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WO2015037009A1 - Protéines isolées capables de se lier à la plexine-a4, et procédés de production et d'utilisation - Google Patents

Protéines isolées capables de se lier à la plexine-a4, et procédés de production et d'utilisation Download PDF

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Publication number
WO2015037009A1
WO2015037009A1 PCT/IL2014/050827 IL2014050827W WO2015037009A1 WO 2015037009 A1 WO2015037009 A1 WO 2015037009A1 IL 2014050827 W IL2014050827 W IL 2014050827W WO 2015037009 A1 WO2015037009 A1 WO 2015037009A1
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Prior art keywords
protein
plexin
cdr3
seq
cdr2
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WO2015037009A8 (fr
Inventor
Yaron GRUPER
Revital ALOYA
Boaz Kigel
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PLEXICURE Ltd
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PLEXICURE Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention in some embodiments thereof, relates to isolated proteins capable of binding plexin- A4 and methods of producing and using same.
  • the plexin family of receptors includes 9 members divided into 4 subfamilies. They are single pass transmembrane receptors characterized by an intracellular G'TPase activating (GAP) domain. The four type- A plexitis function as direct receptors for class- 6 Semaphoring.
  • Plexin- A 1 is a receptor for Sema6D while plexin- A2 and Plexin-A4 are receptors for Seraa.6A and Seraa.6B.
  • Class-6 semaphoring are single pass membrane bound semaphorins thai were initially found to function as axon guidance factors but have recently been found to function outside of the central nervous system loo.
  • Sema6D is the best characterized factor of this semaphorin subfamily. It functions as a promoter of tumorigenesis, immune responses, and tissue remodeling. Interestingly, the sema6D receptor plexin- A.1 forms complexes with the VEGF receptor, VEGFR-2, which undergoes phosphorylation on stimulation with sema6D. in contrast, Sema6A was characterized as an inhibitor of angiogenesis.
  • Plexin- A4 may represent a target for the development of novel anti- angiogenic and anti- tumorigenic drugs.
  • WO 2001/14420 teaches compositions and methods related to newly isolated plexins.
  • Plexin specific binding agents are disclosed and their use in the treatment of oncological diseases is envisaged.
  • Specifically disclosed is the nucleic acid sequence and amino acid sequence of plexin A4.
  • WO 2001/14420 also contemplates suppressing or altering aberrant cell growth involving a signaling between plexin and neuropilin using an agent (e.g., an antibody) which interferes with the binding between a plexin and a neuropilin.
  • an agent e.g., an antibody
  • WO2012/114339 teaches a high affinity molecule which comprises a binding domain which binds a type-A plexin receptor such as Plexin-A4, wherein the binding domain inhibits proliferative signals through the type-A plexin receptor but does not interfere with binding of a neuropilin or semaphorin 6A to the type-A plexin receptor.
  • U.S. 20120251539 teaches treating immune-related disorders by administering an inhibitor of plexin-A4 activity, which results in reducing the plexin- A4 activity.
  • the inhibitor may be for example, a plexin- A4 antibody or a plexin- A4 fusion protein.
  • an isolated protein comprising an antigen recognition domain which specifically binds human Plexin A4, wherein the antigen recognition domain comprises a complementarity determining region (CDR) amino acid sequence as set forth in: Xi-Gln-X 2 -X 3 -X 4 -X5-Pro-X 6 -Thr (SEQ ID NO: 28)
  • CDR complementarity determining region
  • XI is serine or glutamine
  • X2 is a hydroxylated amino acid
  • X3 is Serine or Threonine
  • X4 is Serine or histidine
  • X5 is Tyrosine or valine
  • X6 is a hydrophobic amino acid.
  • the CDR amino acid sequence is on a light chain of the antigen recognition domain.
  • the CDR amino acid sequence is CDR3.
  • the X 2 is serine or tyrosine.
  • the hydrophobic amino acid is selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and cysteine.
  • the hydrophobic amino acid is leucine or tryptophane.
  • the CDR amino acid sequence is as set forth in SEQ ID NO: 29 (QQYSSYPLT) (clone 158). According to some embodiments of the invention, the CDR amino acid sequence is as set forth in SEQ ID NO: 30 (SQSTHVPLT) (clones 75, 151, 139).
  • the CDR amino acid sequence is as set forth in SEQ ID NO: 31 (SQSTHVPWT 86, 69, 60, 146, 30, 20, 25).
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 32, 33, 34, 35 and 36 (vhCDRl-3, vlCDRl-2, respectively) (clone 158).
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 37, 38, 39, 40 and 41 (vhCDRl-3, vlCDRl-2, respectively) (clones 75, 151, 139).
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 42, 43, 44, 45 and 46 (vhCDRl-3, vlCDRl-2, respectively (clones 86, 69, 60, 146, 30, 20).
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 47, 48, 49, 50 and 51 (vhCDRl-3, vlCDRl-2, respectively) (clone 21).
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 52, 53, 54, 55 and 56 (vhCDRl-3, vlCDRl-2, respectively) (clone 25).
  • an isolated protein comprising an antigen recognition domain which specifically binds human Plexin A4, wherein the antigen recognition domain comprises the complementarity determining region (CDR) amino acid sequence set forth in SEQ ID NO: 57: Asp-Tyr-Xi-Met-X 2
  • Xi is any amino acid
  • X 2 is Histidine or Serine.
  • Xi is Tyrosine or Alanine.
  • an isolated protein comprising an antigen recognition domain which specifically binds human Plexin A4, wherein the antigen recognition domain comprises the complementarity determining region (CDR) amino acid sequences set forth in SEQ ID NO: 60 (DYAMS, heavy chain CDRl), 61 (TISG/SGGGYTYYPDSV, heavy chain CDR2), 62 (LDVXiFVDY, heavy chain CDR3), 63 (RSSQSLVHSNGNTYLH, light chain CDRl), 64 (KVSNRFS, light chain CDR2), and 65 (SQSTHVPX 2 T, light chain CDR3).
  • CDR complementarity determining region
  • XI is histidine, tyrosine or asparagine and wherein the X2 is leucine or tryptophane.
  • the isolated protein has the CDR amino acid sequences of:
  • an isolated protein comprising an antigen recognition domain which comprises six complementarity determining region (CDR) amino acid sequences as set forth in:
  • an isolated protein comprising an antigen recognition domain which comprises six complementarity determining region (CDR) amino acid sequences selected from the group consisting of:
  • the protein competes with semaphorin 6B (Sema-6B) binding to Plexin A4.
  • the protein inhibits tumor cell proliferation.
  • the tumor cell is K-Ras mutated.
  • the protein inhibits endothelial cell proliferation.
  • the protein inhibits VEGF- induced Erk phosphorylation. According to some embodiments of the invention, the protein inhibits Sema-6B- induced Erk phosphorylation.
  • the protein synergizes with a chemotherapy to inhibit tumor cell proliferation.
  • the isolated protein is a bispecific antibody.
  • the isolated protein is a monoclonal antibody.
  • the isolated protein is an IgGl antibody.
  • the isolated protein is an antibody fragment.
  • the antibody fragment is selected from the group consisting of a Fab fragment, a (Fab) 2 fragment, an Fv fragment and a single chain antibody.
  • the isolated protein is attached to a pharmaceutical agent.
  • an anti Plexin A4 antibody comprising: providing anti Plexin A4 antibodies;
  • an article of manufacture comprising a packaging material packaging the protein and a chemotherapy.
  • the protein and the chemotherapy are in separate formulations. According to some embodiments of the invention, the protein and the chemotherapy are in a co -formulation.
  • a method of reducing angiogenesis in a tissue comprising contacting the tissue with the protein, thereby reducing angiogenesis in the tissue.
  • a method of reducing cell growth and proliferation in a tissue comprising contacting the tissue with the protein, thereby reducing cell growth and proliferation in the tissue.
  • the contacting is effected ex- vivo.
  • a method of treating an angiogenesis-related disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the protein, thereby treating the angiogenesis-related disorder.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the protein, thereby treating cancer.
  • the tissue comprises a cancer tissue.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and as an active ingredient the protein.
  • the pharmaceutical composition further comprises a chemotherapeutic agent.
  • the cancer is K-Ras mutated.
  • the tumor cell is a pancreatic tumor cell.
  • the protein binds Plexin-A4 with a K D of 20 nM or less.
  • the protein binds Plexin-A4 but does not bind Plexin-Al, Plexin-A2 or Plexin-A3, as determined by FACS.
  • the protein binds the native form of Plexin-A4, as determined by Western blot analysis and FACS.
  • the protein does not bind the denatured form of Plexin-A4, as determined by Western Blot analysis.
  • the method further comprises isolating the protein following the culturing.
  • a method of producing the protein comprising culturing a host cell expressing the protein such that the protein is produced.
  • a method of detecting a plexin-A4 expressing cell comprising contacting a cell suspicious of expressing the plexin-A4 with the protein described herein under conditions which allow an immunocomplex formation, wherein a presence of the immunocomplex is indicative of a plexin-A4 expressing cell.
  • a method of diagnosing cancer in a subject in need thereof comprising contacting a biological sample of the subject, the biological sample comprising cells suspicious of being cancerous, with the protein described herein under conditions which allow an immunocomplex formation, wherein a level of the immunocomplex above that of a control reference sample comprising non-cancerous cells is indicative of cancer.
  • the protein is attached to a detectable moiety, such as described hereinabove.
  • Figure 1 shows sequence alignments of the VLk and VH amino acid sequence of the antibodies of some embodiments of the present invention.
  • Clone 20 is presented by SEQ ID NOs: 3-4
  • Clone 21 is presented by SEQ ID NOs: 9-10
  • Clone 146 is presented by SEQ ID NOs: 13-14
  • Clone 69 is presented by SEQ ID NOs: 21-22
  • Clone 86 is presented by SEQ ID NOs: 25-26
  • Clone 60 is presented by SEQ ID NOs: 19-20
  • Clone 30 is presented by SEQ ID NOs: 11-12
  • Clone 25 is presented by SEQ ID NOs: 7-8
  • Clone 139 is presented by SEQ ID NOs: 17-18
  • Clone 75 is presented by SEQ ID NOs: 23-24
  • Clone 151 is presented by SEQ ID NOs: X15-16XX
  • Clone 158 is presented by SEQ ID NOs: 1-2
  • Clone 27 is presented by SEQ ID NOs: 5-6.
  • (*)
  • Figure 2 is a graphic presentation of the binding affinity of the isolated IgGs to rPlexin A4 as tested by an ELISA assay.
  • Figures 3A-D show the effect of the antibodies of some embodiments of the invention on cell growth and viability.
  • Figures 3A-B show a dose-dependent effect of the antibodies on various cancer cell lines.
  • Figure 3C outlines the characteristics of the cancer cell lines testes (adopted from the Sanger cell line project). Those responsive cells are K-Ras mutated.
  • Figure 3D shows Plexin /Sema6B expression in the tested cell lines, by immunoblot.
  • Figures 4A-B show the effect of the antibodies of some embodiments of the invention on human endothelial cell (HUVEC) growth and Erk phosphorylation in the presence or absence of growth factors, as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • Figures 5A-C show the ability of the antibodies of some embodiments of the invention to compete with Semaphorin 6B (Sema6B) binding to Plexin A4.
  • Figure 5A is a competitive phage ELISA.
  • Figures 5B-C show the inhibitory effect of the antibodies on Sema6B binding to lung cancer, A549, cells and Sema6B -dependent Erk phosphorylation.
  • Figures 6A-B are graphs showing the effect of the antibodies of some embodiments of the invention on chemotherapy-induced cell growth inhibition in A549 cells ( Figure 6 A) and colon cancer, HCT116 cells ( Figure 6B).
  • Figures 7A-B is a graph showing the effect of the antibodies of some embodiments of the invention on cell proliferation as tested by BrdU incorporation into newly synthesized DNA of replicating cells ( Figure 7A) or AlamarBlue staining ( Figure 7B).
  • Figures 8A-D show the effect of the IgG 75 antibodies of some embodiments of the invention on cell growth and viability (A549 and HCT116 cells).
  • Figures 9A-D are graphs showing the ability of some embodiments of the invention to induce internalization of Plexin-A4 as determined by ELISA and by a secondary Saporin conjugate internalization assay.
  • Figure 10 show sensograms of antibody binding to Plexin- A4 as tested by Biacore.
  • Figures 11A-B show that IgG 20, 75, 158 bind the native form of Plexin- A4 and not the denatured form of the protein, as measured by Western blot analysis ( Figure 11 A) and FACS ( Figure 11B).
  • Figures 12 A-B show the specificity of IgG 20, 75, 158 to Plexin A4 and inability to bind other members to the Type A plexin family i.e., Plexin Al, Plexin A2 and Plexin A3.
  • Figure 12A shows stable expression of each of Plexin Al, Plexin A2 and Plexin A3 in PAE cells individually expressing a single member of the family, as demonstrated by Western Blot analysis and FACS.
  • Figure 12B shows cell binding using IgG 20, 75, 158 or control commercial antibodies.
  • Figure 13 is a graph showing time dependent serum concentration of anti plexin A4 IgG 75 following intravenous (i.v.) administration into mice.
  • the present invention in some embodiments thereof, relates to isolated proteins capable of binding plexin- A4 and methods of producing and using same.
  • Plexin A4 binds to neuropilin 1 (Nrpl) and neuropilin 2 (Nrp2) and transduces signals from Sema3A, Sema6A, and Sema6B. Recent studies suggest that inhibitors of plexin- A4 may have anti-tumorigenic and anti- angiogenic functions.
  • the present inventors have immunized mice against plexin- A4 and functionally screened a phage display library expressing total mice mRNA for antibodies which bind plexin-A4, compete with the binding of the tumorigenic and angiogenic ligand semaphorin-6B (sema-6B) and inhibit tumor and endothelial cell proliferation, thereby isolating agents which can be used for the treatment of cancer and reduce angiogenesis.
  • semaphorin-6B semaphorin-6B
  • the present inventors have identified a number of antibodies, all sharing complementarity determining region (CDR) sequences with high level of homology (see Figure 1).
  • the antibodies inhibit tumor and endothelial cell proliferation ( Figures 3A-C and 4A-B), displace sema-6B binding to the receptor ( Figures 5A-C), cooperate with chemotherapy in inhibiting tumor cell proliferation ( Figures 6A-B) and induce internalization of plexin- A4 ( Figures 9A-B, D).
  • Figure 9C shows the affinities of the antibodies to be lower than 20 nM reaching as low as 1 nM, both being clinically relevant.
  • the antibodies bind the plexin- A4 protein in its native form when presented on the cell and not in its denatured form ( Figures 11A-B). Importantly, the antibodies do not bind any of the other known type A plexins as shown in Figures 12A-B. All these findings place these antibodies as potent therapeutic agents.
  • “Plexin A4" refers to the protein that is encoded by the plexin A4 gene. According to a specific embodiment, the protein is the translation product of the human PLXNA4 (SEQ ID No: 77, NP_065962).
  • Semaphorin 6B or “Sema-6B” refers to the protein that is encoded by the Semaphorin 6B gene. According to a specific embodiment, the protein is the translation product of the human SEMA6B (e.g., NM_032108).
  • a protein refers to an isolated polypeptide molecule having a high affinity towards Plexin- A4.
  • a high affinity molecule which is interchangeably referred to as “the protein” or “the isolated protein” refers to a naturally-occurring or synthetic essentially proteinacious molecule, which binds specifically a target protein molecule (i.e., plexin A4) with an affinity higher than 10 ⁇ 6 M. Specific binding can be detected by various assays as long as the same assay conditions are used to quantify binding to the target versus control.
  • the protein is an antibody.
  • the general affinity of the protein is preferably higher than about, 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M and as such is stable under physiological (e.g., in vivo) conditions.
  • the affinity is preferably higher than (i.e., at least) about, 10 "8 M or 10 "9 M, e.g., 1-50 x 10 "9 M, 1-100 x 10 "9 M, 0.5-50 x 10 "9 M or 0.5-100 x 10 "9 M.
  • the term "isolated” refers to a level of purity such that the protein of the invention is the predominant form (e.g., more than 50 %) in the preparation. In other words, other high affinity molecules which are characterized by low or no affinity to Plexin A4 are altogether present in the preparation in less than 50 % of the total high affinity molecules of the preparation.
  • the protein is isolated from the physiological embodiment e.g., from the body (e.g., human or animal).
  • the term isolated also means isolated from a library, such as a phage display library.
  • an isolated protein comprising an antigen recognition domain which specifically binds human Plexin A4, wherein the antigen recognition domain comprises a complementarity determining region (CDR) amino acid sequence as set forth in SEQ ID NO: 28:
  • CDR complementarity determining region
  • XI is serine or glutamine
  • X2 is a hydroxylated amino acid
  • X3 is Serine or Threonine
  • X4 is Serine or histidine
  • X5 is Tyrosine or valine
  • X6 is a hydrophobic amino acid.
  • the CDR amino acid sequence is on a light chain of said antigen recognition domain.
  • the CDR amino acid sequence is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • CDR3 (e.g., on the light chain of the antigen recognition domain).
  • the X 2 is serine or tyrosine.
  • the hydrophobic amino acid is selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and cysteine.
  • the hydrophobic amino acid is leucine or tryptophane.
  • the CDR amino acid sequence is as set forth in SEQ ID NO: 29 (QQYSSYPLT, represented by clone 158).
  • the CDR amino acid sequence is as set forth in SEQ ID NO: 30 (SQSTHVPLT, shared by clones 75, 151, 139).
  • the CDR amino acid sequence is as set forth in SEQ ID NO: 31 (SQSTHVPWT, shared by clones 86, 69, 60, 146, 30, 20).
  • the protein of this aspect of the present invention comprises additional CDR sequences on the light chain and heavy chain of the antigen recognition domain.
  • Such exemplary CDR sequences are provided infra.
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 32, 33, 34, 35, and 36 (vhCDRl-3, vlCDRl-2, respectively), represented by clone 158.
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 37, 38, 39, 40 and 41 (vhCDRl-3, vlCDRl-2, respectively), shared by clones 75, 151, 139.
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 42, 43, 44, 45 and 46 (vhCDRl-3, vlCDRl-2, respectively), shared by clones 86, 69, 60, 146, 30, 20.
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 47, 48, 49, 50 and 51 (vhCDRl-3, vlCDRl-2, respectively), represented by clone 21.
  • the isolated protein further comprises the CDR amino acid sequences set forth in SEQ ID NOs: 52, 53, 54, 55 and 56 (vhCDRl-3, vlCDRl-2, respectively), represented by clone 25.
  • an isolated protein comprising an antigen recognition domain which specifically binds human Plexin A4, wherein the antigen recognition domain comprises the complementarity determining region (CDR) amino acid sequence set forth in SEQ ID NO: 57:
  • CDR complementarity determining region
  • Xi is any amino acid
  • X 2 is Histidine or Serine.
  • the CDR is CDR1 of the heavy chain.
  • Xi is Tyrosine or Alanine.
  • the CDR sequence is as set forth by Asp-Tyr-Tyr-Met-His (SEQ ID NO: 58), represented by clone 158.
  • the CDR sequence is as set forth by Asp-Tyr-Ala-Met-Ser (SEQ ID NO: 59), shared by clones 75, 86, 69, 60, 139, 151, 146, 30, 20, 25 and 21.
  • an isolated protein comprising an antigen recognition domain which specifically binds human Plexin A4, wherein said antigen recognition domain comprises the complementarity determining region (CDR) amino acid sequences set forth in SEQ ID NO: 60 (DYAMS, heavy chain CDRl), 61 (TIS G/S GGGYTYYPDS V, heavy chain CDR2), 62 (LDVXiFVDY, heavy chain CDR3), 63 (RSSQSLVHSNGNTYLH, light chain CDRl), 64 (KVSNRFS, light chain CDR2), and 65 (SQSTHVPX 2 T, light chain CDR3).
  • CDR complementarity determining region
  • Xi of the VH CDR3 is histidine, tyrosine or asparagine and wherein X 2 of the VL CDR3 is leucine or tryptophane.
  • the isolated protein has the CDR amino acid sequences of:
  • an isolated protein comprising an antigen recognition domain which comprises six complementarity determining region (CDR) amino acid sequences selected from the group consisting of: SEQ ID NOs: 35 (CDR1), 36 (CDR2) and 70 (CDR3), (sequentially arranged from N to C on a light chain of said protein) and 32 (CDR1), 33 (CDR2) and 34 (CDR3) (sequentially arranged from N to C on a heavy chain of said protein), represented by clone 158;
  • CDR complementarity determining region
  • proteins of the invention comprise native proteins (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, as long as the function is essentially retained i.e., at least 80 % of the activity e.g., plexin A4 binding.
  • modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification.
  • the isolated protein of any of the above aspects is characterized by at least one or more of the below biological activities.
  • the protein competes with Semaphorin 6B (Sema-6B) binding to Plexin A4. By competing with semaphorin 6B binding the protein serves as a blocking or neutralizing molecule. Binding assays are well known in the art and typically involve the use of detectable moieties such as radioactive isotopes (e.g., 125 I) or fluorescent molecules (e.g., FITC-sema-6B). On the basis of these displacement assays, binding parameters to Plexin-A4 can be obtained and expressed, for example, in a Scatchard plot.
  • detectable moieties such as radioactive isotopes (e.g., 125 I) or fluorescent molecules (e.g., FITC-sema-6B).
  • the protein displaces at least 50 % (e.g., at least 60 %, 70 % or 80 %) of semaphorin-6B binding to plexin-A4.
  • the protein inhibits tumor cell and/or endothelial cell proliferation.
  • Methods of analyzing cell proliferation are well known in the art. Examples include, but are not limited to, the Alamar blue assay, BrdU incorporation assay, the MTT assay and the thymidine incorporation assay.
  • the protein inhibits tumor or endothelial cell proliferation by at least 30 % (e.g., at least 40 %, 50 % or 80 %).
  • endothelial cells refers to the cells that form the endothelium, i.e., the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. According to a specific embodiment, the cells express the surface marker CD31.
  • the tumor cell can be of any tumor i.e., solid or non-solid tumor.
  • the cell can be of a primary tumor or a metastatic tumor.
  • the tumor cell is K-Ras mutated. Specific examples of such K-Ras mutations and representative cells are provided in Figure 3C and described in Almoguera et al. (1988) " Cell 53 (4): 549-54; and Tarn et al. (2006). Clin. Cancer Res. 12 (5): 1647-53.
  • the cell can be a non-cultured cell, a product of primary culturing or a cell line.
  • the protein of the present invention inhibits vascular endothelial growth factor (VEGF)-induced Erk phosphorylation.
  • VEGF vascular endothelial growth factor
  • the protein of the present invention inhibits Sema-6B-induced Erk phosphorylation.
  • Extracellular signal-regulated kinases ERKs 1 and 2 (ERKl/2) are members of the mitogen-activated protein kinase (MAPK) family of cell signaling enzymes controlling cell fates such as embryogenesis, cell differentiation, cell proliferation, and cell death.
  • ERKl/2 are activated via dual phosphorylation on specific tyrosine (Tyr 204 ) and threonine (Thr 202 ) residues by mitogen-activated or extracellular signal-regulated protein kinase (MAPK).
  • Erk also referred to as MAPK
  • Erk phosphorylation kits are typically based on the use of a phospho-specific
  • ERK/MAPK Phospho-Thr 202 and Tyr 204
  • kits are available from various vendors including, but not limited to, Sigma- Aldrich, Perkin-Elmer, Cayman Chemicals and Millipore.
  • the protein induces internalization of the plexin-A4 receptor (e.g., IgG75 and IgG20).
  • Internalization or endocytosis is a process by which cells internalize molecules (endocytosis) by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being internalized.
  • Assays for monitoring internalization are well known in the art and may involve, surface biotinylation, radioactive isotope or fluorescent dyes. For instance, using fluorescent dyes to stain the plasma membrane, it is possible to follow the internalization of surface-plexin A4 by microscopy.
  • the protein induces internalization of more than 30 % (e.g., at least 40 %, 50 % or 70 %) plexin A4 over a period of 30 min.
  • the protein synergizes with a chemotherapy
  • chemotherapeutic agents e.g., small molecules, nucleic acid molecules or antibodies
  • examples of some chemotherapeutic agents are listed in Table 1, below.
  • Cyclophosphamide Acute and chronic lymphocytic Ifosfamide leukemias, Hodgkin 's disease, non-Hodgkin ' s lymphomas, multiple myeloma,
  • Methylmelamines Thiotepa Bladder, breast, ovary
  • lymphocytic lymphocytic, and chronic granulocytic leukemias
  • Pentostatin Hairy cell leukemia mycosis fungoides, chronic lymphocytic leukemia
  • Epipodophyl- Etoposide Testis small-cell lung and other Lotoxins Teniposide lung, breast, Hodgkin's
  • lymphomas acute granulocytic leukemia
  • Kaposi's sarcoma Kaposi's sarcoma
  • Daunorubicin Acute granulocytic and acute lymphocytic leukemias
  • lymphomas acute leukemias, breast, genitourinary, thyroid, lung, stomach, neuroblastoma
  • Bleomycin Testis head and neck, skin, esophagus, lung, and
  • Hodgkin's disease non- Hodgkin ' s lymphomas
  • Plicamycin Testis malignant hypercalcemia Mitomycin Stomach, cervix, colon, breast, pancreas, bladder, head and neck
  • non-Hodgkin ' s lymphomas mycosis fungoides, multiple myeloma, chronic granulocytic leukemia
  • Neoplastic Diseases Section XII, pp 1202-1263 in: Goodman and Gilman's
  • Neoplasms are carcinomas unless otherwise indicated.
  • the chemotherapy is paclitaxel or cisplatin.
  • the chemotherapy is an antibody, such as but not limited to, Ibritumomab, bevacizumab (Avastin), Cetuximab (Erbitux), rituximab (Rituxan), alemtuzumab (Campath), trastuzumab (Herceptin) AND panitumumab (Vectibix).
  • the protein binds Plexin-A4 with a KD of 20 nM or less (e.g., 15 nM or less, 10 nM or less, 5 nM or less, 1 nM or less; e.g., 1-20 nM, 0.1-10 nM).
  • the protein binds Plexin-A4 but does not bind Plexin-Al, Plexin-A2 or Plexin-A3, as determined by FACS.
  • the protein binds the native form of Plexin-
  • A4 e.g., as determined by Western blot analysis and FACS.
  • the protein does not bind the denatured form of Plexin-A4, e.g., as determined by Western Blot analysis.
  • the protein has the CDRs are of clone 20 (IgG20), 75 (IgG 75) or 158 (IgG158).
  • the protein of the invention is an antibody.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv and a single chain Fv that are capable of binding to macrophages.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • the antibody is a monoclonal antibody of any subtype e.g., IgG, IgM, IgA etc. According to a specific embodiment the antibody is IgGl or IgG4.
  • the antibody fragment is a Fab having the CDRs of clone 75.
  • Anti Plexin A4 antibodies of some embodiments of the present invention can be selected from a plurality of antibodies (e.g., antibody library) and screening by testing at least one of:
  • Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11: 1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • bispecific or “bifunctional” antibody refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas. See e.g., Songsivilai and Lachmann (1990) Clin. Exp. Immunol. 79:315- 321; Kostelny et al. (1992) J. Immunol. 148: 1547-1553.
  • the bispecific antibody may bind plexin-A4 and another target which is expected to cooperate with plexin-A4 in biological processes, such an angiogenesis, cell proliferation or Erk activation.
  • the bispecific antibody of the invention binds the plexin-A4 receptor (with the CDRs described herein) and at least one of the FGFRl and the ligand (bFGF) as well as the semaphorin 6B.
  • bFGF ligand
  • the bispecific antibody binds distinct epitopes on Plexin-A4.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the proteins (e.g., antibodies) of the invention can be used in a variety of clinical applications. By virtue of their high affinity to plexin-A4 they can be used in diagnostic applications and in personalized treatments which require the testing of plexin-A4 expression.
  • the protein can be attached to a pharmaceutical agent.
  • a pharmaceutical agent can be a drug (used in therapy) or a detectable moiety.
  • detectable or reporter moieties may be conjugated to the proteins of the invention. These include, but not are limited to, a radioactive isotope (such as [125] iodine), a phosphorescent chemical, a chemiluminescent chemical, a fluorescent chemical (fluorophore), an enzyme, a fluorescent polypeptide, an affinity tag, and molecules (contrast agents) detectable by Positron Emission Tomagraphy (PET) or Magnetic Resonance Imaging (MRI).
  • fluorophores examples include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, PE-Cy5, and the like.
  • PE phycoerythrin
  • FITC fluorescein isothiocyanate
  • Cy-chrome Cy-chrome
  • rhodamine green fluorescent protein
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • Texas red PE-Cy5, and the like.
  • fluorophore selection methods of linking fluorophores to various types of molecules see Richard P. Haugland, "Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994", 5th ed., Molecular Probes, Inc. (1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.; Hermanson, "Bioconjugate Techniques", Academic Press New York, N
  • Fluorescence detection methods which can be used to detect the antibody when conjugated to a fluorescent detectable moiety include, for example, fluorescence activated flow cytometry (FACS), immunofluorescence confocal microscopy, fluorescence in-situ hybridization (FISH) and fluorescence resonance energy transfer (FRET).
  • FACS fluorescence activated flow cytometry
  • FISH fluorescence in-situ hybridization
  • FRET fluorescence resonance energy transfer
  • enzymes may be attached to the antibody of the invention [e.g., horseradish peroxidase (HPR), beta-galactosidase, and alkaline phosphatase (AP)] and detection of enzyme-conjugated antibodies can be performed using ELISA (e.g., in solution), enzyme-linked immunohistochemical assay (e.g., in a fixed tissue), enzyme- linked chemiluminescence assay (e.g., in an electrophoretically separated protein mixture) or other methods known in the art [see e.g., Khatkhatay MI. and Desai M., 1999. J Immunoassay 20: 151-83; wisdom GB., 1994. Methods Mol Biol.
  • HPR horseradish peroxidase
  • AP alkaline phosphatase
  • An affinity tag (or a member of a binding pair) can be an antigen identifiable by a corresponding antibody [e.g., digoxigenin (DIG) which is identified by an anti-DIG antibody) or a molecule having a high affinity towards the tag [e.g., streptavidin and biotin].
  • DIG digoxigenin
  • the antibody or the molecule which binds the affinity tag can be fluorescently labeled or conjugated to enzyme as described above.
  • a streptavidin or biotin molecule may be attached to the antibody of the invention via the recognition sequence of a biotin protein ligase (e.g., BirA) as described in the Examples section which follows and in Denkberg, G. et al., 2000. Eur. J. Immunol. 30:3522-3532.
  • a streptavidin molecule may be attached to an antibody fragment, such as a single chain Fv, essentially as described in Cloutier SM. et al., 2000. Molecular Immunology 37: 1067-1077; Dubel S. et al, 1995.
  • Functional moieties such as fluorophores, conjugated to streptavidin are commercially available from essentially all major suppliers of immunofluorescence flow cytometry reagents (for example, Pharmingen or Becton-Dickinson).
  • drug refers to a therapeutically active ingredient such as a small molecule (e.g., chemotherapy), a protein, a lipid, a carbohydrate or a combination of same.
  • the proteins can be attached (or conjugated) to non- proteinacious moieties which increase their bioavailability and half-life in the circulation.
  • non-proteinaceous moiety refers to a molecule not including peptide bonded amino acids that is attached to the above-described protein.
  • exemplary non-proteinaceous and preferably non-toxic moieties which may be used according to the present teachings include, but are not limited to, polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).
  • Such a molecule is highly stable (resistant to in-vivo proteolytic activity probably due to steric hindrance conferred by the non-proteinaceous moiety) and may be produced using common solid phase synthesis methods which are inexpensive and highly efficient, as further described hereinbelow.
  • recombinant techniques may still be used, whereby the recombinant peptide product is subjected to in-vitro modification (e.g., PEGylation as further described hereinbelow).
  • non-proteinaceous non-toxic moieties may also be attached to the above mentioned proteins to promote stability and possibly solubility of the molecules.
  • Bioconjugation of such a non-proteinaceous moiety can confer the proteins amino acid sequence with stability (e.g., against protease activities) and/or solubility (e.g., within a biological fluid such as blood, digestive fluid) while preserving its biological activity and prolonging its half-life.
  • stability e.g., against protease activities
  • solubility e.g., within a biological fluid such as blood, digestive fluid
  • Bioconjugation is advantageous particularly in cases of therapeutic proteins which exhibit short half-life and rapid clearance from the blood.
  • the increased half- lives of bioconjugated proteins in the plasma results from increased size of protein conjugates (which limits their glomerular filtration) and decreased proteolysis due to polymer steric hindrance.
  • the more polymer chains attached per peptide the greater the extension of half-life.
  • measures are taken not to reduce the specific activity of the protein of the present invention (e.g., plexin A4 binding).
  • Bioconjugation of the protein with PEG i.e., PEGylation
  • PEG PEGylation
  • PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG 2 -NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl- disulfide, carbonyldimidazol-activated PEGs, PEG-thiol, PEG-maleimide.
  • NHS N-hydroxysuccinimide
  • SCM-PEG succinimidyl ester of carboxymethylated PEG
  • PEG-NPC PEG p-nitrophenyl carbonates
  • PEG aldehydes PEG-orthopyridyl- disulfide
  • PEG derivatives are commercially available at various molecular weights [See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle, Ala.)]. If desired, many of the above derivatives are available in a monofunctional monomethoxyPEG (mPEG) form.
  • mPEG monomethoxyPEG
  • the PEG added to the antibody amino acid sequence of the present invention should range from a molecular weight (MW) of several hundred Daltons to about 100 kDa (e.g., between 3-30 kDa). Larger MW PEG may be used, but may result in some loss of yield of PEGylated peptides.
  • PEG purity of larger PEG molecules should be also watched, as it may be difficult to obtain larger MW PEG of purity as high as that obtainable for lower MW PEG. It is preferable to use PEG of at least 85 % purity, and more preferably of at least 90 % purity, 95 % purity, or higher. PEGylation of molecules is further discussed in, e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Dunn and Ottenbrite, eds., Polymeric Drugs and Drug Delivery Systems, American Chemical Society, Washington, D.C. (1991).
  • activated PEG such as PEG-maleimide, PEG- vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide
  • Methods of preparing activated PEG molecules are known in the arts.
  • PEG-VS can be prepared under argon by reacting a dichloromethane (DCM) solution of the PEG-OH with NaH and then with di-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2 gram PEG/mL DCM).
  • DCM dichloromethane
  • PEG-AC is made under argon by reacting a DCM solution of the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM).
  • Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.
  • the antibodies of the invention may be produced using recombinant DNA technology (where a polynucleotide encoding the antibody of the invention is introduced into an appropriate host cell where the antibody is synthesized. Exemplary sequences are provided in SEQ ID NOs: 78-103) or by chemical synthesis such as by solid phase techniques.
  • a method of reducing angiogenesis in a tissue comprising contacting the tissue with the protein (e.g., antibody) or a composition comprising same (e.g., conjugated molecule), as described hereinabove, thereby reducing angiogenesis in the tissue.
  • the protein e.g., antibody
  • a composition comprising same e.g., conjugated molecule
  • a method of reducing cell growth and proliferation in a tissue comprising contacting the tissue with the protein, thereby reducing cell growth and proliferation in the tissue.
  • the tissue is a cancerous tissue and the cell is a cancer cell.
  • cancer refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers.
  • cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • cancer or tumors which can be treated according to the present teachings include, but are not limited to, adrenocortical carcinoma, hereditary; bladder cancer; breast cancer; breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptibility to; breast cancer, type 4; breast cancer, type 4; breast cancer- 1; breast cancer-3; breast-ovarian cancer; Burkitt's lymphoma; cervical carcinoma; colorectal adenoma; colorectal cancer; colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditary nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial carcinoma; esophageal cancer; gastric cancer,
  • the cancer is breast cancer, lung cancer (e.g., non-small cell lung cancer), melanoma, ovarian cancer or colon cancer. Pancreatic cancer and Lymphoma.
  • angiogenesis refers to the growth of new blood vessels originating from existing blood vessels.
  • Angiogenesis refers also to “vasculogenesis” which means the development of new blood vessels originating from stem cells, angioblasts or other precursor cells.
  • Angiogenesis can be assayed as described in the Examples section which follows or by measuring the total length of blood vessel segments per unit area, the functional vascular density (total length of perfused blood vessel per unit area), or the vessel volume density (total of blood vessel volume per unit volume of tissue).
  • angiogenesis related disorder or "a disease associated with undesirable angiogenesis” (used interchangeably herein) refers to a clinical condition in which the processes regulating angiogenesis are disrupted resulting in a pathology. Such a pathology affects a wide variety of tissues and organ systems. Diseases characterized by excess or undesirable angiogenesis are susceptible to treatment with the high affinity molecules described herein. The following provides a non-limiting list of such diseases.
  • Excess angiogenesis in numerous organs is associated with cancer and metastasis, including neoplasia and hematologic malignancies.
  • Angiogenesis-related diseases and disorders are commonly observed in the eye where they may result in blindness.
  • Such disease include, but are not limited to, age- related macular degeneration, choroidal neovascularization, persistent hyperplastic vitreous syndrome, diabetic retinopathy, and retinopathy of prematurity (ROP).
  • angiogenesis-related diseases are associated with the blood and lymph vessels including transplant arteriopathy and atherosclerosis, where plaques containing blood and lymph vessels form, vascular malformations, DiGeorge syndrome, hereditary hemorrhagic telangiectasia, cavernous hemangioma, cutaneous hemangioma, and lymphatic malformations.
  • angiogenesis diseases and disorders affect the bones, joints, and/or cartilage include, but are not limited to, arthritis, synovitis, osteomyelitis, osteophyte formation, and HIV-induced bone marrow angiogenesis.
  • the gastro-intestinal tract is also susceptible to angiogenesis diseases and disorders. These include, but are not limited to, inflammatory bowel disease, ascites, peritoneal adhesions, and liver cirrhosis.
  • Angiogenesis diseases and disorders affecting the kidney include, but are not limited to, diabetic nephropathy (early stage: enlarged glomerular vascular tufts).
  • Excess angiogenesis in the reproductive system is associated with endometriosis, uterine bleeding, ovarian cysts, ovarian hyper stimulation.
  • excess angiogenesis is associated with primary pulmonary hypertension, asthma, nasal polyps, rhinitis, chronic airway inflammation, cystic fibrosis.
  • Diseases and disorders characterized by excessive or undesirable angiogenesis in the skin include psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi's sarcoma in AIDS patients, systemic sclerosis.
  • Obesity is also associated with excess angiogenesis (e.g., angiogenesis induced by fatty diet).
  • Adipose tissue may be reduced by the administration of angiogenesis inhibitors.
  • Excess angiogenesis is associated with a variety of auto-immune disorders, such as systemic sclerosis, multiple sclerosis, Sjogren's disease (in part by activation of mast cells and leukocytes).
  • Undesirable angiogenesis is also associated with a number of infectious diseases, including those associated with pathogens that express (lymph)- angiogenic genes, that induce a (lymph) -angiogenic program or that transform endothelial cells.
  • infectious disease include those bacterial infections that increase HIF-1 levels, HIV-Tat levels, antimicrobial peptides, levels, or those associated with tissue remodeling.
  • Retroviridae e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g.
  • Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronaviridae e.g. coronaviruses
  • Pvhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g. ebola viruses
  • Paramyxoviridae e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g.
  • African swine fever virus African swine fever virus
  • angiogenesis-related disorders include, but are not limited to, hemangiomas, rheumatoid arthritis, atherosclerosis, idiopathic pulmonary fibrosis, vascular restenosis, arteriovenous malformations, meningiomas, neovascular glaucoma, psoriasis, angiofibroma, hemophilic joints, hypertrophic scars, Osier- Weber syndrome, pyogenic granuloma, retrolental fibroplasias, scleroderma, trachoma, vascular adhesion pathologies, synovitis, dermatitis, endometriosis, pterygium, wounds, sores, and ulcers (skin, gastric and duodenal).
  • contacting with the cells or tissue is effected ex- vivo.
  • contacting with the cells or tissue is effected in- vivo.
  • the present invention further provides for a method of treating an angiogenesis- related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein as described herein, thereby treating the angiogenesis-related disorder.
  • the present invention specifically provides for a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount the protein as described herein, thereby treating cancer.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term "preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • the term "subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.
  • proteins e.g., antibodies
  • suitable carriers or excipients e.g., water
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the protein e.g., antibody, accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., the isolated protein e.g., antibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.
  • active ingredients e.g., the isolated protein e.g., antibody
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • Dosage amount and interval may be adjusted individually to provide effective levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, 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 pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • an article of manufacture comprising a packaging material packaging the protein, as described herein, and a chemotherapy.
  • the protein and the chemotherapy are in separate formulations.
  • the protein and the chemotherapy are in a co-formulation.
  • the high affinity and specificity of the proteins of some embodiments of the invention allows their use in the detection of plexin-A4-expressing cells and in diagnostic applications.
  • a method of detecting a plexin-A4 expressing cell comprising contacting a cell suspicious of expressing the plexin-A4 with the protein described herein under conditions which allow an immunocomplex formation, wherein a presence of said immunocomplex is indicative of a plexin-A4 expressing cell.
  • the protein is attached to a detectable moiety, such as described hereinabove.
  • a control sample refers to a sample which comprises cells or preparation thereof (e.g., lysate) expressing normal (non-cancerous) levels of plexin-A4.
  • the cells can be of the suspected tissue (e.g., tumor) or of peripheral blood serving as proxy for cancer onset/progression.
  • diagnosis refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome), classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and/or prospects of recovery and screening of a subject for a specific disease.
  • a pathology e.g., a disease, disorder, condition or syndrome
  • Enzyme linked immunosorbent assay This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
  • Western blot This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents.
  • Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
  • Radio-immunoassay In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I 125 ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
  • a specific antibody and radiolabeled antibody binding protein e.g., protein A labeled with I 125
  • a labeled substrate and an unlabelled antibody binding protein are employed.
  • a sample containing an unknown amount of substrate is added in varying amounts.
  • the decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
  • Fluorescence activated cell sorting This method involves detection of a substrate in situ in cells by substrate specific antibodies.
  • the substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • Immunohistochemical analysis This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies.
  • the substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
  • screening of the subject for a specific disease is followed by substantiation of the screen results using gold standard methods (ultrasound, imaging, biopsy sampling for histological analysis, marker screening etc.).
  • proteins of some embodiments of the invention which are described hereinabove for detecting plexin-A4 expressing cells or cancer may be included in a diagnostic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for use in diagnosing and/or assessing cancer.
  • kit can include, for example, at least one container including at least one of the above described diagnostic proteins (e.g., IgG175, IgG20 or IgG58 antibody)) and an imaging reprotein packed in another container (e.g., enzymes, secondary antibodies, buffers, chromogenic substrates, fluorogenic material).
  • the kit may also include appropriate buffers and preservatives for improving the shelf-life of the kit.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • A549 Lung NS carcinoma cells (ATCC, CCL-185), U-87MG Glioma cells (ATCC, HTB-14), NCI-H460 Lung NS carcinoma cells (ATCC, HTB-177), HCT116 Large intestine carcinoma cells (ATCC, CCL-247), MDA-MB-231 Breast carcinoma cells (ATCC, HTB-26), MDA-MB-435 Malignant melanoma cells (ATCC, HTB-129), SK-OV -3 Ovary carcinoma cells ( ATCC, HTB-77) and SK-MEL-5 Malignant melanoma cells ( ATCC, HTB-70), human colon adenocarcinoma HT-29 (ATCC HTB- 38), Pancreatic adenocarcinoma human Capan-1 (ATCC HTB-79) were cultured in DMEM medium containing 10 % FCS and antibiotics in a humidified incubator at 5 % C0 2 .
  • Human Primary Umbilical Vein Endothelial Cells
  • HUVECs were seeded at a concentration of 3 x 10 cells/well in 96-well dishes coated with PBS-gelatin. Growth factors were then added or not, and the number of adherent cells in each culture determined following 3 days in culture. The induction of proliferation was calculated using AlamarBlue (AbD Serotec) reagent as the fold increase in the number of cells relative to untreated cells. A similar protocol was used for cancer cells which were seeded at a concentration of 2.5 x 10 cells/well in uncoated 96-well dishes and grown in full growth medium (DMEM) containing 10 % FBS without added growth factor s. Each experiment was carried out in triplicates for each antibody concentration and independently three times. Further indication for the outcome was also acquired by Hoechst 33342 (molecular probes) nuclear staining and photograph using a fluorescent microscope. BrdU incorporation
  • the Cell Proliferation ELISA kit (Roche) measure cell proliferation by quantitating BrdU incorporated into the newly synthesized DNA of replicating cells. Detection of BrdU positive cells was done using Roche Labeling and detection kit according to the instructions of the manufacturer. In short, the assay is a cellular immunoassay which uses a mouse monoclonal antibody directed against BrdU, while only proliferating cells incorporate BrdU into their DNA.
  • the procedure involves: culturing the cells in a 96-well microtiterplate as described and pulse-labeling them with BrdU for 16h (between 56 and 72 h of the assay; fixing the cells with a fixation solution (4% PFA), which also denatures the genomic DNA; exposing the incorporated BrdU to immunodetection; locating the BrdU label in the DNA with a peroxidase-conjugated anti-BrdU antibody; and quantitating the bound anti-BrdU-POD with a peroxidase substrate by measuring luminescence.
  • the experiment was performed using standard protocols. In short, 96 well plates were coated with 100 ng of the extracellular portion of plexin-A4 or BSA for 1 hour at 37 °C. The wells were washed with 0.05 % (vol/vol) of PBS-T (Tween 20) and then blocked with 1% BSA solution. Various concentrations of the antibodies were placed for 2 hours at room temperature on the coated wells in the presence of 0.5 % BSA. The wells were washed and an anti-mouse IgGl HRP antibody was added for 1 hour at room temperature. A TMB substrate-chromagen (Dako) was added to initiate a colorimetric reaction that was terminated using 0.2 M sulphuric acid. The absorbance of each well was then measured at 420 nm using a TECAN Infinite M200/pro microplate reader.
  • Plexin A4 antibodies an ELISA assay was used.
  • Plexin A4 coated wells were incubated with 12 ⁇ g/mL rSema6B-hFC for 1 h at room temperature, the wells were washed and incubated with control antibody (ConAb), IgG20 , IgG158 or IgG75 at 8 different concentrations (from 0 nM to 300 nM) for an additional 1 h at room temperature.
  • ConAb control antibody
  • IgG20 , IgG158 or IgG75 at 8 different concentrations (from 0 nM to 300 nM) for an additional 1 h at room temperature.
  • the ELISA assay was performed the other way around, whereby plexin A4 coated wells were incubated with the different anti PlexinA4 Abs (as indicated above) for 1 h at room temperature.
  • Wells were washed and 12 ⁇ g/mL rSema6B-hFC were added for an additional 1 h at room temperature. In both cases, the wells were washed and an anti-human IgGl HRP antibody was added for 1 hour at room temperature and the OD levels representing the Sema6B binding was measured as described above.
  • HUVEC cells were seeded in 6-well gelatinized dishes at a concentration of 1.5 x 10 5 cells/well in a growth medium containing 10 % FCS. Cells were allowed to attach and were incubated for 16 h at 37 °C. The cells were incubated for 1 h with the indicated antibodies. VEGF (10 ng/ml) was added or not and the cells were incubated for additional 15 minutes. A similar protocol was used for A549 cells which were allowed to attach and incubated for 16 h in serum free growth medium at 37 °C. The cells were also incubated with the antibodies for 1 h while Fetal Calf Serum (FCS, Beit Haemek, Israel, at a final concentration of 10%) was added or not for 15 more minutes.
  • FCS Fetal Calf Serum
  • the cells were then washed with ice-cold PBS and lysed with 0.02 ml of RIPA lysis buffer containing 1 mM EDTA, 1% Np-40, 1% deoxycholate, 0.1 % SDS, 150 mM NaCl, 10 mM Tris-HCl and fresh protease and phosphatase inhibitors.
  • the cells were scraped off, nonsoluble debris was removed by high speed centrifugation at 4 °C for lOmin, and aliquots of cell lysates containing 40 ⁇ g of protein were separated on an SDS-PAGE gel. Proteins were blotted onto a nitrocellulose filter and probed with an antibody directed against phosphorylated ERK1/2 (Cell Signaling).
  • the blot was then stripped and re-probed with an antibody directed against total ERK (Cell Signaling). Quantification of band intensity was performed using a Fuji Film image reader LAS- 3000 machine and the ratio between phosphorylated protein and the total amount of a target protein determined using the Multi-Gauge program.
  • rh Plexin A4 (SEQ ID NO: 27 the extracellular portion of Plexin-A4) immunized mice ScFv Phage display library was constructed.
  • the library was constructed from spleen RNA that was RT-PCRed with oligo-dT primers. The first step was to amplify by PCR the VH, V kappa and V lambda genes and to insert them into phagemid one by one. First, the light chain sub-libraries were mixed and subjected to plasmid isolation. The VH DNA pool was then inserted into the mixed plasmid to produce the scFv library of l. lxlO 9 .
  • the library was screened using two functional panning: (1) inhibition of the ScFv binding by rh Semaphorin 6B/Fc (R&D systems, Cat no 2094-S6). (2) internalization of rh Plexin A4 binders into the cells expressing the target.
  • 3 rounds of screening were conducted. In the first two rounds of screening, trypsin digestion was adopted to enrich specific binders. In the 3rd round of screening, the competitive elution was adopted to get the binders inhibited by rh Semaphorin 6B/Fc .To enrich more unique scFvs, both the 2nd round and the 3rd round of screening adopted the competitive eluting strategy.
  • the panning was conducted only by adopting the trypsin digestion. After three rounds of screening, 180 clones were picked up and by DNA sequencing 8 unique scFv genes were identified.
  • An ELISA protocol was used in order to test for single anti Plexin A4 ScFv binding inhibition following incubation with the rSema6B/FC ligand.
  • the wells were washed and an anti- myc tag was added for 1 hour at room temperature to detect the ScFVs and an anti- rabbit HRP antibody for additional 1 hour at room temperature to quantify the signal.
  • the OD levels representing the ScFV binding was measured at 490nM as described above.
  • the Phages of the different clones were incubated with either the target cells PAE (Porcine Aortic Endothelial Cells) stably express hPlexinA4 or the control cells untransfected PAE Cells. After 10 minutes, the cells were washed with serum free medium and surface bound phages were eluted using Glycine-HCL buffer without destroying the cells. Then the cells were lysed by freeze at-80°C and the cell lysates were tittered adopting the ordinary tittering method.
  • PAE Porcine Aortic Endothelial Cells
  • HCT116 and HUVEC cells were resuspended at a density of lxlO 6 cells/mL (100 ⁇ /aliquot) in a medium supplemented with the indicated antibody at a concentration of 15 ⁇ g/mL.
  • the cells were placed at 37 °C for various times along with a negative control kept on ice the entire time.
  • the internalization was stopped after 10, 20, 30. 45 and 60 min for HCT 116 cells or 5, 10, 20 and 30 min for HUVEC cells by adding ice-cold buffer A (PBS with 1% BSA).
  • the cells were washed twice in ice-cold buffer A and the non-internalized IgG were stripped from the cell surface by resuspending the cells in 0.5 ml cold stripping solution (50 mM TCEP, 150 mM NaCl, 1.0 mM EDTA, 0.2% BSA and 20 mM Tris pH 8.6) and incubated on ice for 15 min, with gentle shaking.
  • the cells were spun down and resuspended in 0.5 ml fresh stripping solution and incubated an additional 15 min on ice followed by two washes with 0.5 ml cold buffer A.
  • the cells were lysed in 200 ⁇ lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100 and protease inhibitor cocktail) and incubated for 20 min on ice.
  • the cell lysates were applied (100 ⁇ /well) on ELISA plates. Standard curve for mAb ranging from 0.23 ug/mL to 15 ug/mL was added to the plate.
  • the wells were washed and an anti-mouse IgGl HRP antibody was added for 1 hour at room temperature.
  • a TMB substrate-chromagen (Dako) was added to initiate a colorimetric reaction that was terminated using 0.2 M sulphuric acid.
  • the absorbance of each well was then measured at 420 nm using a TECAN Infinite M200/pro microplate reader.
  • Human PlexinA4 (SEQ ID NO: 27 the extracellular portion of Plexin-A4) was coupled onto three separate FCs: FC2, FC3, and FC4 of a BIAcore CM5 sensor chip (BIAcore, Inc., Piscataway, NJ). FC1 was used as a control. Immobilization was achieved by random coupling through amino groups using a protocol provided by the manufacturer (BIAcore, Inc.). Sensorgrams were recorded for binding of IgG20, IgG158 and IgG75 to these surfaces after injection of a series of the anti PlexinA4 antibodies solutions ranging in concentration from 0.19 nM to 400 nM, in 2-fold increments, at a flow rate of 30 ⁇ /min at 25 °C.
  • Glycine-HCl (10 mM, pH 1.7) was used to regenerate the sensor chip between injections.
  • the signal from the reference FC1 was subtracted from the signals measured in FC2, FC3, and FC4.
  • Kinetic constants were calculated by nonlinear regression fitting of the data according to a 1: 1 Langmuir binding model using BIAcore evaluation software (version 3.2) supplied by the manufacturer.
  • Plexin Al and A3 encoding cDNAs were cloned into the NSPI-CMV lentiviral expression vector.
  • EcoRV restriction enzyme site were added to the 5' end of the CDNA while Sail restriction enzyme site were added to the 3' end.
  • BamHI restriction enzyme site were added to the 5' end of the CDNA while EcoRV restriction enzyme site were added to the 3' end.
  • the myc epitope tag was added in frame upstream to the stop codon of the of Plexin Al cDNA (corresponding to NM_032242).
  • a VSV-G epitope tag was added upstream to the stop codon of Plexin A3 cDNA (corresponding to NM_017514).
  • a pLenti6.2/V5- DEST lentiviral vector (Life technologies) encoding the cDNAs of Plexin A2 (corresponding to NM_025179) and Plexin A4 (corresponding to NM_020911) with the V5 tag in frame upstream to the stop codons.
  • Lentiviruses directing expression of these cDNAs were produced in HEK293 cells and used to infect target cells. Stably infected PAE cells were isolated using puromicin selection for Plexin Al expression and hygromicin for Plexin A3 expression. Cells stably expressing Plexin A2 or A4 cDNA were selected using blasticidine.
  • PBS phosphate-buffered saline
  • EDTA incubated at 4 °C with the first antibody for 60 minutes, washed, and then incubated for 45 minutes with the secondary antibody (Cy5 conjugated goat anti mouse immunoglobulin G [IgG] or Cy3 conjugated donkey anti Rabbit IgG ; Jackson ImmunoResearch Laboratories, PA).
  • the cells were washed with PBS, and fluorescence intensity was measured with a Becton Dickinson Cell Sorter (Becton Dickinson, CA).
  • the membrane was incubated with 5 % dry milk/PBS, probed with the specific antibody, and detected with the appropriate horseradish peroxidase- conjugated secondary antibodies, and enhanced chemiluminescence substrates (biological industries, Beit Haemek).
  • the detection was performed using a Fuji Film image reader LAS -3000 machine.
  • Saporin conjugate to kill cells in contrast to unconjugated Saporin was tested using the ZAP assay (Advanced Targeting Systems CA , cat no KIT-48-Z ). If the primary antibody is internalized, the Saporin is transported into the cell via its binding to the secondary antibody. Once internalized, Saporin separates from its IgG conjugate, it inhibits protein synthesis and ultimately causes cell death.
  • mFab - ZAP is an affinity purified goat anti-mouse Fab that recognizes mouse monoclonal antibodies.
  • HCT116 cells were seeded at a concentration of 2.5 x 10 cells/well in 96-well dishes and grown in full growth medium (DMEM) containing 10 % FBS.
  • DMEM full growth medium
  • the experiments were set up with IgG20, IgG 158 and IgG75 antibody titrations ranging from 200 nM to 10 pM and include primary and secondary antibodies as control.
  • Unconjugated Saporin ranging from luM to lOpM was also an essential control since high levels of Saporin (>luM) causes nonspecific cell death as a result of bulk-phase endocytosis by the treated cells.
  • the mFab-ZAP was added to the appropriate wells at a concentration of 4.5 nM.
  • cell viability was evaluated using AlamarBlue reagent assay as the fold increase in the number of cells relative to untreated cells.
  • Binding affinity of the isolated IgGs to rPlexin A4 was assayed using an ELISA assay, at antibody concentrations ranging from 0.006 - 100 nM. Results shown in Figure illustrate that all our antibodies bind rPlexin A4 receptor with an EC50 value of 0.09- 1.5 nM.
  • the proliferation rate of cancer cells in the presence of the anti-Plexin A4 antibodies was assayed and shown in Figure 3A.
  • A549, H460, HCT116 and SK-OV-3 cells were tested by seeding 2.5x10 cells in 96 well plates in the presence or absence of Control Ab (209-005-088, Mouse Anti-Human IgG (H+L) Jackson ImmunoResearch), IgG20, IgG21, IgG25, IgG27 and IgG158 at lOOug/mL (666nM), 50ug/mL (333nM), 25ug/mL (166.5nM), 12.5ug/mL (83.25nM), 6.25ug/mL (41.6nM), 3.125ug/mL (20.8nM) for 72h.
  • the induction of proliferation was calculated using AlamarBlue (AbD Serotec) reagent as the fold increase in the number of cells relative to untreated cells. It was observed that the anti Plexin A4 antibodies inhibited the cells proliferation at a dose dependent manner and the half maximal inhibitory concentration (IC50) was at 50ug/mL (333nM). IgG27 did not have a significant effect on the cells proliferation rate. It was also evident that not all the cell lines responded to the tested antibodies in the same pattern. While the antibodies inhibited the proliferation of A549, H460 and HCT116 cells at 50 ug/mL, inhibition of proliferation in SK-OV-3 cells was seen only at a much higher concentration (lOOug/mL).
  • Anti-proliferative effect of the anti plexin A4 antibodies was further examined on additional cell lines such as: MDA-MB-231, MDA-MB-435, SK-MEL-5 and U-87- MG ( Figure 3B). While one of our antibody candidates (IgG20) inhibited the proliferation of MDA-MB-231 cells at around 70 %, it reduced the proliferation rate of MDA-MB-435 and U-87-MG cells only at around 20% and had no effect on SK-MEL-5 cells.
  • Figure 3C shows that the responsive cells in which proliferation was inhibited in the presence of the antibodies were K-Ras mutated. It will be appreciated that there was no significant difference in expression of Plexin A4 or Sema6B proteins between the responsive and nonresponsive cell lines ( Figure 3D).
  • the anti-proliferative effect of anti plexin A4 antibodies on HUVEC cells was tested by seeding 3x10 cells in 96 well plates coated with gelatin in the presence of basic FGF (5 ng/niL), VEGF (10 ng/niL) or absence of both. Once the cells attached to the wells, the antibodies were added for 72 h. It was observed that IgG20 and IgG158 treatment at 50ug/mL, compared to the control IgG, resulted in a decrease of up to 35 % in bFGF and VEGF induced HUVEC proliferation. IgG27 had no effect on the cell proliferation rate.
  • the present inventors have further tested the antibodies IgG20, IgG158 and Fab75 anti-proliferative effect by testing phosphorylation of downstream signaling pathway proteins ERK1/2 (Figure 4B).
  • HUVECs cells were incubated for lh with the indicated antibodies followed by a second incubation with or without VEGF (10 ng/niL).
  • Avastin anti VEGF antibody
  • After 15 minutes the cells were lysed, and subjected to Western blot analysis using an antibody against the phosphorylated Thr202/Tyr204 residues of ERKl/2. Blots were then stripped and re-probed with an antibody directed against ERKl/2.
  • FIG. 5A summarizes the results of the competitive ELISA for ScFv clones 20/25/21/27/75 and 158 compared to control M13K07 phages on binding to plexin A4. Adding rSema6B as a competitor resulted in an inhibition of anti-plexin A4 antibodies binding.
  • the results of the competition assay were validated using the full IgG antibodies of the selected candidates IgG20, IgG75 and IgG158 compared to control IgG (see Figure 5B).
  • the results show a significant reduction of the Sema6B binding to plexin A4 in the presence of IgG20, IgG75 and IgG158.
  • the results suggest that the antibodies displaced sema6B from its receptor and support their competitiveness with Sema 6B for the binding to Plexin A4 receptor.
  • the plexin A4 antibodies of some embodiments of the invention exert their activity by inhibiting ERK phosphorylation. Therefore, the present inventors have further tested the antibodies competition ability with Sema6B by testing ERKl/2 phosphorylation downstream to Sema6B binding.
  • A549 cells were incubated for 1 h with the indicated antibodies followed by a second incubation with or without rSema6B (30ug/mL). After 15 minutes the cells were lysed, and subjected to Western blot analysis using an antibody against the phosphorylated Thr202/Tyr204 residues of ERK1/2. Blots were then stripped and reprobed with an antibody directed against ERK1/2.
  • A549 and HCT116 were co treated simultaneously with the combination.
  • the anti-proliferation effects were examined following 72 h of treatment.
  • the three panels in Figure 6A shows treatment of A549 cells: (1) No chemotherapy.
  • Co-treatment of the A549 cells with IgG20 or IgG158 (at 333 nM) and chemotherapy enhances the anti-proliferative effect of the chemotherapy.
  • Cisplatin Using Cisplatin (Fig 6A2) the anti-proliferative effects were elevated from about 25 % to 78 % with IgG20 or from about 16 % to 52 % with IgG158.
  • paclitaxel Fig 6 A3 the anti-proliferative effects were elevated from about 19 % to 58 % with IgG20 or from about 15 % to 45 % with IgG158.
  • Figure 6B shows treatment of HCT116 cells: (1) No chemotherapy. (2) Combination of anti Plexin A4 antibodies and Cisplatin. (3) Combination of anti Plexin A4 antibodies and Paclitaxel. Co-treatment of HCT116 cells with IgG20 or IgG158 (at 333 nM) and chemotherapy also enhances the anti-pro liferate effect of the chemotherapy.
  • Cisplatin Fig 6B2
  • the anti-proliferative effects were elevated from about 26 % to 70 % with IgG20 or from about 15 % to 50 % with IgG158.
  • paclitaxel Fig. 6B3
  • the anti-proliferative effects were elevated from about 23 % to 55 % with IgG20 or from about 20 % to 40 % with IgG158.
  • the anti-proliferate effect of the anti plexin A4 antibodies was examined using BrdU labeling experiments in A549, HCT116 and MDA-MB-231 cells.
  • the indicated anti- Plexin A4 antibodies exert their anti-proliferative activity as fewer cells enter the cell cycle.
  • IgG75 The anti-proliferative effect of IgG75 on cancer cells (A549 Lung NS carcinoma cells and HCT116 Large intestine carcinoma) was tested by seeding 2.5x10 cells in 96 well plates in the presence or absence of Control IgG or IgG75 for 72 h. The induction of proliferation was calculated as the fold increase in the number of cells relative to untreated cells. The results indicate that IgG75 also inhibited the cells proliferation at a dose dependent manner in tumor cell lines and had approx. 60% inhibition effect at 133 nM ( Figures 8A-B). Binding affinity of IgG75 to rPlexin A4 was assayed using an ELISA assay, at antibody concentrations ranging from 0.8 - 333 nM. Results shown in Figure 8C illustrate IgG75 binds rPlexin A4 receptor with an EC50 value of 18 nM ( Figure 8C).
  • Antibodies of some embodiments of the invention were used either as phage associated ScFv or as isolated antibodies to induce internalization of plexin A4 expressed on HCT116, HUVEC and PAE -hA4 cells under conditions which allow for Plexin A4 internalization.
  • Figures 9A-C (HUVEC and HCT116 cells treated with the indicated IgGs) and Table 2 (PAE treated with single chain Fvs) below.
  • the present antibodies (but clone 158) were effective at inducing the internalization of plexin A4.
  • a secondary antibody having toxic moiety active only in the cell was conjugated to the test antibody.
  • an immunotoxin comprising a secondary antibody linked to the ribosome inactivating protein saporin, was used.
  • the test antibody Once the test antibody is internalized, the saporin is transported into the cell via its binding to the secondary antibody. Once internalized, saporin separates from its IgG conjugate, it inhibits protein synthesis and ultimately causes cell death.
  • IgG20 and IgG75 effectively induced the internalization of Plexin A4 and subsequently internalized the Fab- Saporin conjugate leading to an increase in cell death in a concentration dependent manner reaching a peak of approx. 25 % effect at 200 nM compared to primary antibodies alone (Figure 9D).
  • IgG158 exhibited only approx. 10 % effect at 200 nM, in line with previous results.
  • Binding affinity of the anti Plexin A4 Antibodies IgG20, IgG158 and IgG75 to Plexin A4 was assayed using surface plasmon resonance analysis (BIAcore technology).
  • Figure 10 shows that IgG20 and IgG75 binds to Plexin A4 with similar affinity, with K D values ranging between 1.02 nM and 2.03 nM, while IgG158 has a KD value of 20 nM.
  • FIG. 11B are histograms depicting the number of cells counted versus the fluorescence intensity. As shown, there is a significant shift in the florescence intensity of IgG20, 75 or 158 stained cells as compared to cells stained with the secondary antibody only (as negative control). This indicates that antibodies IgG20, 75 and 158 are able to bind the folded Plexin A4 protein which is presented on the cell surface.
  • Lenti viral infection of recombinant human (rh)Plexin Al, A2 or A3 was performed on PAE cells to obtain stably infected cells over expressing each type A Plexin protein on the cells surface.
  • PK STUDY TIME DEPENDENT SERUM CONCENTRATION OF ANTI PLEXIN A4 IgG75 FOLLOWING INTRAVENOUS (I. V.) ADMINISTRATION INTO MICE.
  • mice were randomized into eight groups (five animals per group). Mice received a single i.v. injection of IgG75 at 20 mg/Kg into the tail vein. Blood samples ( ⁇ 0.2 ml) were retrieved from the tail vein predose, 30 min, 4 hr, 12 hr, 24 hr, 48 hr, 72 hr, 7 days and 10 days postdose. Serum was harvested and stored at -80 °C. IgG75 concentration at each time point was determined using ELISA on rPlexin A4 coated wells as previously described. Results of immunoreactive concentration versus time data from mice are presented in Figure 13. EXAMPLE 15
  • TGI Tumor Growth Inhibition
  • TDD Tumor Growth Delay
  • Injection set up CR female NCr nu/nu mice are injected (S.C.) with 1x10 A549 tumor cells in 50 % Matrigel Mice age at start date: 8 to 12 weeks .
  • a pair match is done when tumors reach an average size of 100 - 150 mm 3 , and treatment with the indicated antibodies is begun. Body Weight and Caliper are measured bi-weekly to end.
  • the endpoint of the experiment is a mean tumor weight in Control Group of 800 mm or 33 days, whichever comes first.
  • the study may be converted to TGD when TGI is reached to follow responders.
  • Injection set up CR female NCr nu/nu mice are injected with 5xl0 6 HCT116 tumor cells in 0% Matrigel S.C. Mice age at start date: 8 to 10 weeks.
  • a pair match is done when tumors reach an average size of 90-120 mm 3 , and treatment with the indicated antibodies is begun. Body Weight and Caliper are measured bi-weekly to end.
  • the endpoint of the experiment is a mean tumor weight in Control Group of 1500 mm or 33 days, whichever comes first.
  • the study may be converted to TGD when TGI is reached to follow responders.

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Abstract

L'invention concerne des protéines isolées comprenant un domaine de reconnaissance d'antigène qui se lie spécifiquement à la plexine-A4 humaine. L'invention concerne également des procédés d'utilisation de ces protéines afin de traiter des troubles liés à l'angiogénèse, comme le cancer.
PCT/IL2014/050827 2013-09-16 2014-09-16 Protéines isolées capables de se lier à la plexine-a4, et procédés de production et d'utilisation Ceased WO2015037009A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2020239934A1 (fr) 2019-05-28 2020-12-03 Vib Vzw Lymphocytes t cd8 + dépourvus de plexines et leur application dans le traitement du cancer
WO2020239945A1 (fr) 2019-05-28 2020-12-03 Vib Vzw Traitement du cancer par ciblage des plexines dans le compartiment immunitaire
WO2022063957A1 (fr) 2020-09-24 2022-03-31 Vib Vzw Biomarqueur pour une thérapie antitumorale

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WO2020239934A1 (fr) 2019-05-28 2020-12-03 Vib Vzw Lymphocytes t cd8 + dépourvus de plexines et leur application dans le traitement du cancer
WO2020239945A1 (fr) 2019-05-28 2020-12-03 Vib Vzw Traitement du cancer par ciblage des plexines dans le compartiment immunitaire
US20220228116A1 (en) * 2019-05-28 2022-07-21 Vib Vzw Cd8+ t-cells lacking plexins and their application in cancer treatment
US12472254B2 (en) * 2019-05-28 2025-11-18 Vib Vzw CD8+ T-cells lacking plexins and their application in cancer treatment
WO2022063957A1 (fr) 2020-09-24 2022-03-31 Vib Vzw Biomarqueur pour une thérapie antitumorale

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