 1555 was injected intraperitoneally every other day at a dose of 10 mg/kg body weight.
  • Three grafts of three different donors underwent an identical treatment protocol.
  • mice were sacrificed at day 50 and following excision with surrounding mouse skin the grafts were formalin-embedded. Subsequently, routine hematoxilin+eosin stainings were performed and the grafts were analysed with regard to their pathological changes both qualitatively (epidermal differentiation) and quantitatively (epidermal thickness, inflammatory infiltrate) by a blinded investigator as described previously (Boehncke et al. 1994). Briefly, maximal epidermal thickness was measured from the tip of the rete ridges to the border of the viable epidermis. The values were determined using an ocular micrometer, taking the mean of 10 consecutively measured rete ridges. The density of the inflammatory infiltrate was determined by counting the number of cells in three adjacent high power fields. Means and standard deviations were calculated for each treatment group.
  • EXAMPLE 12 Neutrophil Activation Studies Six antibodies were tested for neutrophil activation: BIRR-1 from CLB (murine), BIRR-1 from Bl (murine), LAC 1555 (In each of IgGl, IgG4 and Fab formats) and LAC 3041 in IgG4 format. The following table provides more detail, as well as their code name:
  • the DCFH oxidation method (see Bass et al. (1983), J Immunol 130: 1910- 1917), as modified for whole blood samples (see Vuorte et al. (1996), Scand J Immunol 43: 329-334), was used to evaluate neutrophil intracellular hydrogen peroxide formation in response to anti-ICAM-1 mAbs. Furthermore, cell surface expression of CD1 lb/CD 18 (Mac-1) was determined via flow cytometry. In detail, the anticoagulant ACD (56mM, final concentration in blood) was added to the polystyrene tubes (Falcon no. 2058, Becton Dickinson Labware, NJ, USA). Each tube was further supplemented with dextran (Rheomacrodex, Product no.
  • DCFH-DA was added into each tube at the final concentration of lOO ⁇ M in blood, and contents were prewarmed in a water bath at 37 °C in the dark.
  • erythrocytes were then lysed with an ice-cold lysing solution containing NH 4 C1 (8,26g/L), potassium bicarbonate (l,0g/L), and tetrasodium EDTA (0,037g/L).
  • the two tested BIRR-1 antibody samples induced neutrophil activation up to 12-fold (Mab 1) and 9-fold (Mab 2) dose-dependent increases in the mean of MFI values of neutrophil CDl lb-PE fluorescence. Consistent dose-dependent increases (up to 5-fold and 4-fold) were observed in the mean of MFI values of cell-associated DCF fluorescence of neutrophils incubated in whole blood with mAbs #1 and #2, respectively. On the contrary, CDl lb-PE and DCF fluorescence intensities (means of MFI) in neutrophils incubated with mAbs #3-6 were comparable to those seen with negative control samples.
  • mAb #4 (at 100 ⁇ g/ml) a moderate increase of CDl lb and DCF fluorescence was observed with only one of the three donors.
  • the murine IgG2a anti-CD3 mAb OKT3 and the bacterial peptide fMLP served as appropriate positive controls, stimulating both cell surface CDl lb and oxidative burst activity in neutrophils.
  • OKT3 induced a 9- and 6-fold increase of neutrophil CDl lb and DCF fluorescence, respectively.
  • fMLP induced a 16- and 6-fold increase of neutrophil CDl lb and DCF fluorescence, respectively.
  • mAbs #1 and #2 readily activated peripheral blood neutrophils in whole blood in vitro.
  • the neutrophil-activating effect was antibody dose-dependent and could be observed in the lowest antibody concentration of 1.0 ⁇ g/mL in blood.
  • mAbs #1 and #2 behaved similarly to the tested positive control antibodies, murine IgG2a anti-CD3 mAb OKT3, whose effector functions are known to be mediated by activation of the classical complement pathway.
  • the powerful effect of fMLP, a well known neutrophil agonist and another positive control compound, on neutrophil CDl lb expression was as expected. Taken together, the data clearly show a neutrophil-activating capacity of mAb R6.5 thus confirming results of Vuorte et al, supra.
  • HuCAL ® IgG4 antibodies 1555, 3041 or the Fab fragment of 1555 were exposed to whole blood, neutrophil CDl lb expression and intracellular DCFH oxidation remained at or near baseline levels of the negative control. In other words, no signs of neutrophil activation in vitro were observed.
  • HuCAL ® IgG4 antibodies 1555 in contrast to BIRR-1, HuCAL ® IgG4 antibodies 1555
  • mAb #4 i.e., mAb 1555 in a human IgGl format (which, when administered, the volunteer exhibited a moderate neutrophil response in terms of both CDl lb [5-fold increase at 100 ⁇ g/ml] and DCF [2-fold increase at 100 ⁇ g/ml]) could be used in vivo if the efficacy effects of such antibody are desired, given that neutrophil activation was much less pronounced compared to the effect induced by mAb R6.5.
  • Antibodies The following anti-ICAM-1 antibodies were used for epitope mappings:
  • the two N-terminal Ig-like domains DI (aa 1-88) and D2 (aa 89-185) of the intercellular adhesion molecule 1 (ICAM-1) were scanned with 13-mer peptides (11 amino acids overlap). This resulted in arrays of 87 peptides covering residues 1-185 of ICAM-1. Peptides #1 to #38 cover domain DI, peptides #39 to #44 consist of residues in DI and D2, peptides #45 to #87 are spanning D2.
  • PepSpot- Analysis The antigen peptides were synthesized on a cellulose membrane in a stepwise manner resulting in a defined arrangement (peptide array) and are covalently bound to the cellulose membrane.
  • Binding assays were performed directly on the peptide array.
  • an antigen peptide array is incubated with blocking buffer for several hours to reduce non-specific binding of the antibodies.
  • the incubation with the primary (antigen peptide-binding) antibody in blocking buffer is followed by the incubation with the peroxidase(POD)-labelled secondary antibody, which binds selectively the primary antibody.
  • a short T(Tween)-TBS- buffer washing directly after the incubation of the antigen peptide array with the secondary antibody followed by the first chemiluminescence experiment is made to get an first overview which antigen peptides do bind the primary antibody.
  • peroxidase-labelled secondary antibodies were used for detection: anti-human: peroxidase conjugate-goat anti-human IgG, gamma chain specific, affinity isolated antibody (Sigma-Aldrich, A6029) anti-mouse: peroxidase conjugate-sheep anti-mouse IgG, whole molecule, affinity isolated antigen specific antibody (Sigma-Aldrich, A5906)
  • Antibody 1555 shows a very weak binding to peptides #14 to #20 in the N-terminal region of the antigen as well as to peptides #35 to #40 with a very low signal intensity. The common motifs are highlighted in bold. The epitope could be described as a discontinuous epitope with regions covered by peptides #14 to #20 and peptides #35 to #40. All other signals (below 17500 BLU) for this epitope mapping can be considered as background. 14.
  • QPKLLGIETPLPK 15. KLLGIETPLPKKE 16. LGIETPLPKKELL 17. IETPLPKKELLLP 18. TPLPKKELLLPGN . 19. LPKKELLLPGNNR
  • antibody 3040 demonstrates stronger binding to peptides #17 to #20 and weak binding to peptides #15 and #16. All other signals (below 17500 BLU) for this epitope mapping can be considered as background.
  • the epitopes can be described as discontinuous epitopes with a dominant region covered by peptides #17 to #20. The common motifs are hightlighted in bold. 15. KLLGIETPLPKKE
  • LGIETPLPKKELL 17.
  • IETPLPKKELLLP 18.
  • TPLPKKELLLPGN 19.
  • LPKKELLLPGNNR 20.
  • Antibody 3043 For antibody 3043 the data were obtained after several washing steps to reduce non-specific binding (see 13.1. "Materials and Methods"). This antibody binds to peptides #17 to #20. The epitope can be described as a linear epitope. The common motif is highlighted in bold. All other signals (below 17500 BLU) for this epitope mapping can be considered as background. 17. IETPLPKKELLLP
  • Antibody PC-1 For antibody PCI the data were obtained after the first short T-TBS-buffer wash, which allows only a vague interpretation of the chemiluminescence results due to broad unspecific binding of the secondary antibody to the cellulose membrane. In contrast to antibody 1555 no chemiluminescence was visually detectable. Although there appears to be some weak binding to different regions, this binding is not considered as specific. Antibody PCI does not show any significant binding pattern on the peptides.
  • Antibodies 3040 and 3041 recognize discontinuous epitopes within domain 1 of ICAM-1 showing similar binding patterns on the antigen peptides.
  • antibody 1555 seems to have a similar discontinuous epitope, however, with a much weaker binding to the antigen peptides.
  • Antibody 3043 shows significant binding to peptides #17 to #20 but in contrast to antibodies 3040 and 3041 no other binding sites.
  • the epitope of 3043 can be described as a linear epitope within domain 1 of ICAM-1, which appears to overlap with the epitopes of 3040 and 3041.
  • For antibody PC-1 no significant binding pattern was detectable indicating that a conformation-dependent epitope might be recognized, which is supported by published data (Rothlein et al., Int Arch Allergy Immunol 1993; 100: 121-127).
  • ICAM-1 (CD54): a counter- receptor for Mac-1 (CDl lb/CD 18). J Cell Biol 111, 3129-3139.
  • Psoriasis as a model for T-cell-mediated disease immunobiologic and clinical effects of treatment with multiple doses of efalizumab, an anti-CD 11a antibody.
  • ICM-1 intercellular adhesion molecule-1
  • LFA-1 lymphocyte function-associated antigen 1
  • Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus. Cell 68, 63-69. Pober.J.S., Gimbrone,M.A., Jr., Lapierre,L.A., Mendrick,D.L., Fiers,W., Rothlein,R., and Springer,T.A. (1986). Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J Immunol 137, 1893-1896.
  • ICAM-1 affects reperfusion injury and graft function after cardiac transplantation. J Surg Res 57, 25-31.
  • ICAM-1 Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell 52, 925-933.
  • Tanaka,Y. (2001). The role of chemokines and adhesion molecules in the pathogenesis of rheumatoid arthritis. Drugs Today (Bare. ) 37, 411-484. Terajima,S., Higaki,M., Igarashi,Y., Nogita,T., and Kawashima,M. (1998). An important role of tumor necrosis factor-alpha in the induction of adhesion molecules in psoriasis. Arch Dermatol Res 290, 246-252.
  • the LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor- mediated activation of resting T cells. J Immunol 144, 4579-4586.
  • Trinucle ⁇ tide phosphoramidites ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600-7. Vuorte,J., Lindsberg,P.J., Kaste,M., Meri,S., Jansson,S.E., Rothlein,R, and

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des régions de liaison antigène, des anticorps et des fragments fonctionnels contenant ces régions de liaison antigène spécifiques de ICAM-1, qui jouent un rôle intégral dans divers troubles ou divers états. Ces anticorps, par conséquent, peuvent être utilisés pour traiter l'arthrite rhumatoïde, le psoriasis, les brûlures dermiques profondes, la rétinopathie diabétique et d'autres troubles divers associés à l'inflammation. Les anticorps de cette invention peuvent aussi être utilisés dans le domaine des diagnostics, ainsi que dans l'investigation du rôle de ICAM-1 dans la progression des troubles associés à un tissu inflammé. Cette invention concerne aussi des séquences d'acides nucléiques codantes pour les anticorps susmentionnés, des vecteurs contenant ceux-ci, des compositions pharmaceutiques et des kits avec des modes d'emploi.
PCT/IB2005/002041 2004-01-26 2005-01-26 Anticorps anti-icam 1 humains et utilisations de ceux-ci WO2005086568A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US53849904P 2004-01-26 2004-01-26
US60/538,499 2004-01-26
US57294804P 2004-05-21 2004-05-21
US60/572,948 2004-05-21

Publications (2)

Publication Number Publication Date
WO2005086568A2 true WO2005086568A2 (fr) 2005-09-22
WO2005086568A3 WO2005086568A3 (fr) 2006-05-04

Family

ID=34976014

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2005/002041 WO2005086568A2 (fr) 2004-01-26 2005-01-26 Anticorps anti-icam 1 humains et utilisations de ceux-ci

Country Status (1)

Country Link
WO (1) WO2005086568A2 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009021548A1 (fr) * 2007-08-10 2009-02-19 Wacker Chemie Ag Expression d'igg pleine longueur et sécrétion dans le milieu de culture de cellules procaryotes
WO2010016652A1 (fr) * 2008-08-08 2010-02-11 Snu R&Db Foundaton Anticorps modulant la différenciation et la fonction de cellules dendritiques par la molécule d'adhésion intercellulaire de liaison-1 et son utilisation
EP2468775A1 (fr) * 2005-12-12 2012-06-27 BioInvent International AB Anticorps anti-ICAM1 pour induction d'apoptose
WO2011160078A3 (fr) * 2010-06-17 2012-07-19 The Johns Hopkins University Utilisation de régions variables de chaîne lourde variable dérivées de camélidés ciblant cd18 et icam-1 en tant que microbicide pour prévenir la transmission de vih-1
EP2563816A4 (fr) * 2010-04-27 2013-10-02 Ca Nat Research Council Anticorps à domaine unique anti-icam-1 et leurs utilisations
WO2014042305A1 (fr) * 2012-09-14 2014-03-20 서울대학교산학협력단 Anticorps d'icam－1 et utilisation associée
WO2018017786A3 (fr) * 2016-07-20 2018-03-01 Janssen Pharmaceutica Nv Anticorps anti-gprc5d, molécules bispécifiques de liaison à l'antigène qui se lient à gprc5d et cd3 et leurs utilisations
WO2018204976A1 (fr) * 2017-05-09 2018-11-15 The Council Of The Queensland Institute Of Medical Research Agents anti-inflammatoires et méthodes de traitement
EP3909979A4 (fr) * 2018-12-31 2022-09-14 Kumho HT, Inc. Anticorps se liant de manière spécifique à icam-1 et utilisation associée
RU2789757C2 (ru) * 2018-12-31 2023-02-09 Кумхо ЭйчТи, Инк. Антитело, специфично связывающееся с icam-1, и его применение
KR20230059469A (ko) * 2021-10-26 2023-05-03 재단법인 아산사회복지재단 암 생존자 유래 항체 및 이의 용도

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030035798A1 (en) * 2000-08-16 2003-02-20 Fang Fang Humanized antibodies

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2468775A1 (fr) * 2005-12-12 2012-06-27 BioInvent International AB Anticorps anti-ICAM1 pour induction d'apoptose
US9803013B2 (en) 2005-12-12 2017-10-31 Bioinvent International Ab Biological materials and uses thereof
US8758749B2 (en) 2005-12-12 2014-06-24 Bioinvent International Ab Biological materials and uses thereof
WO2009021548A1 (fr) * 2007-08-10 2009-02-19 Wacker Chemie Ag Expression d'igg pleine longueur et sécrétion dans le milieu de culture de cellules procaryotes
AU2009280262B2 (en) * 2008-08-08 2012-09-06 Dinona Inc. Antibody modulating the differentiation and function of dendritic cells via binding intercellular adhesion molecule-1 and use thereof
CN102307900A (zh) * 2008-08-08 2012-01-04 狄诺纳有限公司 通过结合胞间粘附分子1调节树突细胞分化和功能的抗体及其用途
CN102307900B (zh) * 2008-08-08 2013-11-27 狄诺纳有限公司 通过结合胞间粘附分子1调节树突细胞分化和功能的抗体及其用途
US8026343B2 (en) 2008-08-08 2011-09-27 Dinona Inc. Antibody modulating the differentiation and function of dendritic cells via binding intercellular adhesion molecule-1 and use thereof
US9115198B2 (en) 2008-08-08 2015-08-25 Dinona Inc. Antibody modulating the differentiation and function of dendritic cells via binding intercellular adhesion molecule-1 and use thereof
WO2010016652A1 (fr) * 2008-08-08 2010-02-11 Snu R&Db Foundaton Anticorps modulant la différenciation et la fonction de cellules dendritiques par la molécule d'adhésion intercellulaire de liaison-1 et son utilisation
EP2563816A4 (fr) * 2010-04-27 2013-10-02 Ca Nat Research Council Anticorps à domaine unique anti-icam-1 et leurs utilisations
WO2011160078A3 (fr) * 2010-06-17 2012-07-19 The Johns Hopkins University Utilisation de régions variables de chaîne lourde variable dérivées de camélidés ciblant cd18 et icam-1 en tant que microbicide pour prévenir la transmission de vih-1
WO2014042305A1 (fr) * 2012-09-14 2014-03-20 서울대학교산학협력단 Anticorps d'icam－1 et utilisation associée
CN109715667A (zh) * 2016-07-20 2019-05-03 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
WO2018017786A3 (fr) * 2016-07-20 2018-03-01 Janssen Pharmaceutica Nv Anticorps anti-gprc5d, molécules bispécifiques de liaison à l'antigène qui se lient à gprc5d et cd3 et leurs utilisations
US10562968B2 (en) 2016-07-20 2020-02-18 Janssen Pharmaceutica Nv Anti-GPRC5D antibodies, bispecific antigen binding molecules that bind GPRC5D and CD3, and uses thereof
CN109715667B (zh) * 2016-07-20 2022-12-30 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
US11685777B2 (en) 2016-07-20 2023-06-27 Janssen Pharmaceutica Nv Anti-GPRC5D antibodies, bispecific antigen binding molecules that bind GPRC5D and CD3, and uses thereof
US11884722B2 (en) 2016-07-20 2024-01-30 Janssen Biotech, Inc. Anti-GPRC5D antibodies, bispecific antigen binding molecules that bind GPRC5D and CD3, and uses thereof
WO2018204976A1 (fr) * 2017-05-09 2018-11-15 The Council Of The Queensland Institute Of Medical Research Agents anti-inflammatoires et méthodes de traitement
EP3909979A4 (fr) * 2018-12-31 2022-09-14 Kumho HT, Inc. Anticorps se liant de manière spécifique à icam-1 et utilisation associée
RU2789757C2 (ru) * 2018-12-31 2023-02-09 Кумхо ЭйчТи, Инк. Антитело, специфично связывающееся с icam-1, и его применение
AU2019419832B2 (en) * 2018-12-31 2023-12-14 Kumho Ht, Inc. Antibody specifically binding to ICAM-1 and use thereof
US12269884B2 (en) 2018-12-31 2025-04-08 Kumho Ht, Inc. Antibody specifically binding to ICAM-1 and use thereof
KR20230059469A (ko) * 2021-10-26 2023-05-03 재단법인 아산사회복지재단 암 생존자 유래 항체 및 이의 용도
KR102711074B1 (ko) * 2021-10-26 2024-10-04 재단법인 아산사회복지재단 암 생존자 유래 항체 및 이의 용도

Also Published As

Publication number Publication date
WO2005086568A3 (fr) 2006-05-04

Similar Documents

Publication Publication Date Title
US11939395B2 (en) Generation and profiling of fully human HuCAL gold-derived therapeutic antibodies specific for human CD38
US9758590B2 (en) Anti-CD38 human antibodies and uses thereof
JP5926568B2 (ja) 抗ｃｄ３８ヒト抗体及びその用途
WO2005086568A2 (fr) Anticorps anti-icam 1 humains et utilisations de ceux-ci
CN1976950B (zh) 抗cd38人抗体及其用途
MX2008004897A (es) Generacion y perfilado de anticuerpos terapeuticos derivados de hucal gold totalmente humanos especificos para cd38 humano

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载