WO2001013937A1 - Peptides capables de moduler la fonction des membres de la famille cd66 (ceacam) - Google Patents

Peptides capables de moduler la fonction des membres de la famille cd66 (ceacam) Download PDF

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Publication number
WO2001013937A1
WO2001013937A1 PCT/US2000/023482 US0023482W WO0113937A1 WO 2001013937 A1 WO2001013937 A1 WO 2001013937A1 US 0023482 W US0023482 W US 0023482W WO 0113937 A1 WO0113937 A1 WO 0113937A1
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peptide
seq
cells
nos
cd66a
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PCT/US2000/023482
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English (en)
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WO2001013937A9 (fr
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Keith M. Skubitz
Amy P. N. Skubitz
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Skubitz Keith M
Skubitz Amy P N
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Priority to EP00957846A priority Critical patent/EP1212075A4/fr
Priority to CA002383562A priority patent/CA2383562A1/fr
Priority to AU69407/00A priority patent/AU6940700A/en
Publication of WO2001013937A1 publication Critical patent/WO2001013937A1/fr
Publication of WO2001013937A9 publication Critical patent/WO2001013937A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • CD66 family members appear to play a role in a wide variety of normal and pathological processes, including: cancer, embryonic development, bacterial infection, viral infection, inflammation, pregnancy, bile transport, and cell adhesion (1-3).
  • CD66 monoclonal antibodies (mAbs) react with members of the carcinoembryonic antigen (CEA) family (4-13).
  • CD66 mAbs belonging to the CD66 cluster are classified according to their reactivity with each family member, as indicated by a lower case letter after "CD66” as follows: CD66a, CEACAM- 1 or biliary glycoprotein (BGP); CD66b, CEACAM-8 or CGM6; CD66c, CEACAM-6 or NCA; CD66d, CEACAM-3 or CGM1; CD66e, CEA; and CD66f, pregnancy specific glycoprotein (PSG) (13, 14).
  • the CD66 (CEA) gene family belongs to the immunoglobulin (Ig) gene superfamily [for review see (1, 2, 15).
  • each of the human CD66 family members contains one amino-terminal (N) domain of 108-110 amino acid residues, homologous to Ig variable domains, followed by a differing number (0-6) of Ig C2-type constant-like domains.
  • the structure of the N- domain is therefore predicted to be a stacked pair of beta-sheets with 9 beta- strands (16).
  • CD66 family members may potentially function as adhesion molecules (12, 17-30).
  • CD66a, CD66c, and CD66e are capable of homotypic and heterotypic adhesion, as shown by use of recombinant CD66a which undergoes homotypic adhesion as well as heterotypic adhesion with CD66c and CD66e (1, 2, 4-12, 17-32).
  • CD66a plays a signaling role and regulates the adhesion activity of CD11/CD18 in human neutrophils (8, 11, 25- 27, 33, 34).
  • CD66a, CD66b, CD66c, and CD66d, but not CD66e are expressed in human neutrophils, where they are "activation antigens" in that their surface expression is increased following neutrophil stimulation with various stimuli.
  • CD66a, CD66b, CD66c, and CD66d mAb binding to the neutrophil surface triggers a transient activation signal that regulates the adhesive activity of CD11/CD18, and increases neutrophil adhesion to human umbilical vein endothelial cells (HUVECs) (8, 11, 25-27, 33, 34).
  • HUVECs human umbilical vein endothelial cells
  • CD66a is frequently down regulated in colon, prostate, breast, and hepatocellular carcinoma, and colorectal adenomas (35-39). Transfection studies have provided evidence that CD66a may act as a tumor suppressor in models of colon cancer (40) prostate cancer (41, 42) breast cancer (43) and bladder cancer (44). CD66a is also important in bacterial infections, since over 95% of pathogenic N. meningitidis and N. gonorrhea interact with CD66a via Opa proteins, and the binding site for these Opa proteins has been localized to the N-domain of CD66a (45-50). An important property of Opa proteins is the stimulation of adhesion and non opsonic phagocytosis of Opa+ bacteria by neutrophils (reviewed in 48).
  • CD66a also appears to function as a receptor for murine hepatitis virus (51-55). Furthermore, CD66a may play a role in angiogenesis since it is selectively expressed on certain endothelial cells (56) and CD66a appears to function as a regulator of bile transport in bile canaliculi (3, 57, 58).
  • CD66a transmits signals (e.g. activation in neutrophils, or growth regulating signals in epithelial cells and carcinomas) are unclear.
  • CD66a is phosphorylated on its cytoplasmic domain, largely on tyrosine with a lower level of phosphoserine, in neutrophils and colon cancer cells (4, 59-61). While at least eight isoforms of CD66a derived from differential splicing have been described (3, 12, 13, 25), only those isoforms with a long cytoplasmic tail can be phosphorylated on tyrosine.
  • associated protein tyrosine kinase and phosphatase activities may be involved in CD66a signaling (59, 62, 63). Summary of the Invention
  • the present invention relates to peptides capable of modulating the function (e.g., signaling or adhesive activities) of CD66 (CEACAM) family members and/or their ligands.
  • the sequences of these peptides are set forth in Tables I-D .
  • Active peptides i.e., those capable of modulating the function of at least one CD66 family member and/or at least one ligand thereof
  • the present peptides described are of about 14 amino acids, peptides containing a relatively large number of amino acid residues, e.g., up to about 100 amino acid residues or more, that contain the described peptides, portions thereof, or similar peptides may have biological activity as well.
  • peptides smaller than those shown in Tables I-IX may also have similar biological activity.
  • peptides with amino acid substitutions or other alterations may block the activities of the described peptides or the parent molecules. Cyclic or otherwise modified forms of the peptides would also be expected to have biological activity.
  • the present invention provides isolated peptides that include an amino acid sequence represented by SEQ ID NOs:l-100 or analogs thereof that modulate the function of at least one CD66 protein (i.e., CD66 family member) and/or at least one ligand thereof.
  • These amino acid sequences can form a part of a larger peptide. Additionally, they can be used in various combinations in any one peptide.
  • the present invention provides isolated peptides represented by SEQ ID NOs:l, 2, 3, 4, 7-15, 17, 22, 32, 33, 35, 37, 39, 41, 47, 53, or 54. It is believed that SEQ ID Nos: 119, 143, 157, 161, 178, and 187 would have activity if they were solubilized or conjugated in a complex.
  • a preferred group of isolated peptides include those having an amino acid sequence represented by SMPFN (SEQ ID NO:101), PQQLF (SEQ ID NO: 102), LPQQL (SEQ ID NO: 103), QQLFG (SEQ ID NO: 104), NRQIV (SEQ ID NO:105), GNRQI (SEQ ID NO:106), IKSDLVNE (SEQ ID NO:107), AASNPP (SEQ ID NO: 108), NTTYLWWVNG (SEQ ID NO: 109), YLWWVNG (SEQ ID NO:l 10), SWLIN (SEQ ID NO:l 11), SWFIN (SEQ ID NO:l 12), AQYSWLIN (SEQ ID NO:l 13), AQYSWFIN (SEQ ID NO:l 14), SWFVN (SEQ ID NO:l 15), AQYSWFVN (SEQ ID NO:l 16), NRQII (SEQ ID NO: 199), GNRQI (SEQ ID NO
  • the present peptides are preferably capable of altering signaling mediated in part by CD66 (CEACAM) family members.
  • the peptides of the present invention modulate at least one of the following (which are functions of CD66 proteins and or ligands thereof): activation of neutrophils; activation or inhibition of T-cells, B-cells, NK cells, LAK cells, dendritic cells, or other immune system cells; proliferation and or differentiation of T-cells, B-cells, NK cells, LAK cells, dendritic cells, or other immune system cells; proliferation and/or differentiation of epithelial cells such as breast or intestinal colonic epithelium cells or keratinocytes.
  • these peptides are preferably capable of altering homotypic and/or heterotypic adhesion among CD66 proteins (i.e., CD66 family members) or adhesion of CD66 proteins to
  • the present invention also provides peptide conjugates.
  • carrier molecules or structures such as microbeads, liposomes, biological carrier molecules, synthetic polymers, biomaterials, and cells, thereby forming peptide conjugates is shown to impart the larger structure with the ability to bind to cells expressing the appropriate CD66 family member.
  • Such peptide conjugates bind to cells expressing a CD66 protein or a CD66 ligand.
  • the peptides or peptide conjugates of the present invention can also include molecules for labeling (i.e., labels such as fluroescence tags, magnetic resonance tags, radioactive tags, enzymatic tags). In this way, these can be used in diagnostic methods to detect specific targets in vivo or in vitro.
  • labels such as fluroescence tags, magnetic resonance tags, radioactive tags, enzymatic tags.
  • the present invention provides a method of activating a neutrophil that includes contacting the neutrophil with a peptide or peptide conjugate (i.e., at least one peptide or peptide conjugate) that includes an amino acid sequence represented by SEQ ID NOs:l, 2, 3, 4, 17, 41, or analogs thereof.
  • a peptide or peptide conjugate i.e., at least one peptide or peptide conjugate
  • the peptide is represented by SEQ ID NOs:l, 2, 3, 4, 17, or 41.
  • the present invention also provides a method of modulating the homotypic and/or heterotypic adhesion of CD66 family members or adhesion of a CD66 protein to a CD66 ligand.
  • the method includes contacting CD66 family members and/or their ligands with a peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:7-15, 17, 22, 32, 33, 35, 37, 39, 47, 53, 54, or analogs thereof.
  • the peptide is represented by SEQ ID NOs:7-15, 17, 22, 32, 33, 35, 37, 39, 47, 53, or 54.
  • the present invention also provides a method of modulating (e.g., activating or inhibiting) immune cell (e.g., T-cells, B-cells, NK cells, LAK cells, or dendritic cells) activation, proliferation, and/or differentiation that includes contacting an immune cell with a peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:14, 53, or analogs thereof.
  • a peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:14, 53, or analogs thereof.
  • the peptide is represented by SEQ ID NOs:14 or 53.
  • some peptides differ from these peptides by one or several amino acids and could compete with these active peptides or the natural CD66 family member or ligand thereof for certain biological activities.
  • the present invention provides a method of blocking the activation of a neutrophil induced by the method described above.
  • This method includes contacting the neutrophil when in the presence of at least one of the peptides used in the method of activating a neutrophil discussed above with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs: 18-21, 28-31, 39, 40, 55-59, 68-71, 84, or analogs thereof.
  • the peptide is represented by SEQ ID NOs: 18-21, 28-31 , 39, 40, 55-59, 68-71, or 84.
  • the present invention provides a method of altering the modulation of the homotypic and/or heterotypic adhesion of CD66 family members or adhesion between a CD66 protein and a CD66 ligand induced by peptides homologous to (e.g., peptides derived from similar regions of similar domains of CD66 family members) those listed above (SEQ ID NOs:7-15, 17, 22, 32, 33, 35, 37, 39, 47, 53, or 54).
  • the method includes contacting CD66 family members and/or ligands thereof with a peptide comprising an amino acid sequence represented by SEQ ID NOs:2, 5, 6, 9, 11, 16, 19, 23-28, 30, 32, 34- 38, 40, 42, 43-47, 49-52, 55, 57, 60-67, 69, 72-100, or analogs thereof, when in the presence of at least one of the peptides listed above (SEQ ID NOs:7-15, 17, 22, 32, 33, 35, 37, 39, 47, 53, or 54).
  • the peptide is represented by SEQ ID NOs:2, 5, 6, 9, 11, 16, 19, 23-28, 30, 32, 34-38, 40, 42, 43-47, 49-52, 55, 57, 60-67, 69, or 72-100.
  • This modulating effect can result, for example from direct binding of one of these peptides to one of the CD66 family members and/or ligands thereof, or from altering the effects of other peptides on the adhesion.
  • Another method of the present invention involves modulating at least one of the following functions of CD66 family members and/or ligands thereof in cells: activation of neutrophils; activation or inhibition of T-cells, B-cells, NK cells, LAK cells, dendritic cells, or other immune system cells; proliferation and/or differentiation of T-cells, B-cells, LAK cells, NK cells, dendritic cells, or other immune system cells; proliferation and/or differentiation of epithelial cells; homotypic and/or heterotypic adhesion among CD66 family members; and adhesion of CD66 family members to other ligands.
  • the method includes contacting cells with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Another method involves delivering a therapeutically active agent to a patient.
  • the method includes administering at least one peptide conjugate comprising a peptide and the therapeutically active agent to a patient wherein the peptide includes an amino acid sequence represented by SEQ ID NOs:l- 100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • the therapeutically active agent is selected from drugs, DNA sequences, RNA sequences, proteins, lipids, and combinations thereof. More preferably, the therapeutically active agent is an antibacterial agent, antiinflammatory agent, or antineoplastic agent.
  • a method of modifying the metastasis of malignant cells includes contacting the malignant cells or normal host tissue with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs: 1-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • a method of altering bacterial or viral binding to cells or a biomaterial includes contacting the cells or biomaterial with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Another method involves altering cell adhesion to a biomaterial.
  • the method includes contacting the biomaterial with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l- 100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Another method involves detecting tumors.
  • the method includes contacting tumor cells or tumor vasculature with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l- 100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Still another method involves detecting inflammation.
  • the method includes contacting inflamed vasculature or leukocytes with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID Os:l-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Yet another method involves detecting a CD66 protein or a ligand thereof.
  • the method includes contacting tissue containing a CD66 protein or a ligand thereof with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Another method involves altering angiogenesis.
  • the method includes contacting endothelial cells, tumor cells, or immune cells with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs: 1-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Yet another method of the present invention involves altering an immune response.
  • the method includes contacting immune system cells with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • Another method of the present invention involves altering keratinocyte proliferation.
  • the method includes contacting keratinocytes with at least one peptide or peptide conjugate that includes an amino acid sequence represented by SEQ ID NOs:l-100, 119, 143, 157, 161, 178, 187, or analogs thereof.
  • the methods described herein can be carried out in vitro or in vivo.
  • the peptides can be used alone or in various combinations as well as in peptide conjugates. They are used in amounts that provide the desired effect. These amounts can be readily determined by one of skill in the art.
  • useful peptides are represented by SEQ ID NOs:l, 2, 3, 4, 7-15, 17, 22, 32, 33, 35, 37, 39, 41, 47, 53, or 54.
  • compositions and methods of the present invention include one or more polypeptides.
  • peptide is said to includes an amino acid sequence represented by SEQ ID NOs: 1-100 or analogs thereof, the peptide can include one or more of the sequences specified.
  • amino acid is used herein to refer to a chemical compound with the general formula: NH 2 -CRH-COOH, where R, the side chain, is H or an organic group. Where R is an organic group, R can vary and is either polar or nonpolar (i.e., hydrophobic).
  • the amino acids of this invention can be naturally occurring or synthetic (often referred to as nonproteinogenic).
  • an organic group is a hydrocarbon group that is classified as an aliphatic group, a cyclic group or combination of aliphatic and cyclic groups.
  • aliphatic group means a saturated or unsaturated linear or branched hydrocarbon group.
  • cyclic group means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group.
  • alicyclic group means a cyclic hydrocarbon group having properties resembling those of aliphatic groups.
  • aromatic group refers to mono- or poly cyclic aromatic hydrocarbon groups.
  • an organic group can be substituted or unsubstituted.
  • polypeptide and peptide are used interchangeably and refer to a polymer of amino acids. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like.
  • isolated as it refers to peptides (i.e., polypeptides) means that the peptides are derived from naturally occurring proteins or other polypeptides and free from other biological material or they are synthesized, either recombinantly or chemically.
  • FIG. 1 Effects of CD66a peptides on neutropliil adhesion to human umbilical vein endothelial cells (HUVECs).
  • HUVECs were grown to confluence in 96 well microtiter plates, and stimulated by adding 50 ng/ml TNF-alpha and 1000 U/ml gamma-interferon (gamma-IFN) and culturing for 48 hours. The wells were then washed and 25 ⁇ l of adhesion buffer containing the indicated CD66a peptide at 167 ⁇ g/ml (final concentration) was added.
  • gamma-IFN gamma-interferon
  • FIG. 1 Effects of various concentrations of the CD66a peptides CD66a-l, CD66a-2, and CD66a-3 on neutrophil adhesion to HUVECs.
  • HUVECs were grown to confluence in 96 well microtiter plates, and stimulated by incubating in the presence of TNF-alpha at 50 ng/ml final concentration for 4 hr at 37°C, and the adhesion of neutrophils was quantitated in the presence of the indicated final concentration of CD66a peptide CD66a-l (circles), CD66a-2 (squares), or CD66a-3 (triangles) and 10 '7 M FMLP as described in Figure 1.
  • FIG. 3 Effects of scrambled CD66a peptides on neutrophil adhesion to HUVECs.
  • HUVECs were grown to confluence in 96 well microtiter plates, and stimulated by incubating in the presence of TNF-alpha at 50 ng/ml final concentration for 4 hr at 37°C. The wells were then washed and 25 ⁇ l of adhesion buffer containing the indicated CD66a peptides (at 167 ⁇ g/ml final concentration) was added.
  • FIG. 5 Effects of CD66a-7 peptide on neutrophil adhesion to HUVECs.
  • HUVECs were grown to confluence in 96 well microtiter plates, and stimulated by adding 50 ng/ml TNF-alpha and culturing for 48 hours. The wells were then washed and 25 ⁇ l of adhesion buffer with or without the CD66a-7 peptide at 167 ⁇ g/ml (final concentration) was added.
  • One hundred microliters ( ⁇ l) of adhesion media containing 10 neutrophils was then immediately added, followed by 25 ⁇ l of adhesion buffer with 6 x 10 "7 M FMLP, and the plates were incubated at 37°C for 30 min in 5% CO 2 .
  • FIG. 7 Effects of CD66e-3 peptide on neutrophil adhesion to HUVECs. Using the method described with respect to Figure 5, the adhesion observed in the presence of the peptide CD66e-3 was statistically greater than that observed with buffer alone (p ⁇ 0.05).
  • FIG. 8 Effects of CD66a peptides on binding of CHO transfectants expressing CD66a (CEACAM 1-4L) to immobilized recombinant human CEACAM 1-Fc using the technique of the Transfectant Binding Assay #1 (Assay #1).
  • Assay #1 Transfectant Binding Assay #1.
  • CD66a-Fc soluble CEACAM 1-4-Fc
  • CD14-Fc negative control constructs CD31(Dl-3)-Fc and CD14-Fc were added and allowed to bind, and the plates were then washed.
  • CHO transfectants were labeled with the fluoresecent tag BCECF-AM and allowed to adhere to these immobilized soluble constructs for 60 min at 37°C.
  • the total fluorescence of each well was then determined using the Cytofluor II fluorescence plate reader.
  • the plates were then washed and the number of cells adhering determined by fluorescence measurements in the Cytofluor II as a percentage of the total cells added per well. The mean +/- SD of 4 determinations are shown.
  • FIG 9 A and 9B Effects of peptides on homotypic adhesion of CD66a-CD66a using Transfectant Binding Assay #2 (Assay #2).
  • Assay #2 Transfectant Binding Assay #2
  • Several peptides blocked binding of CD66a expressing CHO transfectants to immobilized CD66a using the technique of Assay #2. The data is shown as percent of added cells that remained adherent.
  • Control values "0" and "1000" represent the adhesion observed when no CD66a protein is adherent to the well, or when buffer is added without peptide, respectively.
  • Control values "0.01" represent the adhesion observed in the presence of a blocking CD66 antibody.
  • Figure 10A and 10B Figure 10A and 10B.
  • FIG 11 A and 1 IB Effects of peptides on homotypic adhesion of CD66e-CD66e using Transfectant Binding Assay #2 (Assay #2).
  • Assay #2 Transfectant Binding Assay #2
  • Several peptides blocked binding of CD66e expressing CHO transfectants to immobilized CD66e using the technique of Assay #2.
  • the data is shown as percent of added cells that remained adherent. Control values "0" and "1000" represent the adhesion observed when no CD66e protein is adherent to the well, or when buffer is added without peptide, respectively.
  • Control values "0.01" represent the adhesion observed in the presence of a blocking CD66 antibody.
  • FIG 12A and 12B Effects of peptides on heterotypic adhesion of CD66b-CD66c using Assay #2.
  • Several peptides blocked binding of CD66b expressing CHO transfectants to immobilized CD66c using the technique of Assay #2. The data is shown as percent of added cells that remained adherent.
  • Control values "0" and "1000" represent the adhesion observed when no CD66c protein is adherent to the well, or when buffer is added without peptide, respectively.
  • Control values "0.01 " represent the adhesion observed in the presence of a blocking CD66 antibody.
  • Figure 13 A and 13B Binding of microbeads coupled to CD66a-24 to
  • CD66a-24 and CD66a-l peptides were coupled to microbeads and the microbeads were incubated with a suspension of CHO cells expressing CD66a at room temperature for 30 min.
  • the binding of the microbeads to the CHO cells was quantified by counting the number of beads associated with single cells or groups of cells in three cell-group size classes and are reported as the number of microbeads bound to each size group of cells.
  • Figure 13B shows the number of beads associated with single cells, which are reported as the average number of microbeads bound to each single cell.
  • Figure 14 Effect ofCD66 peptides on the activation of T-cells.
  • T-cells were stimulated with anti-CD3 in the presence of various CD66 peptides as indicated and proliferation quantitated using radionucleide uptake expressed as cpm associated with the cells.
  • CD66a Because of the adhesive and signaling properties of CD66a described above, we sought to identify functionally active domains of CD66a by use of synthetic peptides. Peptides of 14 amino acids in length were synthesized. The sequences are set forth in Tables I-D . These were investigated for the ability to modulate the function of CD66 (CEACAM) family members.
  • the present invention provides isolated peptides that include an amino acid sequence represented by (at least one of) SEQ ID NOs: 1-100 or analogs thereof that modulate the function of at least one CD66 protein (i.e., CD66 family member) and/or at least one ligand thereof.
  • Activating or inhibiting neutrophil activation may be useful in treating certain infectious diseases or in cases where the activation of neutrophils results in unwanted effects as in adult respiratory distress syndrome.
  • peptides were also tested for their ability to alter the homotypic adhesion of CD66a to CD66a, CD66c to CD66c, and CD66e to CD66e, as well as the heterotypic adhesion of CD66b to CD66c.
  • a number of the peptides were found to modulate homotypic and/or heterotypic adhesion of CD66 family members. These include peptides having the amino acid sequences represented by SEQ ID NOs:7-15, 17, 22, 32, 33, 35, 37, 39, 47, 53, and 54. It is believed that these may also modulate adhesion between a CD66 protein and other CD66 ligands.
  • peptides that differ from these active peptides by one or several amino acids could compete (i.e., alter their modulation effects) with these active peptides for functional effects or mediate direct binding of the natural CD66 family members.
  • These include peptides having the amino acid sequences represented by SEQ ID NOs:2, 5, 6, 9, 11, 16, 19, 23-28, 30, 32, 34- 38, 40, 42, 43-47, 49-52, 55, 57, 60-67, 69, and 72-84, as well as other homologous peptides (based on domain structure) including SEQ ID NOs:85- 100.
  • Peptides were also tested for their ability to inhibit the activation of T- cells toward proliferation and/or differentiation.
  • One peptide (SEQ ID NO: 14) was found to be a potent inhibitor of T-cell activation while another (SEQ ID NO:53) had weaker activity.
  • Modulating the immune response as for example by activating or inhibiting the proliferation and/or differentiation of T-cells, B- cells, NK cells, LAK cells, dendritic cells, or other immune system cells, may be useful in treating autoimmune diseases, and in transplantation therapies where graft vs. host or host vs. graft effects may be undesirable.
  • the peptides could also be immune stimulants in settings such as cancer, infectious disease, or immunization. Alternatively, they could be immune suppressants. They could also be used to detect inflammation, and preferably modulate inflammation by activating or inhibiting activation of immune or inflammatory cells.
  • a preferred method involves detecting (and preferably modulating) inflammation in tissues such as inflamed vascul
  • the present invention provides isolated peptides represented by SEQ ID Nos:l, 2, 3, 4, 7-15, 17, 22, 32, 33, 35, 37, 39, 41, 47, 53, or 54. It is also believed that peptides represented by SEQ ID Nos: 119, 143, 157, 161, 178, and 187 would have activity if they were solubilized or conjugated in a complex.
  • the present invention provides peptides derived from CD66 (CEACAM) family members that are capable of modulating (i.e., altering by increasing, decreasing, etc.), for example, cell activation, cell adhesion, cell proliferation, cell differentiation, or homotypic and/or heterotypic adhesion among CD66 family members or binding of CD66 family members to their ligands.
  • CD66 CD66
  • compositions comprising the polypeptides of this invention can be added to cells in culture (in vitro) or used to treat patients, such as mammals (in vivo).
  • the polypeptides are used to treat a patient, the polypeptide is preferably combined in a pharmaceutical composition with a pharmaceutically acceptible carrier such as a larger molecule to promote polypeptide stability or a pharmaceutically acceptible buffer that serves as a carrier for the polypeptide or incorporated in a peptide conjugate that has more than one peptide coupled to a single entity.
  • the peptides described herein could be useful for altering the binding of viruses, bacteria, or other pathological etiologic agents to the cells of host tissues, transplanted tissues, or to biomaterials (increase or inhibit binding). They could also be useful for detecting a CD66 protein or a ligand thereof in tissue, whether it be in vitro or in vivo.
  • the biological activity of the peptides identified here suggests that they have sufficient affinity to make them potential candidates for drug localization to cells expressing the appropriate surface structures.
  • This targeting and binding to cells could be useful for the delivery of therapeutically active agents (including targeting drugs, DNA sequences, RNA sequences, lipids, proteins (e.g., human growth factors)) and gene therapy/gene delivery.
  • the therapeutically active agent is an antibacterial agent, antiinflammatory agent, or antineoplastic agent.
  • CD66 family members Since different cells, including specifically many malignant cells, cells of different tissues, growing endothelial cells, including endothelial cells in new vessels in tumors and in diabetic proliferative microvasculature, express different combinations of CD66 family members, it should be possible to generate compounds bearing different combinations of densities of CD66 peptides that would target (bind preferentially) to different desired tissues or cells.
  • the peptide CD66-24 when coupled to microbeads directs the binding of the complexed microbeads to CHO cells expressing CD66a.
  • CD66 family members have been shown to alter metastases of malignant cells and can alter cell differentiation.
  • the peptides described herein could modify the process of metastasis of malignant cells either by altering the behavior of the malignant cells directly, or by altering the physiology of a target tissue (as for example, the liver where CD66e has been shown to alter cytokine production by cells in the liver and also alter the ability of colon cancer cells to metastasize to the liver).
  • the peptides described herein can also be used in detecting tumors.
  • the peptides described herein are believed to be useful for altering angiogenesis. In such a method, endothelial cells, tumor cells, or immune cells are contacted with at least one peptide described herein.
  • CD66 members are expressed in growing keratinocytes at the edge of healing wounds. These peptides may be useful to alter keratinocyte growth or behavior or the behavior of other cell involved in wound healing.
  • peptides may be usefiil in altering the growth or physiology of cells, which are in various disease states, that can express CD66 members, including gut (as for example in inflammatory bowel disease, atrophic states, or cancer), breast, stomach, small bowel, colon, pancreas, thyroid, prostate, lung, kidney, placenta, sebaceous glands, and uterus.
  • gut as for example in inflammatory bowel disease, atrophic states, or cancer
  • breast stomach, small bowel, colon, pancreas, thyroid, prostate, lung, kidney, placenta, sebaceous glands, and uterus.
  • Treatment for these various conditions can be prophylactic or therapeutic.
  • treatment can be initiated before, during, or after the development of the condition.
  • the phrases "inhibition of or "effective to inhibit” a condition includes both prophylactic and therapeutic treatment (i.e., prevention and/or reversal of the condition).
  • molecules/particles with a specific number of specific CD66 peptides would bind specifically to cells/tissues expressing specific ligand combinations, and therefore could have diagnostic and therapeutic use.
  • the peptides of the present invention can be labeled (e.g., fluorescent, radioactive, enzyme, nuclear magnetic) and used to detect specific targets in vivo or in vitro including "immunochemistry" like assays in vitro. In vivo they could be used in a manner similar to nuclear medicine imaging techniques to detect tissues, cells, or other material expressing specific CD66 ligands.
  • the polypeptides of SEQ ID NOs :l-100 can be in their free acid form or they can be amidated at the C-terminal carboxylate group.
  • the present invention also includes analogs of the polypeptide of SEQ ID NOs: 1-100, which typically have structural similarity with SEQ ID NOs:l-100.
  • An "analog" of a polypeptide includes at least a portion of the polypeptide, wherein the portion contains deletions or additions of one or more contiguous or noncontiguous amino acids, or containing one or more amino acid substitutions. Substitutes for an amino acid in the polypeptides of the invention are preferably conservative substitutions, which are selected from other members of the class to which the amino acid belongs.
  • An analog can also be a larger peptide that incorporates the peptides described herein.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic can generally be substituted for another amino acid without substantially altering the structure of a polypeptide.
  • conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Ala, Gly, Ser, Thr, and Pro; Class II: Cys, Ser, Thr, and Tyr; Class III: Glu, Asp, Asn, and Gin (carboxyl group containing side chains): Class IV: His, Arg, and Lys (representing basic side chains); Class V: He, Val, Leu, Phe, and Met (representing hydrophobic side chains); and Class VI: Phe, Trp, Tyr, and His (representing aromatic side chains).
  • the classes also include other related amino acids such as halogenated tyrosines in Class VI.
  • Polypeptide analogs as that term is used herein, also include modified polypeptides. Modifications of polypeptides of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
  • a preferred polypeptide analog is characterized by having at least one of the biological activities described herein. Such an analog is referred to herein as a "biologically active analog” or simply “active analog.”
  • the biological activity of a polypeptide can be determined, for example, as described in the Examples Section.
  • active analogs of SEQ ID NO:l include peptides having an "M” or similar amino acid in the "SMPFN" sequence (SEQ ID NO:101).
  • Active analogs of SEQ ID NO:2 include peptides having a "Q” or similar amino acid in the "PQQLF” sequence (SEQ ID NO: 102), the “LPQQL” sequence (SEQ ID NO: 103), or the “QQLFG” sequence (SEQ ID NO: 104).
  • Active analogs of SEQ ID NO:3 include peptides having an "RQ” sequence or similar amino acid sequence in the "NRQIV” sequence (SEQ ID NO: 105) or the "GNRQI” sequence (SEQ ID NO: 106).
  • Active analogs of SEQ ID NO:4 include peptides having an "IKSDLVNE” portion (SEQ ID NO: 107) of the sequence.
  • Active analogs of SEQ ID NO:9 include peptides having an "AASNPP” portion (SEQ ID NO: 108) of the sequence.
  • Active analogs of SEQ ID NO:22 include peptides having a "NTTYLWWVNG” portion (SEQ ID NO: 109) or "YLWWVNG” portion (SEQ ID NO:l 10) of the sequence.
  • Active analogs of SEQ ID NO:35 include peptides having an "SWLIN” portion (SEQ ID NO:l 11), “SWFIN” portion (SEQ ID NO:l 12), “AQYSWLIN” portion (SEQ ID NO:l 13), or “AQYSWFIN” portion (SEQ ID NO:l 14) of the sequence.
  • Active analogs of SEQ ID NO:47 include peptides having an "SWFVN” portion (SEQ ID NO:l 15) or “AQYSWFVN” portion (SEQ ID NO:l 16) of the sequence.
  • Active analogs of SEQ ID NO:41 include peptides having an "NRQII” portion (SEQ ID NO: 199) or "GNRQI” portion (SEQ ID NO:200).
  • polypeptides of the invention may be synthesized by the solid phase method using standard methods based on either t-butyloxycarbonyl (BOC) or 9- fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodology is described by G.B. Fields et al. in Synthetic Peptides: A User's Guide, W.M. Freeman & Company, New York, NY, pp. 77-183 (1992). The present peptides may also be synthesized via recombinant techniques well known to those skilled in the art. For example, U.S. Patent No.
  • 5,595,887 describes methods of forming a variety of relatively small peptides through expression of a recombinant gene construct coding for a fusion protein which includes a binding protein and one or more copies of the desired target peptide. After expression, the fusion protein is isolated and cleaved using chemical and/or enzymatic methods to produce the desired target peptide.
  • the peptides of the present invention may be employed in a monovalent state (e.g., free peptide or peptide coupled to a carrier molecule or structure).
  • the peptides may also be employed as conjugates having more than one (same or different) peptide bound to a single carrier molecule.
  • the carrier molecule or structure may be microbeads, liposomes, biological carrier molecule (e.g., a glycosaminoglycan. a proteoglycan.
  • albumin or the like
  • a synthetic polymer e.g., a polyalkyleneglycol or a synthetic chromatography support
  • biomaterial e.g., a material suitable for implantation into a mammal or for contact with biological fluids as in an extrcorporeal device
  • ovalbumin, human serum albumin, other proteins, polyethylene glycol, or the like are employed as the carrier.
  • modifications may increase the apparent affinity and/or change the stability of a peptide.
  • the number of peptide fragments associated with or bound to each carrier can vary.
  • the use of various mixtures and densities of the peptides described herein may allow the production of complexes that have specific binding patterns in terms of preferred ligands.
  • polypeptides can be conjugated to other polypeptides using standard methods known to one of skill in the art. Conjugates can be separated from free peptide through the use of gel filtration column chromatography or other methods known in the art.
  • peptide conjugates may be prepared by treating a mixture of peptides and carrier molecules (or structures) with a coupling agent, such as a carbodiimide.
  • the coupling agent may activate a carboxyl group on either the peptide or the carrier molecule (or structure) so that the carboxyl group can react with a nucleophile (e.g. an amino or hydroxyl group) on the other member of the peptide conjugate, resulting in the covalent linkage of the peptide and the carrier molecule (or structure).
  • peptides may be coupled to biotin-labeled polyethylene glycol and then coupled to avidin containing compounds, for instance, as shown in Fig. 13. Peptides are weighed out in aliquots of 0.5 mg and dissolved in a total volume of 500 ⁇ l dimethyl sulfoxide (DMSO,
  • each ReactiVial is individually transferred to a 1.5 mL plastic microfuge tube.
  • Each vial is washed once with 25 ⁇ l DMSO which is also added to the microfuge tube.
  • the volume of DMSO is dried down at room temperature to approximately 20 ⁇ l of remaining solvent in a Savant Speed Vac Plus.
  • HBSS Hanks balanced salt solution
  • Samples are stored at -20°C until coupling to streptavidin-coated beads.
  • Streptavidin-coated 6 ⁇ m diameter polystyrene beads are obtained from Polysciences (Warrington, PA). For each peptide, 100 ⁇ l of suspended beads are aliquoted to a 1.5 ml plastic microfuge tube. As per the manufacturer's directions, the beads are washed three times by sequentially pelleting the beads in a microcentrifuge, decanting the supernatant and redispersing them in 1 ml of fresh phosphate buffered saline (PBS). One third (333 ⁇ l) of the biotinylated peptide from the above preparation is added to the beads in a total volume of 1 ml.
  • PBS fresh phosphate buffered saline
  • the present invention also provides a composition that includes one or more active agents (i.e., polypeptides) of the invention and one or more pharmaceutically acceptable carriers.
  • active agents i.e., polypeptides
  • One or more polypeptides with demonstrated biological activity can be administered to a patient in an amount alone or together with other active agents and with a pharmaceutically acceptable buffer.
  • the polypeptides can be combined with a variety of physiological acceptable carriers for delivery to a patient including a variety of diluents or excipients known to those of ordinary skill in the art. For example, for parenteral administration, isotonic saline is preferred.
  • a cream including a carrier such as dimethylsulfoxide (DMSO), or other agents typically found in topical creams that do not block or inhibit activity of the peptide, can be used.
  • DMSO dimethylsulfoxide
  • suitable carriers include, but are not limited to alcohol, phosphate buffered saline, and other balanced salt solutions.
  • formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Preferably, such methods include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients.
  • the methods of the invention include administering to a patient, preferably a mammal, and more preferably a human, the composition of the invention in an amount effective to produce the desired effect.
  • the peptides can be administered as a single dose or in multiple doses.
  • Useful dosages of the active agents can be determined by comparing their in vitro activity and the in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art.
  • the agents of the present invention are preferably formulated in pharmaceutical compositions and then, in accordance with the methods of the invention, administered to a patient, such as a human patient, in a variety of forms adapted to the chosen route of administration.
  • the formulations include, but are not limited to, those suitable for oral, rectal, vaginal, topical, nasal, ophthalmic, or parental (including subcutaneous, intramuscular, intraperitoneal, intratumoral, intraorgan, intraarterial and intravenous) administration.
  • Formulations suitable for parenteral administration conveniently include a sterile aqueous preparation of the active agent, or dispersions of sterile powders of the active agent, which are preferably isotonic with the blood of the recipient. Absorption of the active agents over a prolonged period can be achieved by including agents for delaying, for example, aluminum monostearate and gelatin.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the active agent as a powder or granules, as liposomes containing the active agent, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, or a draught.
  • Such compositions and preparations typically contain at least about 0.1 wt-% of the active agent.
  • the amount of polypeptide i.e., active agent
  • the amount of polypeptide is such that the dosage level will be effective to produce the desired result in the patient.
  • Nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier such as cocoa butter, or hydrogenated fats or hydrogenated fatty carboxylic acids. Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye. Topical formulations include the active agent dissolved or suspended in one or more media such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations. Examples
  • HBSS Basalt-stained cells
  • Differential cell counts on Wright-stained cells routinely revealed greater than 95% neutrophils.
  • Viability as assessed by trypan blue dye exclusion was greater than 98%.
  • the PE-labeled CD1 lb mAb (Leu 15) and the CD62L mAb (Leu 8) were obtained from Becton Dickenson, Mountain View, CA. Monoclonal antibodies were diluted in PBS containing 1 mg/ml BSA as indicated. N-formyl-met-leu-phe (FMLP) and normal mouse serum (NMS) were purchased from Sigma Chemical Co. (St. Louis, MO). Peptides were diluted in PBS containing 1 mg/ml BSA as indicated. Fluorescence labeling of cells. Neutrophils were labeled with calcein
  • HUVECs (Clonetics Corp., San Diego, CA) were passaged 1 :5 in T-25 flasks (Costar) no more than three times before plating in 96 well microtiter plates at 3000 cells/well. HUVECs were grown to confluence in 96 well microtiter plates in EGM media (Clonetics) and fed every 24 hours. Using the adhesion assay described below, no difference in resting and stimulated neutrophil adhesion was observed, and, as expected (69), no difference in surface expression of CD54 (ICAM-1) or CD62E (E selectin, ELAM-1) in resting or TNF stimulated cells was noted using HUVECs passaged once compared with those passaged five times.
  • IAM-1 CD54
  • ELAM-1 E selectin, ELAM-1
  • the HUVECs were stimulated by culture for the indicated time with the desired cytokines (TNF-alpha (Cetus, Emeryville, CA) or gamma-IFN (gift from Dr. S. Palm, University of Minnesota Medical School)).
  • TNF-alpha Cetus, Emeryville, CA
  • gamma-IFN gift from Dr. S. Palm, University of Minnesota Medical School
  • the wells were then washed four times with adhesion buffer (DMEM + 5% heat inactivated fetal bovine serum (HIFBS)) and 25 ⁇ l of adhesion buffer containing the indicated peptide was added to each well, followed immediately by 100 ⁇ l of adhesion buffer containing 10 5 calcein labeled cells.
  • adhesion buffer DMEM + 5% heat inactivated fetal bovine serum (HIFBS)
  • the mixture was incubated at 0°C for 25 min, and 4 ml of buffer B (PBS, pH 7.4, 0.2% BSA, 0.05% NaN3) (0°C) was then added and the mixture was centrifuged at 400 x g for 5 min at 4°C. The supernatant was removed and the cells were vortexed, and suspended in 1 ml of buffer B (0°C), and 250 ml of fixative (Coulter) (23°C) was then added. Three ml of buffer B (0°C) was then added, and the mixture centrifuged at 400 x g at 4°C for 5 min.
  • buffer B PBS, pH 7.4, 0.2% BSA, 0.05% NaN3
  • the cells were washed with 3 ml of buffer B as above, and resuspended in 200 ml of PBS containing 0.1% NaN 3 (0°C) and stored at 4°C until analysis.
  • Quantitative flow cytometric analysis of surface antigen expression was performed using a FACSTAR Plus (Beckton Dickinson, Mountain View, CA). Forward and right angle light scatter, as well as the peak fluorescence channel, were optimized with fluorescent beads. The cell population studied was determined by forward and right angle light scatter.
  • CD62L down regulation For analysis of CD62L down regulation, purified neutrophils (10 5 in 100 ⁇ l HBSS + 0.02% BSA) were warmed to 37°C for 5 min and then incubated with media containing the indicated peptide (167 ⁇ g/ml) or FMLP (10 "7 M) for 5 min at 37°C. The cells were then cooled to 0°C for 10 min and 5 ⁇ g of the PE-labeled CD62L mAb was added. The cells were then incubated, washed, and analyzed by flow cytometry as above. Peptide selection, synthesis, and purification. CD66a was modeled to conform to the IgV and Ig C2 domains of the heavy and light chains of Fab fragments of immunoglobulin and CD4.
  • Peptides were synthesized as amides by Fmoc solid-phase methodology on a Gilson Automated Multiple Peptide Synthesizer AMS 422. Peptides were purified by preparative reverse phase-HPLC on a Beckman System Gold equipped with a Regis Chemical ODS C18 column (10 ⁇ m particle size, 60 Angstrom pore size, 250 x 21.1 mm). The elution gradient was 12-50% B over 35 min at a flow rate of 5.0 ml/min, where A is water containing 0.1% trifluoroacetic acid, and B is acetonitrile containing 0.1% trifluoroacetic acid. Detection was at 235 nm. Peptide purity and fidelity can be analyzed by amino acid analysis and sequencing or by mass spectrometry.
  • CD66a-6 was not soluble we synthesized peptides from the same region but shifted the center of the peptide in an attempt to generate a soluble peptide.
  • One peptide, CD66a-6L, (Table I) was successfully synthesized, tested, and found to stimulate neutrophil adhesion to HUVECs (Fig. 6). Since only the N-domain peptide of CD66a had activity in the neutrophil activation assay we modeled CD66b, CD66c, CD66d, CD66e, and CD66f N- domains and synthesized appropriate peptides as shown in Tables III- VIII. Of these peptides, only peptide CD66e-3 activated neutrophils (Fig. 7). These results are noteworthy in that many peptides that have only minor differences from active peptides had no biological activity.
  • Method #1 for adhesion assay of CHO transfectant binding to immobilized recombinant CD66a (Assay #1).
  • Stable CHO cell transfectants expressing CD66a (CEACAM1 -4L) CEACAM1 -4S, CEACAM1 -1 S or the neomycin resistance gene (CHO-Neo) (provided by Dr. S. Watt, MRC, Oxford, UK) were grown to 50-70% confluence in Hams-FlO medium containing 10% (v/v) FBS.
  • Adherent cells were detached with PBS containing 1 mM EDTA pH 7.4, washed three times with Hams-FlO medium, and resuspended in Hams-FlO medium at 2 x 10 6 cells/ml.
  • the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5- (and -6)-carboxfluoresceinacetoxymethylester (BCECF-AM); Molecular Probes, Eugene, OR) was dissolved in DMSO at 500 ⁇ g/ml and 20 ⁇ l added per 2 x 10 7 cells for 20-30 min at 37°C. Cells were washed twice with RPMI-1640 medium and twice with PBS containing 0.2% BSA (PBS-0.2% BSA). Cells (5- 10 x 10 4 ) in PBS-0.2% BSA were added to 96 well Immulon 3 flat bottomed microtiter plates (Dynatech) that had been pre-coated as follows.
  • Purified goat anti-human Fc antibody (Sigma Chemical Co.) was added to 96 well flat bottomed Immulon 3 plates at 1 ⁇ g/100 ⁇ l/well at 4°C overnight. The plates were washed 4 times with PBS containing 0.5% BSA (PBS-0.5% BSA) and blocked with PBS-0.5% BSA for at least two hours at room temperature. After washing the plates 4 times with PBS, 50 ⁇ l of soluble recombinant protein containing the Fc fragment of human IgGl attached to CEACAM 1 (CEACAMl-Fc) or other indicated protein (10 ⁇ g/ml) in PBS were added for at least 2 hours at room temperature or overnight at 4°C.
  • the plates were washed 4 times with PBS before the addition of 100 ⁇ l of cells.
  • CHO cell transfectants labeled with BCECF-AM were allowed to adhere for 60 min at 37°C before reading the total BCECF-AM fluorescence in each well on a Cytofluor II plate reader (PerSeptive Biosystems, Hertford, UK) at an excitation wavelength of 485/20 nm, a gain of 70 and an emission wavelength of 530/30 nm.
  • the plates were washed one to three times with PBS-0.2% BSA and the percentage of cells adhering to the constructs estimated from the subsequent fluorescence determinations on the Cytofluor II. Adhesion assays were performed with 4 to 6 replicates in at least two independent experiments.
  • Method #2 for adhesion assay of CHO transfectant binding to immobilized recombinant CD66a (Assay #2.. CHO cells transfected with BGPa cDNA (courtesy Dr. M. Kuroki) were grown in ⁇ -MEM (Gibco Inc., Grand Island, NY) lacking nucleosides with 10% FBS (Bio-Whittaker,
  • PBS 35 ⁇ l
  • 15 ⁇ l (15 ⁇ g) of peptide solution was added into a protein-coated well, and then 5 x 10 4 cells (50 ⁇ l) labeled with dye were added. After vortexing gently, the plate was incubated at room temperature for 25 min in the dark. Each well was gently washed with 100 ⁇ l PBS twice. Remaining cells were solubilized in 100 ⁇ l/well of PBS containing 0.2% NP40 and fluorescence was measured by a microplate reader.
  • Control values "0" and "1000" represent the adhesion observed when no CD66a protein is adherent to the well, or when buffer is added without peptide, respectively.
  • Control values "0.01” represent the adhesion observed in the presence of a blocking CD66 antibody.
  • Assay for binding of peptides coupled to beads to CHO cells expressing recombinant CD66a CHO cells transfected with BGPa cDNA were grown and prepared as in Assay #2. To each tube containing 10 ⁇ l peptide-bound beads (approximately 300,000 beads) 20 ⁇ l of cell suspension was added and mixed gently. The tubes were then incubated for 30 minutes at room temperature.
  • Example 1 Effect of peptides on neutrophil activation determined by adhesion to endothelial cells
  • the CD66a peptides were tested for their ability to alter neutrophil adhesion to human umbilical vein endothelial cells (HUVECs) stimulated for 48 hours with 1000 U/ml gamma-interferon and 50 ng/ml TNF-alpha (Fig. 1).
  • HUVECs human umbilical vein endothelial cells
  • the three peptides that specifically induced neutrophil adhesion were further tested for their effects on the adhesion of neutrophils to TNF stimulated HUVECs.
  • Each of the three CD66a peptides, CD66a-l, CD66a-2, and CD66a- 3 increased neutrophil adhesion to HUVECs at concentrations as low as 50 ⁇ g/ml (approximately 35 ⁇ M) in the presence of FMLP (Fig. 2).
  • three scrambled versions were made of each active peptide (Table II) and tested in the adhesion assay. In contrast to the native peptides, none of the 9 scrambled peptides had activity in the adhesion assay (Fig. 3).
  • CD1 lb expression Effect of CD66a peptides on CD1 lb expression.
  • the effects of the peptides on surface expression of CD1 lb on neutrophils was next examined. While neutrophil adhesion to HUVECs is dependent on the functional activity of surface CD11/CD18, many adhesive stimuli also upregulate the surface expression of CD 11 /CD 18 , and this may play a role in regulating cell adhesion as well (70-72).
  • CD1 lb expression was analyzed by flow cytometry.
  • CD1 lb mAb Since CD11 and CD 18 are translocated to the cell surface only when they are complexed with each other, the use of a directly labeled CD1 lb mAb was used to demonstrate upregulation of CD18 as well as CD1 lb.
  • HBSS HBSS
  • MCF flow cytometry
  • CD1 lb expression also increased, similar to that seen with incubation with 10 "7 M FMLP.
  • CD62L expression Effect of CD66a peptides on CD62L expression.
  • L- selectin recognized by CD62L mAbs, also plays a role in neutrophil adhesion to endothelial cells, and its expression is altered by stimulation (70, 72).
  • Example 3 Modulation of binding of CHO cells expressing recombinant CD66 family members to recombinant CD66 family member proteins in solid phase binding assay #1
  • peptides are their use to target binding of larger structures to specific cells/tissues.
  • the complexing of one or more ofthe described peptides to a larger entity should result in binding ofthe complex to cells expressing the appropriate ligands (for example, CD66a in tumors or CD66a in growing endothelial cells involved in angiogenesis).
  • CD66a-24 and CD66a-l peptides were coupled to microbeads and the microbeads were incubated with a suspension of CHO cells expressing CD66a at room temperature for 30 min.
  • the binding ofthe microbeads to the CHO cells was quantified by counting the number of beads associated with single cells or groups of cells in three cell-group size classes and are reported as the number of microbeads bound to each size group of cells (Fig. 13 A).
  • Fig. 13B shows the number of beads associated with single cells, which are reported as the average number of microbeads bound to each single cell.
  • the lack of binding of CD66a-l coupled beads serves as a negative control for this experiment but does not imply that a different coupling technique would not result in binding.
  • T-cells play an important role in the immune system, and a number of cell-surface molecules have been found to regulate T-cell activation (88, 90, 91, 92).
  • CD66 peptides were tested the effects of CD66 peptides on T-cell activation as determined by proliferation following stimulation by anti-CD3.
  • Peptides were synthesized from regions of CD66 family members that we predict may be exposed on the surface ofthe molecule. Three ofthe peptides were found to have activity in an assay examining stimulated neutrophil adhesion to HUVECs. These same three peptides also stimulated upregulation of CD1 lb/CD 18 and down regulation of CD62L on the neutrophil surface. Scrambled versions of these peptides had no biological activity in either assay, suggesting that the specific amino acid sequence is critical for activity. Thus, the data suggest that peptide motifs from at least three regions of the N-terminal domain of CD66a are involved in the interaction of CD66a with other ligands and can initiate signal transduction in neutrophils. Three other peptides from CD66 family members also stimulated neutrophils.
  • CD66 family members may play important roles, the protein backbone itself appears to have important activity in this and other studies.
  • bacterial fusion proteins free of carbohydrates containing the N or A3B3 domains of CD66e can block CD66e homotypic adhesion, demonstrating that protein-protein interaction is involved in CD66e homotypic adhesion (23).
  • Deglycosylated forms of CD66b and CD66c retain heterotypic adhesion activity (31 ), further demonstrating that carbohydrates are not necessary for their adhesion functions.
  • both recombinant N- terminal domains of CD66a and CD66e expressed in Escherichia coli bind Opa proteins with the same specificities as native CD66 molecules, and deglycosylated forms of CD66e bind bacterial Opa proteins (50) .
  • Site directed mutagenesis studies ofthe related proteins C-CAM-105 and CEA (CD66e) have identified regions important for certain functional activities. For example, the integrity of Arg-98 in the consensus ATPase domain (GPAYSGRET) of C-CAM-105 is essential for homotypic aggregation (58). This arginine is highly conserved in Ig domains, being important in forming a salt bridge with a highly conserved aspartate within the same domain (16). In our model the consensus ATPase domain is present in the sequence of peptide CD66a-5. However, peptide CD66a-5 had no activity in our assay. The finding that these short peptides can stimulate neutrophils, as can
  • CD66a mAbs (26-28, 67, 74, 75) suggests that they have significant affinity for a surface structure, possibly native CD66a. If so, whether the activity derives from binding native CD66a and transducing a signal directly, or by another mechanism will require further study.
  • the ability ofthe synthetic peptides described here to activate neutrophils could be mediated by alterations in
  • CD66a dimerization possibly by disrupting a preexisting association of CD66a with other CD66 members (including CD66a itself in the form of dimers or oligomers already present on the cell surface) or by stimulating dimerization. It has been suggested that CD66a (76) and CD66e (77) exist on the cell surface as dimers. Dimerization of CD66a could potentially occur via interactions between the extracellular domains of CD66a molecules or via other mechanisms. In other receptor systems (e.g. EGF-monomeric, PDGF-dimeric), it is clear that bivalency of ligand is not necessary to induce receptor dimerization (78-81). Finally, the observed functional "stimulation" could reflect either "stimulation" per se or possibly release from a baseline inhibition.
  • CD66 family members transmit signals e.g. activation in neutrophils, immune suppression of T-lymphocytes, or growth regulating signals in epithelial cells and carcinomas
  • CD66a is phosphorylated in neutrophils and colon cancer cells (4, 59-61), and associated protein kinase and phosphatase activity may be involved (59, 62).
  • At least eight isoforms of CD66a derived from differential splicing have been described (3, 12, 13, 25). These isoforms contain one N-domain, either three, two, or no Ig C2-like domains, and either a short or a long cytoplasmic tail.
  • the cytoplasmic domain of neutrophil CD66a contains an immune tyrosine inhibitory motif (ITIM), as well as a motif similar to ITAM (immune tyrosine activating motif) (3, 59). Phosphorylation of ITAMs and ITIMs leads to binding of protein tyrosine kinases and protein tyrosine phosphatases, respectively, which leads to modification of signal transduction (62, 63).
  • ITIM immune tyrosine inhibitory motif
  • Calmodulin has also been found to bind to the cytoplasmic domain of CD66a, causing an inhibition of homotypic self-association of CD66a in a dot-blot assay (82).
  • CD66a has also recently been shown to dimerize in solution, and calcium- activated calmodulin caused dissociation of CD66a dimers in vitro; suggesting that CD66a dimerization is regulated by calmodulin and intracellular calcium (76). It has been suggested that CD66a dimerization could also be influenced by phosphorylation; CD66a is phosphorylated on Thr-453 in the calmodulin binding site by protein kinase C (3). Clearly, dimerization of CD66a could affect binding of other signal regulating molecules.
  • CD66 family members appear to be involved in a wide variety of important biological processes, and their differential expression provides the possibility for diverse interactions.
  • CD66a, CD66b, CD66c, and CD66d are expressed on neutrophils; CD66e is expressed on many tumor cells but not leukocytes; CD66b is expressed on neutrophils but not epithelial cells; CD66c is expressed on both neutrophils and epithelial cells (reviewed in (1) and (13)).
  • CD66a was originally described in biliary canaliculi, it has since been found in carcinomas as well as normal tissues, including: sebaceous glands (83, 84), neutrophils, placenta, stomach, breast, pancreas, thyroid, prostate, lung, kidney, uterus, and colon (reviewed in (1) and (25)).
  • the surface expression of these molecules in other cells may also be regulated; for example, CD66a expression is induced on HUVECs following treatment with gamma-IFN (10).
  • surface expression of CD66 family members may be regulated by other stimuli and this may modify the signal transduction capabilities of cell surface CD66 molecules.
  • CD66 members appear to play an important role in inflammation.
  • Each ofthe CD66 family members expressed on neutrophils, CD66a, CD66b, CD66c, and CD66d, are capable of transmitting activation signals in neutrophils, and neutrophil CD66a and CD66c appear to be able to present CD 15s (a ligand for ELAM-1 or E-selectin) to E-selectin on endothelial cells in a functional way (26).
  • CD 15s a ligand for ELAM-1 or E-selectin
  • E-selectin E-selectin on endothelial cells in a functional way (26).
  • Recent studies have demonstrated the presence of CD66a on T- lymphocytes and a subset of NK cells (CD16-, CD56+) that predominate in decidua (87), and CD66a is upregulated in activated T-cells (87).
  • CD66e expression by tumor cells is correlated with resistance to NK/LAK cell
  • CD66 monoclonal antibodies recognize a phosphotyrosine-containing protein bearing a carcinoembryonic antigen cross-reacting antigen on the surface of human neutrophils. Journal of Immunology 148:852-860.
  • CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members ofthe carcinoembryonic antigen family of adhesion molecules. Blood 78:63-74. 9. Kuroki, M., Y. Matsuo, T. Kinugasa, and Y. Matsuoka. 1992.
  • NCA-95 Three different NCA species, CGM6/CD67, NCA-95, andNCA-90, and comprised in the major 90 to 100-KDa band of granulocyte NCA detectable upon SDS- polyacrylamide gel electrophoresis. Biochemical and Biophysical Research Communications 182:501-506.
  • Monoclonal, anti-domain and anti-peptide antibodies assign the molecular weight 160,000 granulocyte membrane antigen ofthe CD66 cluster to a mRNA species encoded by the biliary glycoprotein gene, a member ofthe carcinoembryonic antigen gene family. Journal of Immunology 150:4978-4984.
  • CD66 identifies the biliary glycoprotein (BGP) adhesion molecule: cloning, expression and adhesion functions ofthe BGPc splice variant. Blood 84:200-210.
  • BGP biliary glycoprotein
  • Biliary glycoprotein a member ofthe immunoglobulin supergene family, functions in vitro as a ca2+-dependent intercellular adhesion molecule. Cell Growth and Differentiation 1:527-533.
  • BGP biliary glycoprotein
  • C-CAM epithelial cell adhesion molecule
  • C-CAM epithelial cell adhesion molecule
  • C-CAM 1 epithelial cell adhesion molecule
  • the N-domain ofthe human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae. Molecular Microbiology 22:929-939.
  • CD66 Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae. Molecular Microbiology 22:941-950. 47. Gray-Owen, S., C. Dehio, A. Haude, F. Grunert, and T. F. Meyer. 1997. CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria Gonorrhoeae and human polymorphonuclear phagocytes. EMBO Journal 16:3435-3445.
  • Binding ofthe coronavirus mouse hepatitis virus A59 to its receptor expressed from a recombinant vaccinia virus depends on posttranslational processing ofthe receptor glycoprotein. Journal of Virology 66:4028-4039. 53. Williams, R. K., G.-S. Jiang, and K. V. Holmes. 1991. Receptor for mouse hepatitis virus is a member ofthe carcijoj embryonic antigen family of glycoproteins. Proceedings ofthe National Academy of Sciences of United States of America 88:5533-5536.
  • CD66a an adhesion molecule ofthe carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues. Journal of Histochemistry and Cytochemistry 44 : 31 -41.
  • Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties ofthe rat liver canalicular bile acid transporter/ecto- ATPase/cell CAM 105 and carcinoembryonic antigen. Journal of Biological Chemistry 271:33095-33104.
  • the fourth immunoglobulin domain ofthe stem cell factor receptor couples ligand binding to signal transduction.
  • Epidermal growth factor induces rapid, reversible aggregation ofthe purified epidermal growth factor receptor. Biochemistry 26:1443-1441.

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Abstract

L'invention concerne des peptides capables de moduler la fonction (p. ex. activités de signalisation ou d'adhésion) des membres de la famille CD66 (CEACAM) et/ou de leurs ligands.
PCT/US2000/023482 1999-08-26 2000-08-25 Peptides capables de moduler la fonction des membres de la famille cd66 (ceacam) WO2001013937A1 (fr)

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EP00957846A EP1212075A4 (fr) 1999-08-26 2000-08-25 Peptides capables de moduler la fonction des membres de la famille cd66 (ceacam)
CA002383562A CA2383562A1 (fr) 1999-08-26 2000-08-25 Peptides capables de moduler la fonction des membres de la famille cd66 (ceacam)
AU69407/00A AU6940700A (en) 1999-08-26 2000-08-25 Peptides capable of modulating the function of cd66 (ceacam) family members

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US60/150,791 1999-08-26
US15250199P 1999-09-02 1999-09-02
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Publication number Priority date Publication date Assignee Title
WO2002012535A1 (fr) * 2000-08-07 2002-02-14 Karolinska Innovations Ab Utilisation d'anticorps anti-ceacam pour la stimulation de cellules b dans la production d'anticorps monoclonaux ou en immunotherapie
WO2003018748A3 (fr) * 2001-08-16 2004-05-27 Kimberly Clark Co Composes anticancereux et de cicatrisation de plaies
EP1472276A2 (fr) * 2001-02-28 2004-11-03 Keith M. Skubitz Peptides de petite taille a capacite de modulation de la fonction des membres de la famille des cd66 (ceacam)
US6852320B2 (en) 1998-04-15 2005-02-08 The Brigham & Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
US7071164B2 (en) 2001-08-16 2006-07-04 Kimberly-Clark Worldwide, Inc. Anti-cancer and wound healing compounds
US7148194B2 (en) 2002-12-30 2006-12-12 Kimberly-Clark Worldwide, Inc. Method to increase fibronectin
US7186693B2 (en) 2001-08-16 2007-03-06 Kimberly - Clark Worldwide, Inc. Metalloproteinase inhibitors for wound healing
US7189700B2 (en) 2003-06-20 2007-03-13 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
WO2007051805A2 (fr) 2005-11-01 2007-05-10 Charitè - Universitätsmedizin Berlin (Charité) Utilisation de matieres specifiques de ceacam8 pour traiter des maladies auto-immunes, et procede de criblage de matieres induisant l'apoptose
WO2007129999A3 (fr) * 2004-07-14 2009-05-07 Biospectrum Inc Peptides d'anti-prolifération et anticorps permettant de les détecter
EP2377883A1 (fr) 2010-04-15 2011-10-19 Universite d'Auvergne Clermont I Antagonistes pour la prévention ou le traitement de maladies intestines inflammatoires et plus particulièrement de la maladie de Crohn
WO2013054331A1 (fr) * 2011-10-11 2013-04-18 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anticorps dirigés contre la molécule d'adhésion cellulaire associée à l'antigène carcinoembryonnaire (ceacam)
US20150174274A1 (en) * 2005-06-09 2015-06-25 Gal Markel Modulation of immunity and ceacam1 activity
US20150315610A1 (en) * 2012-12-25 2015-11-05 Takara Bio Inc. Aav variant
EP1988901A4 (fr) * 2006-02-27 2018-04-18 Gal Markel Agents antibactériens à base de ceacam
EP3210996A4 (fr) * 2014-10-20 2018-07-11 Schweitzer Biotech Company Ltd. Peptide capable de promouvoir la croissance neuronale et application de celui-ci
US10550196B2 (en) 2014-04-27 2020-02-04 Famewave Ltd. Humanized antibodies against CEACAM1
AU2018251839B2 (en) * 2017-04-10 2022-06-30 Immatics Biotechnologies Gmbh Peptides and combination thereof for use in the immunotherapy against cancers
US11427647B2 (en) 2014-04-27 2022-08-30 Famewave Ltd. Polynucleotides encoding humanized antibodies against CEACAM1

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US5965710A (en) * 1993-08-21 1999-10-12 Imperial Cancer Research Technology Limited Monoclonal antibodies for use in diagnosis and treatment of colorectal cancer

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SKUBITZ ET AL.: "Stimulation of neutrophil adhesion to endothelial cells by synthetic peptides of CD66a", MOLECULAR BIOLOGY OF THE CELL, vol. 10, no. SUPPLEMENTAL, November 1999 (1999-11-01), pages 78A, ABSTRACT 452, XP002936888 *
SKUBITZ ET AL.: "Synthetic peptides of CD66a stimulate neutrophil adhesion to endothelial cells", JOURNAL OF IMMUNOLOGY, vol. 164, no. 8, 15 April 2000 (2000-04-15), pages 4257 - 4264, XP002936887 *
TEIXEIRA ET AL.: "The N-domain of the biliary glycoprotein (BGP) adhesion molecule mediates homotypic binding: Domain interactions and epitope analysis of BGPc", BLOOD, vol. 84, no. 1, 1 July 1994 (1994-07-01), pages 211 - 219, XP002936886 *

Cited By (38)

* Cited by examiner, † Cited by third party
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US8110189B2 (en) 1998-04-15 2012-02-07 The Brigham And Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
US6852320B2 (en) 1998-04-15 2005-02-08 The Brigham & Women's Hospital, Inc. T cell inhibitory receptor compositions and uses thereof
US7132255B2 (en) 1998-04-15 2006-11-07 The Brigham And Women's Hospital, Inc. Identification of compounds that bind biliary glycoprotein and affect cytotoxic T lymphocyte activity
WO2002012535A1 (fr) * 2000-08-07 2002-02-14 Karolinska Innovations Ab Utilisation d'anticorps anti-ceacam pour la stimulation de cellules b dans la production d'anticorps monoclonaux ou en immunotherapie
EP1472276A2 (fr) * 2001-02-28 2004-11-03 Keith M. Skubitz Peptides de petite taille a capacite de modulation de la fonction des membres de la famille des cd66 (ceacam)
EP1472276A4 (fr) * 2001-02-28 2007-05-09 Keith M Skubitz Peptides de petite taille a capacite de modulation de la fonction des membres de la famille des cd66 (ceacam)
US7071164B2 (en) 2001-08-16 2006-07-04 Kimberly-Clark Worldwide, Inc. Anti-cancer and wound healing compounds
US7186693B2 (en) 2001-08-16 2007-03-06 Kimberly - Clark Worldwide, Inc. Metalloproteinase inhibitors for wound healing
WO2003018748A3 (fr) * 2001-08-16 2004-05-27 Kimberly Clark Co Composes anticancereux et de cicatrisation de plaies
US7148194B2 (en) 2002-12-30 2006-12-12 Kimberly-Clark Worldwide, Inc. Method to increase fibronectin
US7189700B2 (en) 2003-06-20 2007-03-13 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
US7795225B2 (en) 2003-06-20 2010-09-14 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
WO2007129999A3 (fr) * 2004-07-14 2009-05-07 Biospectrum Inc Peptides d'anti-prolifération et anticorps permettant de les détecter
US10188760B2 (en) * 2005-06-09 2019-01-29 Gal Markel Modulation of immunity and CEACAM1 activity
US11793894B2 (en) 2005-06-09 2023-10-24 Gal Markel Modulation of immunity and CEACAM1 activity
US20150174274A1 (en) * 2005-06-09 2015-06-25 Gal Markel Modulation of immunity and ceacam1 activity
US8309091B2 (en) 2005-11-01 2012-11-13 Charite-Universitatsmedizin Berlin CEACAM8-related method for treating autoimmune diseases
WO2007051805A2 (fr) 2005-11-01 2007-05-10 Charitè - Universitätsmedizin Berlin (Charité) Utilisation de matieres specifiques de ceacam8 pour traiter des maladies auto-immunes, et procede de criblage de matieres induisant l'apoptose
EP1988901A4 (fr) * 2006-02-27 2018-04-18 Gal Markel Agents antibactériens à base de ceacam
US11433113B2 (en) 2006-02-27 2022-09-06 Gal Markel CEACAM based antibacterial agents
US10765719B2 (en) 2006-02-27 2020-09-08 Gal Markel CEACAM based antibacterial agents
WO2011128429A1 (fr) 2010-04-15 2011-10-20 Universite D'auvergne Clermont I Antagonistes pour la prévention ou le traitement de maladies inflammatoires de l'intestin, en particulier de la maladie de crohn
EP2377883A1 (fr) 2010-04-15 2011-10-19 Universite d'Auvergne Clermont I Antagonistes pour la prévention ou le traitement de maladies intestines inflammatoires et plus particulièrement de la maladie de Crohn
CN103987729A (zh) * 2011-10-11 2014-08-13 堤乐哈修门医学研究基础建设及服务有限公司 癌胚抗原相关细胞粘附分子(ceacam)的抗体
AU2012322272C1 (en) * 2011-10-11 2017-11-02 Ramot At Tel-Aviv University Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
US9771431B2 (en) 2011-10-11 2017-09-26 Ccam Biotherapeutics Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
US11891453B2 (en) 2011-10-11 2024-02-06 Famewave Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
AU2012322272B2 (en) * 2011-10-11 2017-08-17 Ramot At Tel-Aviv University Ltd. Antibodies to carcinoembryonic antigen-related cell adhesion molecule (CEACAM)
WO2013054331A1 (fr) * 2011-10-11 2013-04-18 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anticorps dirigés contre la molécule d'adhésion cellulaire associée à l'antigène carcinoembryonnaire (ceacam)
CN103987729B (zh) * 2011-10-11 2017-05-31 堤乐哈修门医学研究基础建设及服务有限公司 癌胚抗原相关细胞粘附分子(ceacam)的抗体
US9938541B2 (en) * 2012-12-25 2018-04-10 Takara Bio Inc. AAV variant
US20150315610A1 (en) * 2012-12-25 2015-11-05 Takara Bio Inc. Aav variant
US11427647B2 (en) 2014-04-27 2022-08-30 Famewave Ltd. Polynucleotides encoding humanized antibodies against CEACAM1
US10550196B2 (en) 2014-04-27 2020-02-04 Famewave Ltd. Humanized antibodies against CEACAM1
US11866509B2 (en) 2014-04-27 2024-01-09 Famewave Ltd. Humanized antibodies against CEACAM1
US10407465B2 (en) 2014-10-20 2019-09-10 Schweitzer Biotech Company Ltd. Peptides enhancing neuronal outgrowth and application thereof
EP3210996A4 (fr) * 2014-10-20 2018-07-11 Schweitzer Biotech Company Ltd. Peptide capable de promouvoir la croissance neuronale et application de celui-ci
AU2018251839B2 (en) * 2017-04-10 2022-06-30 Immatics Biotechnologies Gmbh Peptides and combination thereof for use in the immunotherapy against cancers

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EP1212075A4 (fr) 2005-11-23
WO2001013937A9 (fr) 2002-09-06

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