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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
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  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3084—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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  • 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
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  • C07—ORGANIC CHEMISTRY
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  • 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
  • C07K2317/734—Complement-dependent cytotoxicity [CDC]
• • 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

Definitions

• Anti-Gb3 Antibodies useful in the treatment of angiogenesis-associated diseases include:
• the present invention is in the field of new anticancer therapies, more specifically in the field of anti-angiogenic molecules.
• it relates to anti-Gb3 antibodies having specific CDR sequences, as well as to the use of anti-Gb3 antibodies that are not coupled to a therapeutic molecule in the treatment of diseases associated with angiogenesis.
• Cancer cells because of their genetic instability, can acquire resistance to cancer treatments, which is at the origin of therapeutic failures or cancer recurrences.
• the endothelial cells of the tumor's blood vessels are genetically stable. In addition, they are essential for neovasculansation without which the tumor can not continue to grow, for lack of nutrients. Therefore, a strategy to inhibit angiogenesis, i.e., proliferation of tumor endothelial cells, would not be sensitive to the genetic variability of the tumor, with endothelial cells being genetically stable.
• Targeting endothelial cells also has other benefits
• the tumor cells are difficult to reach. Indeed, with traditional anti-cancer therapies, the penetration of the treating agents into the tumor tissues is diminished because of the high interstitial pressure in most tumors. This is not the case in vessels that are a more easily accessible target. Because of this accessibility, endothelial cell targeting allows efficient distribution and rapid accumulation in the tumor vessels. Therefore, targeting the vasculature is a strategy that can be applied to the majority of tumor types.
• the vast majority of endothelial cells are in a quiescent state.
• the endothelium of the tumor networks is composed of endothelial cells presenting a proliferating "angiogenic" phenotype that it is theoretically possible to target.
• the vessels that irrigate the tumors have structural and functional characteristics different from those of the normal vessels. It is thus expected that this strategy is selective and causes few side effects on so-called "physiological" angiogenesis.
• targeting proliferating endothelial cells has many advantages, both in terms of accessibility of targeted cells, the number of tumors that can be treated, reduction of side effects if only proliferating endothelial cells are targeted, and prevention of metastases.
• Globotriasosylceramide or globotriaosylceramide (Gb3), also called CD77, P k , ceramide trihexoside (CTH), and Burkitt Lymphoma Antigen (BLA) is a glycosphingolipid in the form of Galactoseal ⁇ 4Galactose i ⁇ 4Glucose i ⁇ Cerami de, synthesized by the enzyme Lactosylceramide. 4-alpha-galactosyltransferase (A4GALT). It is expressed by the HUVEC endothelial cell line, more when this line is proliferating than when the cells are confluent and are no longer in the exponential phase of growth. It therefore constitutes a marker of the endothelial cells involved in angiogenesis (Heath-Engel et al., Obrig et al).
• the ceramide the hydrophobic part of the molecule anchored in the outer leaflet of the plasma membrane, is formed of a fatty acid chain composed of 16 to 28 carbon atoms, linked by an amide linkage to a sphingoid base, generally sphingosine. . It can have variations in the length but also in the number of unsaturation of its chain of fatty acids. In particular, the main possible variations for the sphingoid base and the fatty acid chain are shown in Table 1 below.
• Gb3 is a natural receptor of certain bacterial toxins such as Shiga toxin, produced by Shigella dysenteriae, and verotoxins produced by Escherichia coli. These toxins are composed of two subunits A and B, the subunit B being involved in the binding to Gb3, while the subunit A corresponds to the toxic part inhibiting the protein synthesis.
• the binding of these bacterial toxins to Gb3 leads to the transport of toxin from the plasma membrane to the endoplasmic reticulum via early endosomes and the Golgi apparatus.
• Subunit A produces its toxic effects in the endoplasmic reticulum.
• Shiga toxin and verotoxins have therefore been proposed to exert cytotoxic activity on endothelial cells, in particular on proliferating endothelial cells (Heath-Engel et al., Obrig et al., WO98 / 51326).
• Verotoxins and Shiga toxin have also been proposed as a therapeutic agent in the treatment of tumors expressing Gb3 on their surface, the subunit B for targeting, while the A subunit plays a cytotoxic role.
• verotoxin 1 has been shown to be capable of inducing apoptosis of Burkitt lymphoma cells (Tétaud et al).
• Gb3 is expressed in many other tissues, such as the tissue of the stomach, esophagus, prostate and kidney. Since Shiga toxin and verotoxins are small molecules (about 68 kilodaltons (kDa)), they can leave the bloodstream and enter healthy tissues, where they have the same cytotoxic properties for Gb3-expressing cells as for tumor cells. Because of their lack of specificity of action, these toxins lead to unacceptable side effects.
• Bast et al show that two hours after administration to rabbits, verotoxin 1 is no longer present in the bloodstream and can be found in target organs expressing Gb3.
• the small molecular weight of these toxins and their exit from the bloodstream also reduces their duration of action on the cells of the blood vessels, thus also decreasing their effectiveness.
• Monoclonal antibodies can be generated for many types of ligands, including glycosphingolipids, although some are immunogenic. Monoclonal antibodies directed against Gb3 were already used for labeling Gb3-expressing cells, in particular monoclonal antibody 38.13 (Bordron et al., Chark et al).
• this monoclonal antibody 38.13 does not bind Gb3 in the same way as verotoxins (Chark et al).
• an anti-Gb3 monoclonal antibody obtained from ascites 1A4 induced apoptosis of Burkitt's lymphoma cells by a mechanism different from that of verotoxin 1 (Tétaud et al). It was therefore unlikely that an anti-Gb3 monoclonal antibody could have the same anti-angiogenic effect as verotoxins.
• W098 / 51326 which describes the use of Gb3-binding molecules as anti-angiogenic agents, provides as such agents verotoxins and anti-Gb3 antibodies coupled to a toxic molecule, such as toxins. The authors of this document therefore considered that an anti-Gb3 antibody alone would not have an anti-angiogenic effect.
• anti-Gb3 monoclonal antibodies have anti-angiogenic activity on proliferating endothelial cells.
• These monoclonal antibodies can therefore be used in the treatment of diseases associated with angiogenesis, and in particular solid tumors, with all the previously mentioned advantages that this targeting comprises (less risk of resistance, applicable to any type of tumor, targets easily). reached, prevention of metastases).
• this targeting comprises (less risk of resistance, applicable to any type of tumor, targets easily). reached, prevention of metastases).
• Shiga toxin and verotoxins have several advantages over Shiga toxin and verotoxins:
• the antibodies Depending on their isotype, the antibodies have a molecular weight that varies between 150 (IgG) and 1000 (IgM) kDa, and therefore have a higher molecular weight than the Shiga toxin and verotoxins. This should allow them to stay longer in the bloodstream and therefore have a longer duration of action than these toxins on the endothelial cells of tumor neovessels.
• the isotypes IgG (in particular IgG1) and IgM are the most advantageous. Indeed, the IgM have the largest molecular weight, about 80% remaining in the bloodstream, and they have an in vivo half-life of about 10 days.
• Mouse antibodies obtained by the inventors can be humanized, so as to minimize any immune response of the recipient, which is not possible for bacterial toxins, which generate an immune response may reduce their effectiveness.
• the present invention therefore relates to an antibody directed against the glycosphingolipid membrane globotriaosylceramide (Gb3), or a functional fragment or a derivative thereof, characterized in that it has at least one complementarity determining region (CDR) having a amino acid sequence selected from SEQ ID NO: 1 to 42, or having an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 1 to 42.
• CDR complementarity determining region
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has a heavy chain comprising at least one complementarity determining region (CDR) having an amino acid sequence selected from SEQ ID NO 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 31 to 33, and 37 to 39, or having an amino acid sequence of at least 80%, preferably at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 31 to 33, and 37 to 39.
• CDR complementarity determining region
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has a light chain comprising at least one complementarity determining region (CDR) having an amino acid sequence chosen from SEQ ID NO: 4 to 6, 10 to 12, 16 to 18, 22 to 24, 28 to 30, 34 to 36, and 40 to 42, or having an amino acid sequence of at least 80%, preferably at least 85% %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 4 to 6, 10 to 12 , 16 to 18, 22 to 24, 28 to 30, 34 to 36, and 40 to 42.
• CDR complementarity determining region
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has: a heavy chain comprising at least one complementarity determining region (CDR) having an amino acid sequence selected from SEQ ID NO: 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 31 at 33, and 37 to 39, or having an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least
• CDR complementarity determining region
• a light chain comprising at least one complementarity determining region (CDR) having an amino acid sequence selected from SEQ ID NO: 4 to 6, 10 to 12, 16 to 18, 22 to 24, 28 to 30, 34 at 36, and 40 to 42, or having an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%), at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 4 to 6, 10 to 12, 16 to 18, 22 to 24, 28 to 30, 34 to 36, and 40 to 42.
• CDR complementarity determining region
• the anti-Gb3 antibody, functional fragment or derivative according to the invention may also advantageously have a heavy chain comprising three CDR-H (heavy chain CDR) having the following amino acid sequences, or sequences having at least 80% preferably at least 85%, at least 90%, at least 95%, at least 96%), at least 97%, at least 98%, at least 99% identity with the following sequences:
• CDR1-H-3E2 SEQ ID NO: 1
• CDR2-H-3E2 SEQ ID NO: 2
• CDR3-H-3E2 SEQ ID NO: 3
• CDR1-H-25C10 SEQ ID NO: 25, CDR2-H-25C10: SEQ ID NO: 26, CDR3-H-25C10: SEQ ID NO: 27, f) CDR1-H-11E10: SEQ ID NO: 31, CDR2-H-11E10: SEQ ID NO: 32, CDR3-H-11E10: SEQ ID NO: 33, or
• CDR1-H-16G8 SEQ ID NO: 37
• CDR2-H-16G8 SEQ ID NO: 38
• CDR3-H-16G8 SEQ ID NO: 39.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention may also advantageously have a light chain comprising three CDR-L (light chain CDR) having the following amino acid sequences, or sequences having at least 80% preferably at least 85%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the following sequences: a) CDR1- L-3E2: SEQ ID NO: 4, CDR2-L-3E2: SEQ ID NO: 5, CDR3-
• L-3E2 SEQ ID NO: 6
• CDR1-L-15C11 SEQ ID NO: 16
• CDR2-L-15C11 SEQ ID NO: 17
• CDR3-L-15C11 SEQ ID NO: 18
• CDR3-L-11E10 SEQ ID NO: 36, or
• CDR1-L-16G8 SEQ ID NO: 40
• CDR2-L-16G8 SEQ ID NO: 41
• CDR3-L-16G8 SEQ ID NO: 42.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has heavy and light chains respectively comprising CDR-H and CDR-L having the following amino acid sequences, or sequences having at least 80 %, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the following sequences:
• CDR1-H-3E2 SEQ ID NO: 1
• CDR2-H-3E2 SEQ ID NO: 2
• H-3E2 SEQ ID NO: 3, CDR1-L-3E2: SEQ ID NO: 4, CDR2-L-3E2: SEQ ID NO: 5, CDR3-L-3E2: SEQ ID NO: 6, b) CDR1-H-14C11: SEQ ID NO: 7, CDR2-H-14C11: SEQ ID NO: 8, CDR3-H-14C11: SEQ ID NO: 9, CDR1-L-14C11: SEQ ID NO: 10, CDR2-L-14C11: SEQ ID NO: 11, CDR3-L-14C11: SEQ ID NO: 12,
• CDR1-H-15C11 SEQ ID NO: 13
• CDR2-H-15C11 SEQ ID NO: 14
• CDR3-H-15C11 SEQ ID NO: 15
• CDR1-L-15C11 SEQ ID NO: 16
• L-15C11 SEQ ID NO: 17
• CDR3-L-15C11 SEQ ID NO: 18
• CDR1-H-22F6 SEQ ID NO: 19
• CDR2-H-22F6 SEQ ID NO: 20
• CDR3-H-22F6 SEQ ID NO: 21
• CDR1-L-22F6 SEQ ID NO: 22
• CDR2-L-22F6 SEQ ID NO: 23
• CDR3-L-22F6 SEQ ID NO: 24,
• CDR3-H-25C10 SEQ ID NO: 27, CDR1-L-25C10: SEQ ID NO: 28, CDR2-L-25C10: SEQ ID NO: 29, CDR3-L-25C10: SEQ ID NO: 30,
• CDR1-H-16G8 SEQ ID NO: 37
• CDR2-H-16G8 SEQ ID NO: 38
• CDR3-H-16G8 SEQ ID NO: 39
• CDR1-L-16G8 SEQ ID NO: 40
• CDR2-L-16G8 SEQ ID NO: 41
• CDR3-L-16G8 SEQ ID NO: 42.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has a heavy chain comprising a variable region having a sequence chosen from SEQ ID NO: 43 to 49 or a sequence having at least 80% preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 43 at 49.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has a light chain comprising a variable region having a sequence chosen from SEQ ID NO: 50 to 56 or a sequence having at least 80 %, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 50 to 56.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention has: a heavy chain comprising a variable region having a sequence selected from SEQ ID NO: 43 to 49 or a sequence having at least 80%, preferably at least 85%>, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 43 to 49, and
• a light chain comprising a variable region having a sequence selected from SEQ ID NO: 50 to 56 or a sequence having at least 80%), preferably at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 50-56.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention may be chosen from among the anti-Gb3 monoclonal antibodies generated by the inventors or variants thereof, which have heavy chains. and light ones whose variable regions have the following amino acid sequences or sequences having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%), at least 99% identity with the following sequences:
• Antibody 3E2 heavy chain: SEQ ID NO: 43, light chain: SEQ ID NO: 50,
• Antibody 15C11 heavy chain: SEQ ID NO: 45 light chain: SEQ ID NO: 52,
• 22F6 antibody heavy chain: SEQ ID NO: 46, light chain: SEQ ID NO: 53,
• g) 16G8 antibody heavy chain: SEQ ID NO: 49, light chain: SEQ ID NO: 56.
• antibody or “immunoglobulin” is meant a glycoprotein composed of two types of glycopolypeptide chains called “heavy chain” and “light chain”, an antibody consisting of two heavy chains and two light chains, linked by disulfide bridges. Each chain consists of a variable region and a constant region. The constant region of a particular isotype of heavy or light chain is normally identical from one antibody to another of the same isotype, except somatic mutations. In contrast, the variable region varies from one antibody to another.
• the genes coding for the heavy and light chains of the antibodies are generated by recombination of respectively three and two distinct gene segments called VH, DH and JH-CH for the heavy chain and VL and JL-CL for the light chain.
• the CH and CL segments do not participate in the recombination and form the constant regions of the heavy and light chains respectively.
• the recombinations of the VH-DH-JH and VL-JL segments form the variable regions of the heavy and light chains, respectively.
• the VH and VL regions have 3 hyper-variable areas or complementarity determining regions (CDRs), called CDR1, CDR2 and CDR3, the CDR3 region being the most variable, since it is located at the recombination zone.
• CDRs complementarity determining regions
• the CDR1, CDR2 and CDR3 regions are each preceded by the FR1, FR2 and FR3 regions respectively, corresponding to the framework regions (FR region framework) which vary at least from one VH or VL segment to another.
• the CDR3 region is also followed by an FR4 framework region.
• the CDRs of an antibody are defined from the amino acid sequence of its heavy and light chains with respect to criteria known to those skilled in the art.
• Various methods for determining CDRs have been proposed, and the portion of the amino acid sequence of a heavy or light chain variable region of an antibody defined as a CDR varies depending on the method chosen.
• the first one The method of determination is that proposed by Kabat et al (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed., US Department of Health and Human Services, NIH, 1991, and later editions).
• CDRs are defined by looking for the amino acids responsible for antigen binding of the antibody.
• a 2nd method was proposed by IMGT, this time based on the determination of the hypervariable regions.
• Table 2 summarizes the amino acid sequences of the CDRs and variable regions of the heavy and light chains of the anti-Gb3 antibodies generated by the inventors:
• Variable Region SEQ ID NO 43 Variable Region SEQ ID NO 47
• Variable Region SEQ ID NO 50 Variable Region SEQ ID NO 54
• Variable Region SEQ ID NO 44 Variable Region SEQ ID NO 48
• Variable Region SEQ ID NO 51 Variable Region SEQ ID NO 55
• Variable Region SEQ ID NO 45 Variable Region SEQ ID NO 49
• Variable Region SEQ ID NO 52 Variable Region SEQ ID NO 56
• SEQ ID NO 53 variable region By “functional fragment” is meant an antibody fragment retaining the antigen-binding domain and therefore having the same antigenic specificity as the original antibody, such as Fv, ScFv, Fab, F (ab fragments). ') 2, Fab', scFv-Fc or di-antibodies ("diabodies").
• derivative of an antibody is meant a binding protein formed of a carrier peptide and at least one of the CDRs of the original antibody to preserve its ability to recognize Gb3.
• the antibodies, functional fragments or derivatives according to the invention can be obtained by genetic recombination or by chemical synthesis, according to technologies well known to those skilled in the art.
• the antibody according to the invention is a monoclonal antibody.
• monoclonal is meant an antibody obtained from a substantially homogeneous antibody population, i.e. the antibodies forming this population are essentially identical except for possible natural mutations that may be present in minor quantities. These antibodies are directed against a single epitope and are therefore very specific.
• epitope is meant the site of the antigen to which the antibody binds. Regardless of the antigen, an epitope is a three-dimensional region formed by portions of the antigen that may or may not be adjacent in a linear, non-dimensional structure of the antigen.
• the antibodies, functional fragments or derivatives are of course not in a natural form, but isolated, obtained by purification from a natural source, by genetic recombination or by chemical synthesis.
• the "percent identity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or amino acids between the two compared sequences, obtained after optimal alignment of the two sequences. . This percentage is purely statistical and the differences between the two sequences are distributed randomly over their entire length.
• the comparison of two nucleic acid or amino acid sequences is generally performed after optimally aligning them, the comparison being possible by segment or using an "alignment window".
• the optimal sequence alignment can be achieved using different software well known to those skilled in the art, including BLAST NR (nucleic acid) or BLAST P (protein) software.
• the percentage identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally aligned sequences, in which the compared nucleic acid or amino acid sequence may have deletions or insertions by compared to the reference sequence.
• the percent identity is calculated by determining the number of positions at which the nucleotide or amino acid is identical between the two sequences, preferably between the two complete sequences, and dividing it by the total number of positions in the window. alignment (preferably the complete sequences) and multiplying the result by 100. This percentage of identity can be calculated easily using for example the BLAST software with default parameters.
• the CDR or the variable region of an antibody according to the invention has an amino acid sequence which is not 100% identical to one of those described above and in the sequence listing (reference sequences ) but which has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with at least such a reference sequence, it may have insertions, deletions or substitutions with respect to the reference sequence.
• the substitution is preferably carried out by an "equivalent" amino acid, that is to say any amino acid whose structure is close to that of the original amino acid and is therefore unlikely to alter the biological activities of the antibody. Examples of such substitutions are shown in the following Table 3:
• Gb3 is represented by immunolabeling with a doublet corresponding to the presence of 2 different fatty acid chains at the ceramide level. Porcine cells also express two forms of Gb3, represented by a doublet.
• the 3E2 antibody recognizes both forms of Gb3 of the porcine doublet, as well as the upper band, but not the lower band, of the doublet expressed by HMEC-1 cells.
• the 22F6 antibody also recognizes this upper band of the Gb3 doublet expressed by the HMEC-1 cells and should therefore be particularly specific for proliferating endothelial cells, as well as any antibody having the same CDR1, CDR2 and CDR3 or the same variable regions. .
• the antibody, functional fragment or derivative according to the invention is the antibody 3E2 or 22F6 as defined above by the sequences of the variable regions of their heavy and light chains, advantageously the 3E2 antibody, or an antibody of the same antigenic specificity.
• the antibody, functional fragment or derivative according to the invention may have at least one complementarity determining region (CDR) having an amino acid sequence chosen from SEQ ID NO: 1 to 6 and 19 to 24, or having an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 1 to 6 and 19 to 24.
• CDR complementarity determining region
• the antibody, functional fragment or derivative according to the invention may have a heavy chain comprising at least one complementarity determining region ( CDR) having an amino acid sequence selected from SEQ ID NO: 1 to 3, and 19 to 21, or having an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO: 1 to 3, and 19 to 21 and / or has a light chain comprising at least one complementarity determining region (CDR) having an amino acid sequence selected from SEQ ID NO: 4 to 6, and 22 to 24, or having an amino acid sequence having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with one of SEQ ID NO 4 to 6, and 22 to 24.
• CDR complementarity determining region
• CDR-H heavy chain comprising three CDR-H (heavy chain CDRs) having the following amino acid sequences, or sequences having at least 80%), preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the following sequences: a) CDR1-H-3E2: SEQ ID NO: 1, CDR2-H-3E2: SEQ ID NO: 2, b) CDR1-H-22F6: SEQ ID NO: 19, CDR2-H-22F6: SEQ ID NO: 20, CDR3-H-22F6: SEQ ID NO: 21,
• CDR-L light chain CDR
• amino acid sequences or sequences having at least 80%, preferably at least 85%,> 90%, at least minus 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the following sequences:
• CDR1-L-3E2 SEQ ID NO: 4
• CDR2-L-3E2 SEQ ID NO: 5
• CDR3-L-3E2 SEQ ID NO: 6
• CDR3-L-22F6 SEQ ID NO: 24.
• the anti-Gb3 antibody, functional fragment or derivative according to the invention can have:
• a heavy chain comprising a variable region having a sequence selected from SEQ ID NO: 43 and 46 or a sequence having at least
• a light chain comprising a variable region having a sequence selected from SEQ ID NO: 50 and 53 or a sequence having at least
• the anti-Gb3 antibody, functional fragment or derivative according to the invention can be chosen in particular from the anti-Gb3 monoclonal antibodies generated by the inventors or variants thereof, which possess chains.
• heavy and light whose variable regions have the following amino acid sequences or sequences having at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97% , at least 98%), at least 99% identity with the following sequences: a) Antibody 3E2: heavy chain: SEQ ID NO: 43, light chain: SEQ ID NO: 50, and
• 22F6 antibody heavy chain: SEQ ID NO: 46, light chain:
• the most advantageous isotypes are the IgG isotypes, in particular IgG1, and IgM, more particularly the IgM isotype, which has the highest molecular weight. Therefore, any antibody according to the invention is preferably of IgG or IgM isotype, preferably IgM.
• any antibody, functional fragment or derivative according to the invention may advantageously be chimeric or humanized. Indeed, this makes it possible to avoid the immune reactions of the patient against the administered antibody.
• the antibody according to the invention may advantageously be any of the chimeric or humanized versions of the antibodies 3E2, 14C1 1, 15C11, 22F6, 25C10, 11E10, and 16G8 described above, advantageously 3E2 and 22F6 antibodies. , and in particular of the antibody 3E2.
• chimeric antibody an antibody which contains a naturally occurring variable (light chain and heavy chain) derived from an antibody of a given species in association with the constant light chain and heavy chain regions of an antibody of a heterologous species to said given species.
• the chimeric type antibodies according to the invention can be prepared using genetic recombination techniques.
• the chimeric antibody may be made by cloning a recombinant DNA comprising a promoter and a sequence coding for the variable region of a non-human monoclonal antibody, in particular murine monoclonal antibody, according to the invention and a sequence coding for the constant region. human antibody.
• a chimeric antibody of the invention encoded by such a recombinant gene will for example be a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from murine DNA and its isotype determined by the constant region derived from the murine DNA. Human DNA. This will notably be the case for the chimeric antibodies obtained from the murine monoclonal antibodies 3E2, 14C11, 15C11, 22F6, 25C10, 11E10, and 16G8 described in the present description. For methods of preparing antibodies For example, it is possible to refer to Verhoeyn et al. (BioEssays, 8: 74, 1988).
• humanized antibody is meant an antibody which contains CDRs regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or more) human antibodies.
• some of the skeletal segment residues (referred to as FR) may be modified to maintain binding affinity (Jones et al., Nature, 321: 522-525, 1986, Verhoeyen et al., Science, 239: 1534-1536 1988, Riechmann et al., Nature 332: 323-327, 1988).
• the humanized antibodies according to the invention may be prepared by techniques known to those skilled in the art such as “CDR grafting", “resurfacing”, Superhumanization, "Human string content", “FR libraries” technologies. ",” Guided Selection ",” FR shuffling “and” Humaneering "as summarized in the review by Almagro et al.
• the anti-Gb3 antibody, or functional fragment or derivative thereof according to the invention may also have been optimized for certain effector functions.
• it may include mutations increasing its affinity for the Fc receptor to which its isotype binds. It can also be produced under specific conditions (cells, medium, etc.) to obtain a particular glycosylation of the antibody.
• certain substitutions of the Fc ⁇ portion and low or no fucosylation make it possible to increase the affinity for the Fc ⁇ RIII receptor (see in particular Shields et al., Journal of Biological Chemistry, Vol 276, No. 9 , Issue of March 2, pp. 6591-6604, 2001, EP1176195A1, EP1331266A1, WO 01/77181).
• the antibody can also, especially when it is an IgG isotype and in particular an IgG1 isotype, have been modified to limit its ability to fix the complement and therefore its activity of complement-dependent cytotoxicity (CDC), while preserving its antibody-dependent cellular cytotoxicity (ADCC) functions.
• CDC complement-dependent cytotoxicity
• ADCC antibody-dependent cellular cytotoxicity
• an anti-GD2 antibody it has been shown that a point mutation in the constant part of the human IgG1 region (replacement of the lysine at position 322 of the human Fc constant part of the light chain ⁇ by alanine) allowed to reduce its complement fixation while maintaining its ADCC properties, and to reduce some side effects associated with the administration of this antibody (US7432357B2).
• the anti-Gb3 antibody according to the invention is an antibody with a low CDC activity, that is to say that in a CDC activity test in vitro, the CDC activity of the antibody at a concentration of 0, 1 ⁇ g / ml is not significantly different from that of the control without antibodies.
• an advantageous embodiment relates to a humanized anti-Gb3 IgG1 isotype antibody, comprising an ⁇ isotype light chain, in which the lysine at position 322 of the human Fc constant part of the ⁇ light chain is replaced by an alanine or an equivalent amino acid, preferably an alanine.
• the invention also relates to a nucleic acid (also called nucleic or nucleotide sequence) coding for any of the anti-Gb3 antibodies, or functional fragments or derivatives according to the invention as described above.
• nucleic acid also called nucleic or nucleotide sequence
• All the different nucleic sequences, because of the degeneracy of the genetic code, encoding a particular amino acid sequence are within the scope of the invention.
• Such nucleic sequences may have been optimized to promote its expression in a host cell of interest.
• Particular sequences of interest are those of the variable regions of the heavy and light chains of the antibodies generated by the inventors, represented as follows:
• Antibody 3E2 heavy chain: SEQ ID NO: 57, light chain: SEQ ID NO: 64,
• 14C11 antibody heavy chain: SEQ ID NO: 58, light chain: SEQ ID NO: X65
• 15C11 antibody heavy chain: SEQ ID NO: 59, light chain: SEQ ID NO: 66,
• Antibody heavy chain: SEQ ID NO: 61 light chain: SEQ ID NO: 68,
• 11E10 antibody heavy chain: SEQ ID NO: 62, light chain: SEQ ID NO: 69,
• the invention also relates to a vector comprising at least one of the nucleic sequences described above.
• a vector comprises the elements necessary for the expression of said nucleic sequence, and in particular a promoter, a transcription initiation codon, termination sequences, and appropriate transcriptional regulatory sequences. These elements vary according to the host serving for the expression and are readily selected by those skilled in the art in view of his general knowledge.
• the vector may especially be plasmidic or viral. It is used to clone or express the nucleic sequences according to the invention.
• the invention also relates to a host cell comprising one or more nucleic sequences or one or more vectors according to the invention.
• the host cell may be of procary or eukaryotic origin, and may especially be selected from bacterial cells, insect cells, plants, yeast or mammals.
• the antibody, functional fragment or derivative according to the invention can then be produced by culturing the host cell under appropriate conditions. It can also be obtained by chemical synthesis, in particular in the solid or semi-solid phase.
• the antibodies, functional fragments or derivatives according to the invention have anti-angiogenic properties as such.
• they exhibit cytostatic and cytotoxic activity on proliferating endothelial cells. They can therefore be used alone in the treatment of diseases associated with angiogenesis, as defined below.
• the antibodies according to the invention described above are not coupled to a therapeutic molecule, in particular a cytotoxic, cytostatic or anti-angiogenic molecule such as a toxin, in particular an angiogenesis-inhibiting toxin.
• an antibody is "coupled" to a molecule if there is a covalent bond between the antibody and the molecule.
• the fact that the antibody according to the invention is not coupled to a therapeutic molecule means that it is not bound by a covalent bond to such a molecule.
• this does not prevent the antibody, which is not coupled to a therapeutic molecule, from being administered in combination (simultaneously or sequentially) with another therapeutic molecule, including a cytotoxic, cytostatic or anti-angiogenic molecule in the case of tumors.
• the two molecules can act independently of one another.
• the anti-Gb3 antibodies, or functional fragments or derivatives thereof according to the invention as described above can nevertheless also be coupled to another therapeutic molecule.
• the present invention also relates to an antibody, directed against the Gb3 membrane glycosphingolipid and not coupled to a therapeutic molecule, for its use as a medicament in the treatment of angiogenesis-associated diseases. It also relates to the use of an antibody directed against the Gb3 membrane glycosphingolipid and not coupled to a therapeutic molecule, for the preparation of a medicament for the treatment of diseases associated with angiogenesis. It also relates to a method of treating angiogenesis-associated diseases in a subject in need, comprising administering an effective amount of an antibody directed against Gb3 membrane glycosphingolipid and not coupled to a therapeutic molecule. It also relates to a method for inhibiting angiogenesis in a subject in need, comprising administering an effective amount of an antibody directed against Gb3 membrane glycosphingolipid and not coupled to a therapeutic molecule.
• diseases associated with angiogenesis any disease whose evolution requires angiogenesis to develop, that is to say the formation of new vessels from a pre-existing vascular network.
• diseases associated with angiogenesis include solid tumors; psoriasis; angiomas; prominent eye diseases, including age-related macular degeneration (AMD), diabetic retinopathy, or neovascular glaucoma; autoimmune diseases, including lupus or rheumatoid arthritis, as well as atherosclerosis-related diseases; obesity or Alzheimer's disease. Indeed, the development of all these diseases involves angiogenesis.
• blood cell diseases such as leukemias or lymphomas
• cytotoxic, cytostatic or anti-angiogenic is used in the treatment of solid tumors.
• Solid tumors for which anti-Gb3 antibodies are useful include adenomas, sarcomas, and carcinomas; and in particular adenocarcinomas, ovarian, breast, pancreatic, skin, lung, brain, kidney, liver, nasopharyngeal cavity, thyroid, central nervous system (neuroblastoma by example), prostate, colon, rectum, cervix, testes, or bladder.
• the anti-Gb3 antibody used is any of those described above in the section concerning the antibodies as such.
• the anti-Gb3 antibody recognizes the upper band but not the lower band of the Gb3 doublet expressed by HMEC-1 cells.
• it may be antibodies 22F6 and 3E2, or an antibody possessing at least one CDR or all the CDRs, or even one and / or the other of the variable regions of these antibodies.
• the anti-Gb3 antibody may be administered in combination with another treatment, including surgical resection of the tumor, radiotherapy treatment or chemotherapy with another therapeutic molecule, provided that another therapeutic molecule is not coupled to the antibody.
• the treatment radiotherapy / radiotherapy / other therapeutic molecule
• the combination may also be administered first (simultaneously or sequentially) and then the treatment continued with only one of the two therapies.
• a therapeutic molecule is not coupled to the antibody, it can be selected from all other anticancer molecules, whether they also target angiogenesis or the tumor itself.
• FIG. 1 HPTLC profiles of growth phase (lane 1) or confluent (track 2) HMEC-1 cells, rat brain ganglioside markers (lane 3) and standard neutral glycolipids (lane 4).
• B HPTLC profiles of HMEC-1 cells incubated in depleted (lane 1) or complete (lane 2) culture media, ganglioside markers of rat brain (lane 3), and a mixture of standard neutral glycosphingolipids (lane 4).
• C HPTLC profiles of HMEC-1 cells treated with PET (lane 1) or S1P (lane 2), rat brain ganglioside markers (lane 3), and standard neutral glycolipids (lane 4).
• the migration solvent used is C / M / H 2 O, 0.2% CaCl 2 , 55: 45: 10 v / v / v. All bands are revealed by orcinol.
• FIG. 1 Protocol for obtaining antibodies against HMVEC-L endothelial cells cultured in the presence of T84 human adenocarcinoma cells.
• Figure 3 Principle of 96-well microplate limiting dilution cloning.
• Panel B represents the theoretical number of cells remaining after dilution.
• FIG. 1 Electrophoretic analysis of IgM 3E2 mAb on a 6% polyacrylamide gel under non-reducing conditions (A) on a 12% polyacrylamide gel under reducing conditions (B).
• FIG. 1 Analysis of flow cytometric labeling of HMEC-1, Raji and NXS2 cells with purified anti-Gb3 monoclonal antibodies 3E2.
• C A
• 25C 10.a B
• 16G8.h C
• ld D
• 15C7.2e E
• All cells were labeled with 10 ⁇ g / ml of antibody. Percentages of positive cells are noted in the panel figures A, B, C, D, E and F.
• FIG. 6 Study of the Specificity of the 3E2 Antibody by ELIS A (Panel A) Fixation of the 3E2 Antibody Measured by ELISA on Raji Cells, HMEC-1 Cells and IMR32 Cells (Panel B) Attachment of the 3E2 antibody and the 38.13 anti-Gb3 rat monoclonal antibody to the HMEC-1 cells measured by ELISA. The initial concentration of the antibodies is equal to 10 ⁇ g / ml (3 independent analyzes).
• Figure 7. Immunostaining on HPTLC of HMEC-1 cells with mAb 3E2, mAb 3, 8, and supernatant of uncleaved hybridoma 3E2.
• FIG 8. Immunostaining on HPTLC of HMEC-1 cells by mouse mAbs 3E2 and A4.
• Rat brain ganglioside markers (lane M), neutral glycolipid mixture (lane 1), purified porcine Gb3 (lane 2) and glycolipid extracts of HMEC-1 cells (lane 3).
• the lines are chemically revealed by orcinol.
• Patent B Immunostaining is performed with 5 ⁇ g / ml of MAb 3E2 or 1 A4.
• Figure 9 Analysis of the specificity of the antibody 3E2 by flow cytometry on HMEC-1, Raji and NXS2 cells. Expression of Gb3 on HMEC-1, Raji and NXS2 cells by flow cytometry with 10 ⁇ g / ml mAb 3E2 (A), 1A4 (B) and IgM isotype control, ⁇ (C). Percentages of positive cells are noted in Table 10.
• FIG. 10 HPTLC glycolipid profile revealed in orcinol of HMEC-1 (4), Raji (5), NXS2 (6) and T84 (7) cells.
• FIG. 11 Expression of Gb3 on HMEC-1, HMVEC-L and HUVEC cells by flow cytometry with 10 ⁇ g / ml mAb 3E2 (A), 1 A4 (B) and isotypic IgM control, ⁇ (C) . Percentages of positive cells are noted in FIG. 11 and the fluorescence intensity average values are listed in Table 11.
• Figure 13 Nucleotide sequences of the variable regions of the V H (panel A) and V L (panel B) gene segments of the 3E2 antibody.
• Figure 14 Flow cytometric analysis of Gb3 expression of HMEC-1 cells incubated for 24 h in depleted media (MA) and in complete medium (MC). Fixation was measured using mAb 3E2 (Panel A) and mAb 1 A4 (Panel B). The percentage of positive cells and the fluorescence intensity average values are listed in Table 14 (3 independent experiments).
• FIG. 15 Flow cytometric analysis of Gb3 expression of HMEC-1 cells incubated in depleted media supplemented with S ⁇ SIP or PET. Fixation was measured using mAb 3E2 at 24 h (Panel A) and 72 h (Panel B). The percentage of positive cells and the average fluorescence intensity values are listed in Table 15 (3 independent experiments).
• Figure 16 Cell viability of HMEC-1, Raji and NXS2 cells measured by MTT after 24 h incubation of mAb 3E2 and 1A4. Cell viability of HMEC-1 cells in the presence of mAb 3E2 and A4 (Panel A) and Raji and NXS2 cells in the presence of ⁇ mAb 3E2 (panel B), 3 independent experiments.
• Figure 18 Representation of the number of HMEC-1 cells as a function of the incubation time of the mAb 3E2 at 20 ⁇ .
• Figure 19 Percentage of HMEC-1 cells in division and estimation of their cell division time in the presence of the antibody 3E2.
• (Panel A) Microvessels in development from transverse sections of murine aortic rings. Photo 1, growth of microvessels at implantation of aortic sections (J0). Photo 2, microvessel growth after five days after incubation in complete culture medium. Photos 3 and 4, aortic rings treated with respectively 20 ⁇ g / ml and 40 ⁇ g / ml of mAb 3E2.
• Figure 21 Evaluation of the CDC activity of mAb 3E2 and 1A4. Specific lysis of the cells was determined by flow cytometry by measuring the percentage of cells having incorporated propidium iodide. The CDC activity is measured in the presence of decomplemented human serum (') or not decomplemented (c').
• CDC activity performed on HMEC-1 cells,
• Panel B Raji cells and
• Panel C on NXS2 cells.
• Figure 22 Nucleotide sequences of the variable regions of the heavy chain of 7 anti-Gb3 antibodies.
• Figure 23 Nucleotide sequences of the variable regions of the anti-Gb3 antibody light chain.
• FIG. 24 Reduction of the growth of a subcutaneous tumor by the 3E2 antibody.
• Female AI mice were inoculated subcutaneously in the right flank with 10 6 NXS2 cells (neuroblastoma line) alive (viability> 95%) resuspended in 150 ⁇ l of PBS.
• the tumors reached a volume V of 100-150 mm 3
• the mice received a single injection of 3E2 antibody directed against the upper band of the doublet Gb3 expressed by the HMEC-1 cells, anti-GD2 14G2a antibody, IgM isotype control antibody or PBS alone.
• the graph represents the tumor volume, expressed as a percentage of the tumor volume during the injection of the treatment, as a function of the duration after treatment.
• FIG. 25 The 3E2 antibody inhibits the growth of metastases in vivo.
• A Representative photograph of mouse livers at day 28 after intravenous injection of NXS2 cells. The scale bars represent 1 cm.
• Endothelial cells HMVEC-L, HMEC-1, HUVEC.
• HMVEC-L cells are primary human microvascular endothelial cells from lungs (Cambrex, Clonetics ®, USA). Each ampoule corresponds to a single Caucasian donor. They were seeded at a density of 5 10 4 cells / cm 2 in 6-well plates, then cultured in a humid atmosphere enriched with C0 2 at 37 ° C in the middle of specific EGM -2 ® MV Culture (Cambrex, USA) having 25 ml of FCS (fetal calf serum), 0.2 ml of hydrocortisone, 2 ml of hFGF-B, 0.5 ml of VEGF, 0.5 ml of R3-IGF-1, 0.5 ml of ascorbic acid, 0.5 ml of hEGF and 0.5 ml of GA-1000, per 500 ml of medium.
• FCS fetal calf serum
• hydrocortisone 2 ml of hFGF-B
• VEGF 0.5 m
• the line FDVIEC-1 human microvascular endothelial cells
• Pr. FJ Candal Center for Disease Control, Atlanta, Ades et al., 1992
• a plasmid containing the coding region of the SV40 virus T antigen retain the main characteristics specific to microvascular endothelial cells (Ades et al., 1992) and are cultured between passages 13 and 20 in order to limit phenotypic drift related to cell culture.
• HUVEC cells are human macrovascular primary cells extracted from umbilical cord veins (Cambrex). Each ampoule corresponds to several donors. They are seeded at a density of 3.5 ⁇ 10 4 cells / cm 2 in 6-well plates and then cultured in a humid atmosphere.
• EGM -2 enriched in C0 2 at 37 ° C in the specific culture medium EGM -2 (Cambrex, USA) according to: 10 ml of FCS, 0.2 ml of hydrocortisone, 2 ml of hFGF-B, 0.5 ml of VEGF, 0.5 ml of R3-IGF-1, 0.5 ml of ascorbic acid, 0.5 ml of hEGF, 0.5 ml of GA-1000 and 0.5 ml of heparin, per 500 ml of middle. They can be grown from the first to the sixth pass.
• Tumor lines Raji, NXS2, IMR32 and T84.
• the Raji human lymphoma cells and the IMR32 human neuroblastoma cell line are from ATCC (Rockville, USA).
• the NXS2 cells of murine neuroblastoma were provided by Prof. H. Lode (Charity Children's Hospital, Berlin, Germany).
• the Raji cells are inoculated at 0.1 ⁇ 10 6 cells / ml in RPMI 1640 medium (Invitrogen) supplemented with 10% inactivated FCS, 2 mM L-glutamine, 100 IU / ml penicillin and 100 ⁇ g / ml. streptomycin.
• the IMR32 cells are cultured in the same medium but are inoculated at 2 ⁇ 10 4 cells / cm 2 .
• the NXS2 cells inoculated at a rate of 2 ⁇ 10 4 cells / cm 2, are maintained in DMEM medium containing 4.5 g / l of glucose (Sigma), 10% of inactivated SVF, 2 mM of L-glutamine, 100 IU / ml of penicillin and 100 ⁇ g / ml of streptomycin.
• the T84 epithelial cells of a human colon adenocarcinoma were provided by Dr. M. Neunlist (INSERM UMR913, France).
• Gb3 was rationally given to us as ascites by Dr. J. Wiels (Institut Gustave Roussy, Villejuif, France). It was purified by affinity chromatography using a mannan column (Pierce, Rockford, USA) and conditioned at 4 ° C in PBS.
• the 38.13 anti-Gb3 rat IgM monoclonal antibody and isotype control of mice (clone 11E10) were obtained in purified form from Beckman Coulter (Fullerton, CA, USA).
• Biotinylated secondary goat antibodies such as mouse anti-IgG + IgM (H + L) antibodies, mouse anti-IgG (H + L) antibodies, mouse anti-mouse antibodies were provided by Jackson Immunoresearch (Laboratories, Westgrove, PA, USA).
• Peroxidase-coupled goat antibodies specific for the rat ⁇ chain and biotinylated goat secondary antibodies specific for mouse subclasses Fc Y i, Fc Y 2a, Fc Y 2b, Fc Y 3
• Biotinylated goat antibodies specific for mouse light ( ⁇ and ⁇ ) chains were provided by Southernbiotech (Birmingham, AL, USA).
• the streptavidin-R-phycoerythrin complex was obtained from Biosource (Camarillo, CA, USA), the streptavidin-horseradish-peroxidase complex and the streptavidin-FITC complex were obtained from Beckman Coulter.
• the mixture of neutral glycosphingolipids comprising lactosylceramide (LacCer), galactosylceramide (GalCer), ceramide trihexoside (Gb3) and globoside (Gb4) as well as purified Gb3 were obtained from Matreya (Pleasant Gap, PA, USA). These glycolipids are either of porcine origin (Gb3 and Gb4) or of bovine origin (LacCer and GalCer).
• the purified Gb3 was redissolved with 1 ml of a chloroform: methanol (2: 1 v / v) mixture so as to obtain a final concentration of 1 ⁇ g / ⁇ l.
• Sphingosine-1-phosphate S1P was obtained from Biomol International (Plymouth Meeting, PA, USA) and rehydrated (Morita et al., 2000) in order to obtain a concentration of 10 ⁇ in the PET diluent consisting of 5% of polyethylene glycol, 2.5% ethanol and 0.8% Tween-80.
• the mixture of gangliosides markers standards (GMi, GDi has GDi, GTi b) was prepared in the laboratory by extraction of rat brain gangliosides, according to the technique described by Folch (Folch et al. 1951). 1.2. Extraction and purification of glycolipids.
• the glycolipids were extracted from cell pellets according to the method of S. Ladish (Ladish and Li, 2000). The cells are trypsinized, washed twice in PBS pH 7.3 and then incubated at -20 ° C. overnight. They then undergo hypotonic lysis in 1 ml of distilled water. Aliquots are then removed and centrifuged at 15,000 g, 4 ° C, for 15 min to remove insoluble particles, debris cell and DNA. The protein concentration of the aliquots is then determined by absorbance at 280 nm (Nanodrop ND-1000, Labtech, Palaiseau, France) in order to deposit on a thin layer of silica a quantity of glycolipids corresponding to 0.5 - 2.5 mg of protein equivalent per sample.
• DIPE / 1-butanol partition The total lipid extract is redissolved in 5 ml of an organic mixture containing DIPE / 1-butanol (60:40, v / v). Series of vortex and sonication (Diagenode's Bioruptor, Sparta, USA) are alternated and repeated until an opalescent solution is obtained. By adding and mixing 2.5 ml of 0.1% NaCl saline, a partition is obtained after centrifugation at 750 g for 10 min at 4 ° C. The upper organic phase containing neutral lipids and phospholipids is pipetted and can be stored at -20 ° C.
• the lower aqueous phase and the interface containing the acidic glycolipids and the more hydrophilic neutral glycolipids are again partitioned with a new volume of DIPE / 1-butanol (60:40, v / v).
• the glycolipids contained in the aqueous phase are evaporated in a speed-vac (Eppendorf, Hamburg, Germany).
• Second step desalting by gel filtration. In order to remove salts and contaminants such as proteins that can be co-extracted at this stage, the dry residues are redissolved and vortexed in 500 ⁇ of solvent A (chloroform: methanol: H 2 O (30: 60: 8).
• glycolipids recovered are taken up in a small volume of chloroform: methanol (1: 2, v / v) so as to deposit, with the aid of a TLC ATS4 deposition automaton (Camag, Muttenz, Switzerland) 2 to 2, 5 mg of protein equivalent per glycolipid sample.
• the glycolipids are separated on HPTLC plates consisting of a silica gel 60 covering an aluminum foil (Merck, Darmstadt, Germany). Before deposition, these silica plates are subjected to a first migration in a glass vessel (Camag) containing a mixture of chloroform: methanol (1: 1, v / v), in order to rid them of possible contaminants.
• a first migration system consisting of chloroform: methanol solvent (2: 1, v / v) makes it possible to migrate and eliminate very apolar lipids which can be coextracted and which can interfere with the separation of glycolipids of interest.
• the latter are then separated during a final development in a saturated tank for 4 h with 70 ml of chloroform: methanol: 0.2% aqueous CaCl 2 (55: 45: 10, v / v / v).
• the strips are chemically exposed at 150 ° C. after spraying the plate with a solution of orcinol previously prepared by dissolving 0.2 g of orcinol (Sigma-Al drich, St. Louis, USA) in 40 ml of water.
• glycolipids can then be quantified by ImageQuant 5 ' 2 densitometry software (GE Healthcare, Waukesha, USA).
• the specificity of the antibodies can be determined on glycolipids separated by HPTLC. After separation by chromatography of glycolipids for which 2 to 2.5 mg of protein equivalent per sample is exposed to orcinol and 0.5 mg of protein equivalent for the samples which will be labeled by the antibodies, the HPTLC plates are cut into strips and plasticized by incubation for 1 min in 0.1% polyisobutyl methacrylate dissolved in hexane, then saturated at room temperature for 1 hour with 1% PBS-BSA. The strips are then incubated overnight at 4 ° C. with the antibodies (either directly incubated with the hybridoma supernatants or incubated with 5 ⁇ g / ml of purified antibody diluted in 0.1% PBS-BSA).
• the antibody binding is detected by two successive incubation steps, a first incubation step of 1 h at room temperature of the biotinylated secondary antibodies (diluted 1: 2000 in PBS). 0.1% BSA), followed by incubation of the streptavidin-horseradish-peroxidase complex, diluted 1: 2000 for 1 hour at room temperature.
• the bound peroxidase is revealed by a solution of 4-chloro-1-naphthol (Sigma) which is prepared extemporaneously at the rate of 1 mg of product dissolved in 1 ml of methanol, taken up in 20 ml of PBS. and added 30 ⁇ of hydrogen peroxide 30 volumes. 1.5. Analysis of antibody specificity by enzyme immunoassay
• the desiccated cell plates were prepared in the following manner. The cells are trypsinized and washed three times with cold PBS, and then diluted to obtain a cell concentration of 2 ⁇ 10 6 cells / ml. Is then deposited 50 ⁇ of this suspension to the bottom of each of the 96 wells of a microtiter plate Immuno-Plate (Nunc, Maxisorp ®, Denmark). After desiccation of the cells overnight in an oven at 37 ° C, the plates are either used immediately or stored at room temperature for several months under aluminum.
• the antibodies diluted in 0.1% PBS-BSA (10 ⁇ g / ml of initial concentration) are deposited in each of the triplicate wells, with a volume of 100 ⁇ of antibody in a dilution range of 1: 256.
• the plates are incubated for 2 hours at room temperature and then washed. three times with PBS.
• the binding of the antibody is detected successively by a first incubation for 1 h at room temperature with the biotinylated secondary antibodies (diluted 1: 4000 in PBS-BSA 0.1%), then after washing, by incubation.
• streptavidin-horseradish-peroxidase complex (diluted 1: 4000, 1 hr at room temperature). After several washes with PBS, the fixation of the peroxidase is revealed by a solution of ABTS (Merck) which reveals a progressive green coloration. The reaction can then be blocked by the addition of 10% SDS. The measurement of the optical density is determined by reading the plate with a spectrophotometer at 405 nm (Multiskan Thermo Electron reader, Illkirch, France).
• the cells are then directly incubated with 100 ⁇ l of primary antibodies diluted to 10 ⁇ g / ml in PBSF for 45 minutes on ice. After incubation, the cells are washed 3 times with cold PBS and then fixed with 4% paraformaldehyde (Electron Microscopy Science, Washington, USA) in PB S, on ice for 15 minutes, in order to prevent the internalization of the cells. antigen-antibody complexes. After three weeks at PB S, antibody fixation is first detected by incubation for 30 minutes on ice of biotinylated secondary antibodies (diluted 1: 400 in PBSF) followed by washes and then incubation. streptavidin-phycoerythrin complex (diluted 1: 400, 30 minutes on ice).
• the cells are seeded in their culture medium on glass slides 14 mm in diameter (Thermo Scientific, Hudson, USA) in a 24-well plate, 24 to 48 hours before labeling, at a rate of 2 ⁇ 10 5 cells. / cm 2 .
• the non-specific sites are blocked by incubating 5% inactivated human serum on ice and then the cells are incubated with the antibodies diluted to 40 ⁇ g / ml in PBSF for 45 minutes on ice. After labeling, the cells are washed 3 times with cold PBS and then fixed with 4% paraformaldehyde on ice for 15 minutes.
• the cells are again washed 3 times with PBS and the binding of the antibody is detected first by a 30 minute incubation on ice of the biotinylated secondary antibodies (diluted 1: 400 in PBSF) followed by 30 minutes of ice incubation of streptavidin-FITC complex (diluted 1: 400). After staining of the cell membrane, the nuclei are labeled for 15-20 min at room temperature, Drag5 (Biostatus, Leicestershire, UK) diluted 1: 1000 in PBS.
• the slides are assembled in Fluoromount-G medium (Southernbiotech, Birmingham, AL, USA) and the cell labeling is observed under the confocal microscope TCS-SP1 (Leica, Mannheim, Germany, PICell platform, IFR 26, Inserm, France) at a magnification of 63 X.
• mice are raised at the animal center of the INSERM U892 unit (under the control of AFSTAL, French association of Sciences and Techniques of Laboratory Animal).
• Raji cells and IMR32 cells (2.5 ⁇ 10 5 cells) diluted 1: 2 in Matrigel highly concentrated in growth factors (BD Biosciences, Bedford, USA) were injected subcutaneously into the flank of mice.
• the mice were sacrificed by elongation as soon as small blood capillaries appeared on the xenografted tumors. Directly after excision, the tumors were pre-immersed in the isopentane and then immersed for a few seconds in liquid nitrogen to be stored at -80 ° C.
• the frozen sections are incubated successively with 50 ⁇ g / ml of antibody anti-Gb3 or mouse IgM isotype control antibody (clone 1 1E10) for 1 h at room temperature, and then incubated after several washes at PB S, with biotinylated goat anti-mouse IgM secondary antibody, diluted at 1: 100. Detection is performed using a chromogenic substrate.
• the sections are incubated for 30 minutes with VECTASTAIN ® Elite ABC reagent (Vector Laboratories). The peroxidase activity is detected with the DAB ® Menarini Kit substrate (Rungis, France) diluted 1:50, resulting in the deposition of a brown pigment.
• the sections are then counter-stained with hematoxylin and observed at 40X magnification with a DM IRB (Leica) microscope. 1.9. Obtaining anti-Gb3 antibody 3E2 (IgM.
• HMVEC-L cells Five Balb / c @ BYJ Rj mice, 6 weeks old, were immunized with HMVEC-L cells previously co-cultured with T84 human colonic adenocarcinoma cells via membranes allowing the exchange of soluble factors between the cells. two cell compartments (Gaugler et al., 2007). HMVEC-L cells were seeded in 6-well plates in an amount of 5 10 4 cells / cm 2 in their culture medium EGM -2 ® MV.
• the T84 cells (1 ⁇ 10 5 cells / cm 2 ) were seeded in their DMEM: F 12 culture medium, on the porous membrane filter (pores of 0, 4 mm) of a Transwell 6-well Transwell-Clear insert (Corning, The Netherlands) for 72 hours.
• FDVIVEC-L cells were then trypsinized, washed in PBS, fixed with 4% paraformaldehyde and stored at 4 ° C in incomplete Freund's adjuvant (Sigma, St. Louis, USA).
• mice were injected intraperitoneally with five injections at 0, 8, 10, 32, and 60 weeks of 2, 1-2.8 10 6 HMVEC-L cells previously co-cultured, as well as one dose. at 65 weeks of 1.5 ⁇ 10 6 cells (stored in PBS without adjuvant), 6 days before sacrifice.
• the kinetics of the humoral response of the mice was analyzed during the immunization period by ELISA tests carried out on desiccated HMVEC-L cells but also on desiccated HMEC-1 cells in order to envisage the use of these transformed cells. for antibody screening studies.
• mouse splenocytes were fused to SP2 / 0 murine myeloma cells at a 5/1 fusion ratio: 2.5 splenocytes were fused with polyethylene glycol (PEG 1500, Sigma) at 0.5 ⁇ 10 8 murine myeloma cells SP2 / 0 (cultured in RPMI 1640).
• the cells were taken up in 200 ml of RPMI 1640 medium supplemented with 20% inactivated SVF, 2 mM L-glutamine, hypoxanthine-aminopterin-thymidine HAT IX medium (Sigma-Aldrich) and inoculated at 100 ⁇ into 35 microplates of 96 wells. After 15 days, the first hybridomas were visible and could be amplified in 12 well microplates in RPMI 1640 medium supplemented with 20% inactivated serum, 2 mM L-glutamine, 100 IU / ml penicillin and 100 ⁇ g / ml of streptomycin.
• the selected hybridomas were cloned by the limiting dilution method (Fig. 3). Each clone obtained was then tested again by ELISA using the anti-IgG + IgM detection antibodies, then the isotype of the positive monoclonal antibodies was determined using anti-isotype detection antibodies ( ⁇ , ⁇ , y2a). , y2b and ⁇ 3) and anti-light chain ( ⁇ and ⁇ ).
• the eluates are selected after measurement of the absorbance at 280 nm (Nanodrop) and pooled to be dialysed in PBS, filtered through 0.22 ⁇ and stored at 4 ° C.
• the degree of purity of the antibodies is assessed by an electrophoretic analysis under denaturing conditions (SDS-PAGE) that is reducing with 12% acrylamide (375 mM Tris pH 8.8, SDS 0.1%, APS 0.1%, TEMED), which is non-reducing with 6% acrylamide.
• SDS-PAGE electrophoretic analysis under denaturing conditions
• the migration on the gel is carried out with 5 ⁇ g of antibody per condition, at 90 V for 45 h in Tris-Glycine-SDS buffer (Biorad, Hercules, USA).
• the electrophoretic profile is detected by staining with Coomassie Blue.
• the antibodies are first labeled with iodine 125 in the presence of an oxidant, iodogen.
• an oxidant iodogen.
• 200 ⁇ g of anti-Gb3 antibody 3E2 is deposited, and the tube is then supplemented with 100 ⁇ l of phosphate buffer at 0, 1M, pH 7.2.
• Iodine 125 Perkin Elmer, Waltham, USA
• the reaction mixture is incubated for 30 minutes with gentle stirring at room temperature.
• the purity of the radiolabelled antibody was monitored by thin layer chromatographic analysis. For this, 2 ⁇ l of the reaction mixture were deposited on an ITLC-SG strip (PALL Science, Iris Parkway, USA). By migrating in 10% trichloroacetic acid, the free 125 I separates from 125 I bound to the antibody. It is thus possible to evaluate the level of labeled antibody which must be greater than 90% and which can be reinforced by purification on a NAP-5 TM column (GE Healthcare, Waukesha, USA) and be evaluated again by thin layer chromatography. ITLC-SG.
• the number of Gb3 antigenic sites was determined according to the Scatchard technique, on Raji lymphoma lines and on HMEC-1 endothelial cells.
• HMEC-1 cells the number of antigenic sites was evaluated in particular after 24 h of incubation in depleted medium (MCDB 131 medium supplemented with 0.1% decomplemented FCS, 2 mg / ml of hydrocortisone, 2 mM of L-glutamine, 100 IU / ml penicillin and 100 mg / ml streptomycin) and after 24 h incubation in complete medium (MCDB 131 medium supplemented with 15% FCS decomplemented, 10 ng / ml EGF, 2 mg ml of hydrocortisone, 2 mM L-glutamine, 100 IU / ml penicillin and 100 mg / ml streptomycin).
• the tubes After centrifugation at 15,000 g for 3 minutes, the tubes are immersed in liquid nitrogen, cut into two portions and immediately placed in hemolysis tubes for radioactive counting. The lower end containing the cells constitutes the bound fraction and the upper end containing the supernatant constitutes the unbound fraction. Measurements are made at the ⁇ counter (1480 Wizard 3, Perkin Elmer, Finland) and the results are analyzed with GraphPad Prism software (GraphPad Software Inc., San Diego, USA). 1.11. Biological properties of the 3E2 antibody.
• the MTT assay (Mosmann, 1983) is based on the transformation of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) into blue crystals of formazan by a mitochondrial enzyme. succinate dehydrogenase. The formazan crystals formed by the cells are solubilized chemically and detected spectrophotometrically at a wavelength of 570 nm. This test is used to compare the viability of control cells with that of cells treated with molecules.
• the assay is performed in 24-well plates with cells seeded at a density of 2 ⁇ 10 4 cells / cm 2 .
• HMEC-1 cells were seeded in a 24-well microplate at 2 ⁇ 10 5 cells / cm 2 and then maintained for 12 hours in their complete medium at 37 ° C., 5% CO 2 .
• the 3E2 antibodies and the isotype control were then diluted to 20 ⁇ g / ml in complete culture medium and incubated with the cells in triplicate.
• digital images were recorded for 24 to 72 h in order to produce an accelerated film (Leica, DMI6000B / PICell platform). Observations were on the number of cells per time interval, the rate of dividing cells per time interval, and the cell division time (mean of observations from three different wells). Tests of the aortic rings.
• mice aged 4 weeks were provided by the Janvier breeding farm (St Berthevin, France). Abdominal aorta were isolated when the mice were 6 to 8 weeks old. They were then cleaned in complete MCDB 131 medium and cut into cross sections 0.5-1 mm wide. These rings were then deposited at the bottom of the wells of a 96-well microplate previously treated with 30 ⁇ l of Matrigel (ECM Gel, Sigma-Aldrich, St. Louis, USA) diluted half in PB S. The rings were then were covered with 20 ⁇ l of undiluted Matrigel and then kept for 5 days at 37 ° C., 5% of C0 2 in complete medium containing 0, 20 and 40 ⁇ g / ml of antibody.
• Matrigel ECM Gel, Sigma-Aldrich, St. Louis, USA
• the culture medium with or without antibodies was renewed every 2 days. After 5 days of incubation, the formation of vascular buds was observed by microscopy (IRB DM microscope, Leica). In order to objectively measure the formation of these buds, we asked a 5-person jury to independently participate in a blind notation of images of the aortic rings recorded at 10X magnification and asked them to assign each image a score, based on the number, length and density of microvessels formed (scale from 0 to 5, the minimum value of 0 corresponding to the absence of buds), in order to evaluate the anti-angiogenic activity of the antibodies.
• the CDC activity is measured by flow cytometry using a solution of propidium iodide (PI) capable of incorporating the DNA of the cells.
• PI propidium iodide
• the cells are trypsinized, taken up in culture medium and then placed in conical bottom 96-well plates (Nunc, Denmark) at a rate of 0.2 10 6 cells / well. The plates are then centrifuged to remove the supernatant, and the cells are incubated with the antibodies to give a final concentration of 0.1 to 10 ⁇ g / ml.
• Fresh filtered serum is collected 24 h after the blood sample in the absence of anti-coagulant. A fraction is decomplemented at 56 ° C for 45 min, filtered again and stored at 4 ° C.
• Human serum is then added which represents 1: 5 of the final volume, either in its decomplemented form or in its non-decomplemented form.
• the plates containing the cells, the antibodies and the serum are then incubated for 2 h at 37 ° C., 5% CO 2 .
• the cell pellets are then collected by brief centrifugation of the plate and the cells taken up with 0.6 ⁇ g of a solution of propidium iodide in a volume of 100 ⁇ . After an incubation of 30 min at 37 ° C in the dark, the cells are analyzed directly using the cytometer at a rate of 1.10 5 cells per analysis. 1.12. Nucleotide sequencing of variable regions of heavy and light chains of anti-Gb3 antibodies.
• RNA of the mouse hybridomas was extracted according to the method described by Chomczynski and Sacchi (Chomczynski and Sacchi, 1987) with the RNAble ® reagent (Eurobio, Les Ulis, France).
• the cells are homogenized in a denaturing solution containing guanidinium isothiocyanate and phenol.
• the nucleic acids are denatured, the proteins dissociated thanks to the formation of complexes between RNA and guanidine isothiocyanate which makes it possible to break the hydrophilic interactions between the DNA and the proteins.
• the DNA and proteins are extracted from the aqueous phase while the RNA remains in this phase.
• the cell pellet of hybridomas (10 10 6 cells) is washed twice in PB S and then taken up in 2 ml of RNAble.
• the cells are lysed by repeated pipetting to dissociate the nucleoprotein complexes.
• the homogenate is mixed vigorously for 15-30 seconds. This step makes it possible to separate, by differential solubilization, the nucleic acids (the RNAs being insoluble in phenol) from the proteins.
• the mixture is incubated on ice for 5 minutes and then centrifuged for 15 minutes at 12,000 g at 4 ° C.
• the aqueous phase containing the RNA is removed and transferred to a microtube.
• RNA concentration is determined spectrophotometrically by measuring the absorbance at 260 nm of an aliquot as well as its purity at 280 nm by the ratio of the absorbances at 260 nm / 280 nm for a value of about 2.
• the RNA thus extracted are either stored at -80 ° C or directly treated with DNAse.
• the DNAse is treated according to the protocol of the supplier.
• the reaction is carried out in a final volume of 10 ⁇ l comprising 2 ⁇ g of total RNA, a 20 mM Tris-HCl buffer solution, pH 8.4, 2 mM MgCl 2 , 50 mM KCl, and 1 U of DNAse 1. (Sigma, St-Quentin-Favier, France).
• the DNAse is inactivated by the addition of 1 ⁇ l of a 25 mM EDTA solution, followed by incubation at 65 ° C. for 10 minutes.
• the treated RNAs can also be stored at -80 ° C, or be directly subjected to a reverse transcription step.
• RNA in complementary DNA RNA
• the cDNAs are synthesized from 0.2 ⁇ g of RNA in a final volume of 40 ⁇ . According to the supplier's protocol (Invitrogen), the total RNAs are denatured by incubation at 65 ° C. for 5 min. 500 ng of poly- (dT) i2-is oligonucleotides and 1 nM of each dNTP in 12 ⁇ l of H 2 0 wg QSP. After incubation, the RNAs are placed rapidly on ice to prevent refolding.
• reaction medium consisting of reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15 mM MgCl 2), 0.2 ⁇ DTT, 40 U of RNaseOUT ® and 200 U of M-MLV (Moloney Murine Leukemia Virus Reverse Transcriptase). After incubating for 50 minutes at 37 ° C., the reaction is stopped by incubation for 15 minutes at 70 ° C. The cDNAs thus obtained can be stored at -20 ° C. or directly amplified by PCR.
• V H and V L DNAs Amplification of V H and V L DNAs by PCR.
• the PCR gene amplifications of the V H and V L DNAs are carried out using specific oligonucleotide pairs described in Table 4 (Clackson et al., 1991, Lefranc and Lefranc, 1997). On the 5 'side, the oligonucleotides hybridize to the FR1 region and to the 3' side, to the different 1 ⁇ 2 or J L segments.
• Table 5 proposes four different amplification conditions for the amplification of the heavy chain variable regions (a, b, c and d) and two different conditions for the amplification of the light chain variable regions (e and f). .
• For each of the conditions there are primer mixtures that hybridize on the 5 'side (primers back) and 3' (forward primers).
• primer mixtures that hybridize on the 5 'side (primers back) and 3' (forward primers).
• PCR reactions were carried out in a final volume of 25 ⁇ l containing 1 ⁇ l of cDNA, each primer (10 ⁇ M), each dNTP (10 nM), MgCl 2 (62.5 nM), reaction buffer, 5 U GoTaq ® DNA polymerase (Promega, Charbonippos-les-Bains, France) and VH 2 mQ 25 ⁇ qs.
• Thirty cycles of amplification (Gene Amp ® PCR-System 2700, Applied Biosystems, Courtaboeuf, France) were performed after a denaturation step of 94 ° C for 5 min. Each of the cycles included a step of 10 minutes at 72 ° C to complete the synthesis of the DNA strands.
• the amplification products were then visualized on a 1% agarose gel (QA-Agarose-TM, MP Biomedicals, France) containing 0.1% GelRed TM (FluoProbes, Montlucon, France).
• RNA extractions and three independent PCR amplifications were performed in order to perform three independent sequencing of the heavy and light chain variable regions.
• the amplification conditions chosen for each hybridoma are listed in Table 5.
• the nucleotide sequencing was performed by the platform of "Sequencing DNA and Genotyping" of the IFR 26 of the University of France, according to the method of Sanger (Sanger et al., 1977) with an AB3730 sequencer of 48 capillaries (Applied Biosystems). ). For each sequencing, 1 ⁇ l of appropriate primer back (Table 6) and 5 ⁇ l of PCR product V H OR V L were used .
• the PCR products were purified by the ExoSAP-IT process (Amersham GE Healthcare) which allows, under the action of a enzyme degrade single strand fragments less than 100 bp and eliminate nucleotide primers and excess dNTPs.
• the three independent sequencing made from three different RNA extractions enabled us to compare and determine the exact sequences of the heavy and light chain variable regions of each hybridoma.
• the nucleotide sequences obtained were then aligned with data from the International Immunogenetics Information System® (IMGT) database, http: //www.imgt.org, in order to study the repertoire of V H and V L genes used in mice. .
• IMGT International Immunogenetics Information System®
• HMEC-1 cells were transformed with SV40 virus, so as to make them more easily used in culture while allowing them to retain their microvascular endothelial cell characteristics. They were then placed under different culture conditions close to the stimuli that the endothelial cells can undergo in a tumor microenvironment, which involves the sending of chemical signals by the tumor as growth factors that will induce their proliferation.
• Figure 1A shows the HPTLC analysis of glycolipids extracted when the HMEC-1 cells were in the growth phase (lane 1), or at confluence (lane 2). The cells were also analyzed by densitometry.
• HMEC-1 endothelial cells are in the proliferating phase two or three days after seeding, or having reached confluence after five days, a difference in glycolipid expression is observed, especially for the more apolar complex gangliosides. (No. 1 and 2) but also for glycolipid Gb3 (No. 12, Fig. 1). Densitometric analysis showed that the Gb3 content is almost twice as high for proliferating cells as for confluent cells (value equal to 1.716 ⁇ 0.069, two independent analyzes). Glycolipid expression profile of HMEC-1 cells following the culture medium composition.
• FIG. 1B shows the HPTLC analysis of HMEC-1 cells which have been seeded at 20,000 cells / cm 2 and then incubated for 24 hours, as soon as the subconfluence state is reached, in depleted culture medium (lane 1) or in complete culture medium (lane 2). The cells were also analyzed by densitometry.
• Figure 1C shows the FIPTLC analysis of HMEC-1 cells that were seeded at 20,000 cells / cm 2 and cultured in complete medium, until the subconfluency state is reached, and then incubated for 24 h in depleted medium supplemented with either sphingosine-1-phosphate 1 ⁇ l (lane 2) or its diluent, PET (lane 1).
• glycolipids are derived from the aqueous phase of a partition DIPE / 1-butanol, 60:40 v / v, NaCl 0, 1% and revealed by orcinol, which revealed 14 bands of variable intensity that correspond to at least 7 molecular species.
• DIPE / 1-butanol 60:40 v / v, NaCl 0, 1%
• orcinol which revealed 14 bands of variable intensity that correspond to at least 7 molecular species.
• This glycolipid Gb3 is in particular present in the form of a doublet corresponding to two types of ceramide fatty acid chains.
• the cells In the presence of depleted media, the cells enter the GO phase of the cell cycle, which is a quiescent non-divisional stage.
• This stage of non-division is reversible because under the influence of growth factors that order the cell to divide, they can then return to the Gl phase cell cycle to continue a normal progression of the cell cycle and proliferate.
• the Gb3 content of the proliferating cells is greater than that of the quiescent cells.
• a proangiogenic factor such as sphingosine-1-phosphate
• the Gb3 content of the cells increases by 50%, whereas in complete medium it is twice as much. higher.
• the increase of the content in Gb3 more important in the presence of complete medium can be explained in particular by a synergistic action of growth factors present in the fetal calf serum of the complete medium.
• mice To generate mouse monoclonal antibodies against glycolipids of intratumoral endothelial cells, we chose to immunize Balb / c mice with endothelial cells that had previously been cultured in the presence of tumor cells in a co-culture model. mimicking the tumor microenvironment.
• HPTLC revealed the presence of at least seven more or less expressed glycolipid molecular species, including a neutral glycolipid present in the form of a doublet corresponding to glycolipid Gb3.
• ELISA glycolipid antigens
• HMVEC-L human primary lung endothelial cells were co-cultured in the presence of T84 human colonic adenocarcinoma cells through porous membranes allowing the exchange of soluble factors between the two compartments (6-well Transwell-Clear, 0.4 mm pores, Corning, the Netherlands). Once Isolated and fixed with paraformaldehyde, these endothelial cells were used to immunize five Balb / c mice.
• This co-culture system has been developed in the laboratory and has been previously described to study the influence of endothelial cell irradiation on non-irradiated T84 cells co-cultured with irradiated endothelial cells (Gaugler et al., 2007). .
• the endothelial cells were proliferating at the time when the adenocarcinoma cells were implanted three days later and were still proliferating at the time they were trypsinized and then 4% paraformaldehyde, for immunization.
• mice Five Balb / c mice were immunized with these endothelial cells co-cultured and fixed with 4% PFA. Sera were collected before each injection and the kinetics of the mouse humoral response was analyzed by desiccated ELISA on both HMVEC-L primary endothelial cells and SV40-transformed HMEC-1 endothelial cells. .
• For the anti-IgM response we used polyclonal biotinylated goat antibodies directed against the ⁇ chain of mouse antibodies and for the anti-IgG response, polyclonal biotinylated goat antibodies directed against the (H + L) antibody fragments mouse.
• the immunoglobulin isotype analysis shows that this is an early ⁇ response followed by a lower ⁇ response. Indeed, for the anti-IgM response, antibody levels are detectable from the first immunization (week 8) and for the anti-IgG response, antibody levels are detectable after the second injection (week 10) . We also observe a cross-reaction of these antibodies on HMEC-1 endothelial cells that allows us to consider the use of these SV40-transformed cells for screening studies.
• Somatic hybridization yielded 1086 hybridomas. Their supernatants were removed for screening and stored at 4 ° C and these 1086 hybridomas were taken up in SVF containing 10% dimethylsulphoxide, to be stored at -80 ° C.
• the generated hybridomas were selected using goat detection antibodies directed against both mouse IgM ( ⁇ ) and IgG (H + L) and in two approaches.
• the hybridoma supernatants were tested by ELISA on desiccated HMEC-1 cells in order to preferentially screen for antibodies directed against antigens of glycolipidic nature.
• the antibodies selected by ELISA were then screened by flow cytometry and the nature of the antigens was determined by immunolabeling glycolipids of HMEC-1 cells separated by HPTLC (iTLC).
• iTLC immunolabeling glycolipids of HMEC-1 cells separated by HPTLC
• the cell membranes are damaged and the antibodies selected by ELISA can be directed against intracellular antigens while we are looking for antigens present on the surface of the membranes.
• flow cytometry we were able to isolate 13 hybridomas and in order to determine the nature of the recognized antigen, we immunolabeled HPTLC-separated glycolipids with these hybridoma supernatants (data not shown).
• the supernatants of the 13 hybridomas recognize for 6 of them (25C10 (1), 1E10 (3), 3E2 (4), 15C1 1 (5), 16G8 (10) and 22F6 (12)), the same antigen of glycolipidic nature present in the form of a doublet that migrates above GM1, previously identified as Gb3.
• Immunolabeling shows that Gb3 is present as a doublet corresponding to two types of ceramide fatty acid chains. It is found that some antibodies recognize both bands of Gb3 (antibodies 3E2 and 15C11), others recognize rather the upper band (antibody 22F6) and others the lower band (antibodies 25C10, 1 1E10 and 16G8). It has already been shown that the composition of the ceramide fatty acid chain can influence the conformation of a glycolipid and thus modify the reactivity of the antigen for its antibody.
• the 13 selected hybridomas were cloned by limit dilution, again tested by ELISA on desiccated HMEC-1 cells, and their isotype was determined by a new ELISA using anti- ⁇ , ⁇ , y2a, y2b and ⁇ 3 antibodies. and their type of light chain with anti- ⁇ and ⁇ antibodies.
• the hybridomas selected by this first screening approach are listed in the following Table 7. This table notably presents the isotypes of each of the selected antibodies as well as the name of the corresponding monoclonal antibody. Table 7. Hybridomas selected by the first screening strategy.
• Immunolabeling on HPTLC is a sensitive detection method that can directly detect purified glycolipids extracted from about 10 6 to 30 6 6 cells while in flow cytometry, whole cells are analyzed at a rate of 0. , 4 10 6 cells. Immunostaining on HPTLC would thus make it possible to detect antibodies that bind to minority glycolipids which, due to their low content, could be undetectable by flow cytometry.
• the hybridomas were cloned by limiting dilution and isotyped by ELISA using anti-isotype detection antibodies ( ⁇ , ⁇ , y2a, y2b and ⁇ 3) and detection antibodies directed against the light chains (K and ⁇ ).
• the SDS-PAGE profile of the purified antibody 3E2 is shown in Figure 4.
• the monoclonal antibody 3E2 is pure and undenatured.
• the migration of the monoclonal antibody 3E2 on a polyacrylamide gel under denaturing conditions allows us to visualize, under reducing conditions, three bands corresponding to the heavy (H) and light (L) chains and to the characteristic J segment of ⁇ -type immunoglobulins. Due to its high molecular weight, equal to 900,000 Da, IgM can not migrate in the 6% acrylamide gel under non-reducing conditions. Analysis of the specificity of purified anti-Gb3 antibodies by affinity chromatography.
• Anti-Gb3 3E2 Antibody C. 25C10.a. 16G8.h. 14Cl l. ld and 15C7.2e.
• Fig. 5 shows the cell labeling in flow cytometry of the purified anti-Gb3 monoclonal antibodies selected by the first screening strategy (3E2.c, 25C 10.a, 16G8.h) or by the second screening strategy (HC ll. and 15C7.2e). Tagging was performed on HMEC-1 cells, Raji lymphoma cells for which Gb3 is a marker, and NXS2 cells of murine neuroblastoma that do not express Gb3, which we had previously verified with an ELISA on NXS2 cells desiccated using anti-Gb3 antibody 38.13 (data not shown).
• 3E2 antibodies. C, 25C10.a, 16G8.h and 14Cl. ld (A, B, C and D) we used secondary antibodies specific for the ⁇ chain of mouse antibodies and for the 15C7.2e (E) antibody which would be a light chain dimer we used secondary antibodies directed against IgG (H + L).
• Antibodies from the second screen appear to have a lower affinity for Gb3 than antibodies from the first screening as shown by the percentages of positive cells obtained with the 14C1 antibody. ld and 15C7.2e on HMEC-1 cells (respectively 25.3 and 11.7%) and with the 14C1 1 antibody. ld on Raji cells (10.0%).
• HMEC-1 cells When the labeling is carried out with the antibody 3E2.c, 68.2% of the HMEC-1 cells and 87.9% of the Raji cells are positive. Unlike Raji cells, the labeling of HMEC-1 cells is heterogeneous and shows that some cells strongly express Gb3 and others express it more weakly. For the 25C10.a and 16G8.h antibodies resulting from the first screening, the HMEC-1 cells are more weakly homogeneously labeled (respectively 50.7% and 15.4%) and the Raji cells are labeled at 87, 1% and 51, 1%.
• the 12E10.1c antibody of IgG1 isotype which was fixed on the Gb3 HMEC-1 cells by immunostaining on HPTLC.
• the IgG1 isotype antibodies are capable of significantly activating the complement pathway, which gives them interesting cytotoxic properties.
• the hybridoma was therefore cloned and purified and the antibodies were tested by flow cytometry on HMEC-1, NXS2 cells and Daudi cells, which are human Burkitt lymphoma cells expressing Gb3.
• HMVEC-L in proliferation that had previously been co-cultured in the presence of tumor cells through a membrane system allowing the passage and exchange of growth factors between cells, in order to mimic the tumor microenvironment. Since the immunogenic power of lipids is low, we first verified the serum kinetics of the antibody response by an ELISA test on desiccated cells, which allows antibodies to be screened preferentially. directed against desiccation-resistant antigens such as glycolipids. We obtained an antibody response against HMVEC-L primary cells and then a cross-reaction on HMEC-1 cells that allowed the use of these SV40 transformed cells as a cell model for further work.
• This co-culture model had already been developed in the laboratory (Gaugler et al., 2007) and it had previously been demonstrated in a similar co-culture system that the presence of tumor cells amplified cell proliferation and migration.
• primary endothelial cells by acquiring phenotypic and genotypic modifications (Khodarev et al., 2003).
• the ELIS A technique which uses desiccated cells, was developed in the laboratory and is based on preliminary studies with other cell types, such as the screening of monoclonal antibodies against membrane components of human mononuclear cells.
• mouse pancreatic cells have been used in a desiccated form for the detection and quantification of antibodies present in ELISA. in the serum of insulin-dependent diabetics.
• our antibody generation strategy we chose to immunize Balb / c mice with whole endothelial cells. Many anti-glycolipid antibodies have already been generated as a result of the immunization of whole tumor cells and in this way new molecular species of glycolipidic nature have been identified.
• HMVEC-L cells fixation of HMVEC-L cells with 4% paraformaldehyde was chosen to screen hybridoma supernatants on cells treated under the same conditions as those used for immunization, but in this way also The plasma membrane of HMVEC-L cells could be stabilized and the cells for the different immunizations during the 65 weeks could be from the same co-culture.
• the glycolipids being weakly immunogenic, they can be assimilated to molecules such as haptens incapable of triggering an immune reaction by themselves. To make them more immunogenic, they can be coupled to a "carrier" protein molecule whose role will be to expose, more importantly, the hapten molecule.
• the somatic hybridization made it possible, from the 1086 hybridomas obtained, to preselect the first 139 hybridomas by ELISA on dried cells, which were then screened according to two strategies.
• the first which was to screen hybridomas on living cells by flow cytometry, allowed 13 of them to be selected and by HPTLC immunolabeling of glycolipid extracts from cells, we determined that 6 of them recognized the glycolipid Gb3.
• a second screening strategy which consisted of screening by immunostaining the 139 hybridomas obtained by ELISA, made it possible to select many other antibodies, mostly directed against Gb3 but also against glycolipids being characterized. After purification, it was found that the antibodies generated by the second screen had a lower affinity.
• monoclonal antibodies IgM, ⁇ directed against Gb3: monoclonal antibodies 25C10 and 16G8 which, by immunostaining on HPTLC, instead recognize the lower band of the doublet Gb3 ; which is shown in a minority manner in HMEC-1 cells, and the monoclonal antibody 3E2 which has the best immunostaining binding profile on HPTLC and in flow cytometry.
• Gb3 is a globotriaosylceramide, also called Gb3 / CD77, or CTH (ceramide trihexoside). He first identified as an antigen of a rare blood group, the Pk group on the surface of erythrocytes and is also known as a marker of Burkitt lymphoma (BLA, Burkitt Lymphoma Antigen). On the surface of endothelial cells, its expression is regulated by pro-inflammatory cytokines such as T F- ⁇ involved in tumorigenesis processes.
• BLA Burkitt Lymphoma Antigen
• this glycolipid is known as a specific receptor for bacterial toxins and Preliminary studies have shown that verotoxin binding to Gb3 could inhibit in vitro angiogenesis (Heath-Engel and Lingwood, 2003). These results support us in the interest of developing therapeutic antibodies targeting Gb3, especially since the content of this glycolipid seems to be modulated when the endothelial cells are proliferating (see Example 2). In addition, the vast majority of anti-glycolipid antibodies generated as a result of somatic hybridization were directed against Gb3, suggesting that this glycolipid is quite immunogenic in Balb / c mice.
• the affinity of the antibody 3E2 was evaluated at 30 nM.
• the number of Gb3 sites is of the order of 2 ⁇ 6 sites for both cell types but there are slightly more sites on Raji cells than on HMEC-1 cells.
• flow cytometry FIGG 9, Example 3
• the distribution of Gb3 was homogeneous for Raji cells for which 87.9% of the cells are positive, and heterogeneous for the HMEC-1 cells for which 68 2% of the cells are positive. Indeed, in the population of HMEC-1 cells, some cells weakly express Gb3 while others express it more strongly, unlike Raji cells.
• mAb 3E2 was evaluated by an ELISA test on Raji, HMEC-1 desiccated cells and IMR32 cells of human neuroblastoma that do not express Gb3, using an anti-detection antibody.
• Mouse ⁇ chain (Fig. 6).
• the 3E2 antibody does not bind to the IMR32 cells. It binds to Gb3-positive HMEC-1 and Raji cells but their ELISA binding profile is different (A). Indeed, the antibody binds more weakly on HMEC-1 cells. This difference could be explained by the average number of Gb3 sites which is slightly larger for Raji cells. Moreover, in the population of HMEC-1 cells, it is observed by flow cytometry that there is a heterogeneity of labeling with the 3E2 antibody and that some cells express Gb3 more weakly (Fig. 5, Example 3). It could also be envisaged that the Gb3 of HMEC-1 cells is less well recognized when it is present in a desiccated form, since it is recognized that the conformation of a glycolipid can influence its recognition by an antibody.
• the binding of the antibody 3E2 is revealed by the successive incubation of a biotinylated anti-mouse secondary antibody followed by the incubation of a streptavidin-peroxidase complex, whereas that of the antibody 38.13 is revealed by the incubation of a secondary antibody anti- ⁇ rat directly coupled to peroxidase.
• Antibody 38.13 recognizes Gb3 of HMEC-1 cells as a doublet (lane 3), whereas the 3E2 antibody recognizes only the upper lane (lane 4).
• the antibodies that is to say before cloning the hybridomas, it is found that when immunolabeling with the supernatant of the antibody 3E2, it also recognized the Gb3 in the form a doublet (track 5). It is thus assumed that cloning has made it possible to select, in a mixture of several hybridomas of different specificities, a monoclonal antibody which binds only to the upper band of the Gb3 doublet.
• the mouse monoclonal antibodies 3E2 and 1A4 have different chromatographic binding profiles: the 3E2 antibody recognizes the Gb3 upper band of the HMEC-1 cells whereas the 1A4 antibody recognizes the Gb3 in the form of a doublet (B) . In contrast, both the 3E2 and the A4 antibodies recognize the porcine source purified Gb3 (Matreya) as a doublet. Moreover, it is found that the binding of the antibody 3E2 is weaker than that of the antibody A4, probably because the antibody 1A4 has a better affinity for Gb3 than the antibody 3E2. Immunofluorescence cell staining study in flow cytometry.
• Table 10 Mean fluorescence intensity after analysis in flow cytometry.
• the binding profile of the 3E2 antibody is different from that of the 1A4 antibody: 68.2% of the cells are positive with the 3E2 antibody (A) and 90.8% of the cells are positive with the antibody 1A4 (B).
• the average fluorescence intensity value is 568.3 for the 3E2 antibody and 984.2 for the 1A4 antibody (Table 10).
• Raji cells The labeling of Raji cells is homogeneous and the percentage of positive cells is comparable between the two antibodies (87.9% of positive cells and 423.8 of average fluorescence intensity for antibody 3E2, and 93.6% positive cells and 498, 1 mean fluorescence intensity value for the antibody 1A4).
• Raji cells strongly express Gb3 homogeneously within the cell population and both antibodies seem to recognize the glycolipid in comparable affinities.
• HMEC-1 cells the upper band of Gb3 is major.
• the antibody 3E2 preferentially recognizes this form with respect to the antibodies 38, 13 and 1 A4 which recognize Gb3 as a doublet (Fig. 10).
• flow cytometry after labeling the HMEC-1 cells with the antibody 3E2 (Fig. 10, A), the presence of a predominantly less marked population was observed.
• the molecular species corresponding to the upper band of Gb3 is found on a high proportion of cells that express it more weakly than a more marked minority population.
• flow cytometry after labeling HMEC-1 cells with the 1A4 antibody (Fig. 10, B), the presence of a predominantly more marked population was observed. There would then be a small proportion of HMEC-1 cells that strongly express the lower form of Gb3 or both forms undifferentiated.
• the 3E2 antibody does not recognize non-co-cultured HMVEC-L cells (3.4% positive cells). This lack of fixation is due to an overall decrease in the Gb3 doublet observed by HPTLC, because even if the overall Gb3 content is low, the upper band remains predominant in HMVEC-L cells. Since the 1A4 antibody binds to the Gb3 of HMVEC-L cells (26.1% positive cells) and since the upper band is predominant, the difference in binding is due to a difference in affinity between the two antibodies. The affinity of the 1A4 antibody for Gb3 seems superior to that of the 3E2 antibody, since the low Gb3 content that can be visualized in HPTLC can be detected by flow cytometry only with the 1A4 antibody. . In HUVEC cells, almost no Gb3 is detected by HPTLC. In flow cytometry, only 7.5% of the cells are positive with the A4 antibody instead of 2.5% positive cells with the 3E2 antibody.
• HPTLC analysis shows that the overall content of neutral glycolipids is limited in HUVEC cells (lane 6).
• HMVEC-L cells it is the proportion of Gb3 within the total neutral glycolipid fraction which is limited (lane 5) since these cells strongly express a glycolipid present in the form of a doublet probably corresponding to glycolipid Gb4.
• HMEC-1 it is the lower band of Gb4 which is maj oritaire.
• Primary cells have higher levels of complex gangliosides migrating below GDi a .
• ganglioside migrating above GM1 would be the most abundant of the total ganglioside fraction. Since the preponderance of GM 3 in the total ganglioside fraction has already been demonstrated in human endothelial cells (Obrig et al., 1993, Muthing et al., 1999, Kanda et al., 2004), it is likely that this predominant ganglioside HMEC-1, HMVEC-L and HUVEC cells are GM 3 .
• the 3E2 antibody is an immunohistochemical tool that seems to meet expectations. It recognizes the presence of Gb3 which is localized at the membrane level of Gb3-positive tumors (Raji) and shows no non-specific binding on non-expressing tumors (IMR32).
• the antiparasitic antibody is also suitable for immunohistochemical studies because it does not exhibit specificity.
• RT-PCR the nucleotide sequences of the variable regions VH and VL in order to align them with the data of the IMGT (International Immunogenetics Information System®) bank.
• the primers used for heavy and light chain sequencing are listed in Table 12.
• the nucleotide sequences of the heavy and light chains are shown in FIG. 13 and the genes used for these variable regions are listed in Table 13.
• Monoclonal antibody 3E2 has more than 99% homology with the germ-line IGHV4 gene (genomic DNA). There are two nucleotides that are mutated in the FR1 region. The antibody has 65.96% homology with the IGHJ2 gene and 78.57% with the IGHD1 gene. The VK of the antibody has a homology of 90.91% on 220 nucleotides with the IGKV14 gene and has 100% homology with the IGKJ5 gene. Due to the differences observed with the germinal configuration genes, it is thus demonstrated that the antibody has been subjected to a maturation process revealed by several somatic mutations. 4.2. Conclusion
• hybridoma lines were obtained by somatic hybridization, including the antibody 3E2 (IgM, ⁇ ) directed against the neutral glycolipid Gb3.
• IgM antibody 3E2
• Gb3 is a marker
• IMR32 human and murine NXS2 neuroblastoma cells that do not express Gb3, and primary endothelial cells
• HMVEC- microvascular lung cells L
• macrovascular umbilical cord cells HUVEC macrovascular umbilical cord cells
• the antibody has a good affinity of the order of 30 nM.
• the membrane localization of Gb3 was demonstrated by immunocytochemistry, followed by immunohistochemistry, its ability to specifically bind to Gb3 on sections of frozen Raji tumors.
• the 3E2 antibody thus constitutes a new tool for analyzing Gb3, in addition to the antibodies that have already been generated previously, such as the rat IgM antibody 38.13 and the mouse IgM antibody 1A4 that were obtained in the team. Pr. S. Hakomori.
• Gb3 was used to establish Gb3 as a marker of Burkitt's lymphoma, to show that Gb3 was involved in the apopototic phenomena of Gb3-positive lymphoma B cells and that Gb3 targeting by Gb3 antibodies (Taga et al 1997, Tetaud et al., 2003) or recombinant verotoxin B subunit (Mangeney et al., 1993) could also induce an apoptotic response.
• a third antibody, BGR-23 obtained more recently (Kotani et al., 1994) was used to study the tissue distribution of Gb3 in Fabry disease (Askari et al., 2007).
• These antibodies are specific for the Gakx1 ⁇ 4Gai terminal motif and can not recognize (1) the Gakx1 ⁇ 3Gai motif present at the terminal end of the oligosaccharide chain of isoglobotriosylceramide (iGb3) and (2) the Galal ⁇ 4Gai motif present at the of the oligosaccharide chain of the Gb 4 globoside (GalNac 1 ⁇ 3Gakx1 ⁇ 4Gal 1 ⁇ 4Glc 1 ⁇ Cer).
• the 3E2 antibody recognized on HPTLC the band of the Gb3 doublet of HMEC-1 cells and the porcine Gb3 as a doublet, unlike the anti-Gb3 antibodies of rat 38. 13 and mouse A4, which recognized the two molecular species of Gb3 cells HMEC-1 and porcine Gb3.
• the molecular species corresponding to this upper band is expressed heterogeneously on HMEC-1 cells.
• Antibodies 3E2 and 1A4 equally recognized Gb3 of Raji cells, which express on their surface the superior form in a largely major and homogeneous manner because all the cells are marked extensively. Moreover, in an in vitro model, it has been very clearly shown that incorporation of GDi a into HUVEC cells makes these cells more sensitive to low levels of VEGF. They thus propose that, in the tumor microenvironment, glycolipids of tumor cells released and incorporated into the endothelial cells by a shedding phenomenon, would promote tumor progression. Since T84 cells do not express Gb3, the antibodies generated as a result of the immunization were directed against Gb3 expressed by HMVEC-L endothelial cells derived from co-culture.
• HBMECs Human microvascular brain endothelial cells express 2 times more Gb3 than umbilical cord endothelial cells (HUVEC) (612 ⁇ 185 and 269 ⁇ 62 ng / mg protein) (Kanda et al., 2004).
• Tumor transformation may also influence glycolipid content, as evidenced by the abnormal overexpression of GD 2 disialoganglioside acid in neuroblastomas, most melanomas and some other tumors, or the overexpression of Gb3 on several tumor cell lines such as lymphoma. Burkitt (Wiels et al., 1981), breast cancer cells, ovarian cancer cells astrocytic tumors (Gariepy 2001, LaCasse et al., 1999), epithelial carcinoma cells of the upper digestive tract (Marques Filho et al., 2006) or in human tumor tissues such as breast cancer (Johansson et al., 2009). ), colorectal tumors or metastases (F aiguines et al., 2008).
• the biochemical changes observed during the malignant transformation of cells also affect the biosynthesis of ceramide. These modifications are manifested as well by variations in the length of the aliphatic chain, the number of substitutions or the degree of unsaturation. If in healthy tissues, the chains of fatty acids are shorter (C14: 0 to C18: 0), gangliosides of tumor cells have longer chains of fatty acids (C22: 0, C22: 1 and C24: 1 ) (Hakomori and Kannagi, 1983). Aberrant ⁇ -hydroxylation of the fatty acids of tumor gangliosides can also be observed.
• the porcine Gb3 obtained from Matreya consists of a mixture of two types of ceramide chains (C: 16 and C: 20) which are composed of 70% saturated fatty acids and 30% unsaturated fatty acids. .
• M immunoglobulins would mimic the pentameric structure of these toxins and collect naturally occurring glycolipids in the form of rafts lipid levels, based on synthetic plasma membrane models. These data notably explain why an IgG1 isotype antibody such as the 12E10.1c antibody selected by the second screening strategy had a bad affinity for Gb3, as shown by the weak binding of this antibody to Gb3 of the HMEC-cells. 1.
• Verotoxin binding also depends on the length of the fatty acid chain of Gb3 ceramide. Glycolipids with medium or long fatty acid chains (Cl 6 and C24) are preferentially recognized, while short chains (Cl 2 and Cl 4) have minimal binding. C20: 0 and C22: 1 fatty acid chains have greater binding capacity and the presence of unsaturated fatty acids significantly increases verotoxin binding. Hydroxylation of fatty acids also increases binding. The binding of the verotoxin to the terminal Gal (al-4) Gal motif of glycolipids thus depends on the entire structure of the molecule and its molecular environment at the level of the plasma membrane of the target cells. These results highlight that glycolipids are involved in a cellular tropism closely related to their structure.
• the antibody 3E2 thus targets an original glycolipid present on the endothelial cells. It would be important to determine the structure of the two molecular species of Gb3 present in the HMEC-1 and HMVEC-L cells, in order to highlight the structural variations that are responsible for the differential specificity of the 3E2 and 1 A4 antibodies.
• Gb3 expression was heterogeneous in HMEC-1 cells and appeared to be modulated according to the state of proliferation of these cells. In particular, it should be investigated whether these results can be corroborated with biological activity, in order to determine the therapeutic potentialities of the antibody.
• Table 15 Percentage of positive cells and average fluorescence intensity of the cells after flow cytometry analysis.
• the Gb3 content at the surface of HMEC-1 cells increases slightly. In flow cytometry, this content is detectable only after 72 hours whereas this increase was visualized as early as 24 hours in HPTLC (FIG. The cells are positive at 42.0 + 0.3% after incubation with S1P, whereas only 24.4 + 1.6% are positive in the presence of depleted medium supplemented with PET diluent.
• the increase in Gb3 content is greater (Fig. 14).
• the fetal calf serum present in the complete medium comprises a mixture of growth factors and this increase may be due to a synergistic action of the growth factors.
• the inhibition of cell viability induced by the antibodies 3E2 and 1A4 is comparable and leads to a saturation plateau of 20 to 40 ⁇ g / ml of antibody, for which values equal to 14.6 ⁇ 1.6% and 16.3 ⁇ 3, 1% inhibition.
• No inhibition of cell viability is observed with the isotypic control antibody on these HMEC-1 cells.
• This inhibition is more important for Raji cells (B) which have more Gb3 sites (2.0 10 6 sites against 1.7 10 6 sites for HMEC-1 cells) and which express Gb3 homogeneously. Within the HMEC-1 cell population, some cells express Gb3 more weakly, which may explain why the inhibition of viability is lower. No inhibition is observed for NXS2 cells that do not express Gb3. Inhibition of cell viability is therefore dependent on Gb3.
• Fig. 18 represents the number of HMEC-1 cells as a function of the incubation time with 20 ⁇ g / ml of 3E2 antibody or isotype control antibody. It is observed that there are fewer HMEC-1 cells in the presence of the 3E2 antibody at 24 h of incubation. This decrease is significant from 6 hours of incubation (A). After 24 hours, there is a decrease in the number of cells that can be evaluated at about 18%, which is close to the 14.6% inhibition of cell viability that was observed at 24 hours in the morning. MTT (Fig. 16).
• Figure 19 shows the proportion of cells that enter cell division per time interval (A) and their division time during these 24 hours of cumulative incubation (B).
• the dividing cells can be detected and counted: the cells lose their adhesion, become detached from the support, become more refractive and adopt a circular morphology whereas they exhibit cytoplasmic expansions when they are adherent. It is thus possible to identify dividing cells and also their dividing time until both daughter cells adhere back to the support.
• the 3E2 antibody exhibits cytostatic activity: it induces an inhibition of cell viability at 24 h, a decrease in the number of cell divisions with an extension of the dividing time. It also induces inhibition of vascular bud formation on explants of aortic cross sections in culture. These observations were obtained in the absence of complement.
• CDC complement dependent cytotoxicity
• Fig. 21 presents the CDC activity of the antibodies 3E2, 1A4 and the isotype control (10 ⁇ g / ml of antibody) on the FDVIEC-1, Raji and NXS2 cells, in the presence of decomplemented human serum and not decomplemented.
• the 3 ⁇ 2 antibody exhibits CDC activity on HMEC-1 cells (Table 17, 51.3 ⁇ 9.2% lysed cells) and Raji cells (71.8 ⁇ 5.5%). lysed cells). The activity can be detected at low concentrations of the order of 0.5 ⁇ ⁇ of antibodies (20.6 ⁇ 3.2 of lysed HMEC-1 cells and 36.4 ⁇ 6.8% of lysed Raji cells). ).
• the 1A4 antibody also has a significant CDC activity at 10 ⁇ g / ml (65.2 ⁇ 13.2% of HMEC-1 cells positive and 82.9 ⁇ 7.7% of Raji positive cells). The CDC activity can be detected as soon as 0.1 ⁇ g / ml of antibody (37.2 ⁇ 12.6% of HMEC-1 positive cells and 25.8 ⁇ 3.9% of Raji positive cells).
• NXS2 cells which do not express Gb3, are not lysed in the presence of the 3E2 and 1 A4 antibodies and the HMEC-1 and Raji cells, which express Gb3, are not lysed in the presence of isotype control: thus the observed cytotoxic activity is clearly dependent on Gb3.
• the NXS2, HMEC-1 and Raji cells are not lysed in the presence of non-decomplemented serum, so there is no activation of the alternative complement pathway which plays a role. important in innate nonspecific immune defenses.
• HMEC-1 and Raji cells are not lysed in the presence of the antibodies A4 or 3E2 when they are incubated in the presence of decomplemented serum or in the absence of human serum: they do not observe apoptosis-type cell death or necrosis.
• the cytolytic activity observed is therefore very dependent on complement activation, via the binding of antibodies to Gb3.
• Antibodies act primarily in three ways that can be cumulative. By direct fixation, they can induce cytostatic activities (cell cycle blocking type) or cytotoxic activities (apoptosis type, necrosis). By CDC, they can induce the activation of complement proteins by binding of the Clq protein complex to the Fc region of antibody bound to their target, resulting in the formation of the membrane attack complex and cell lysis. By ADCC, the Fc ⁇ R receptors bind to the Fc region of antibody bound to their target thereby leading to lysis or phagocytosis induced by an effector cell of the immune system.
• the various biological tests used show that the antibody 3E2 initially has a complement-independent cytostatic activity.
• MTT is observed to inhibit the cell viability of FDVIEC-1 cells equal to 14.6 ⁇ 1.6% for 20 ⁇ g / ml of antibody as early as 24 hours (Fig. 16). This result is confirmed by a video-kinetic study of HMEC-1 cells.
• FIG 18 When the cells are incubated with 20 ⁇ g / ml of antibody, there is a decrease in the overall number of cells close to the inhibition value obtained in MTT (FIG 18). There are fewer cells dividing and their dividing time is generally longer.
• the cell viability inhibition of HMEC-1 cells is 22.2 ⁇ 9.1%.
• mitotic aberrations In the presence of the antibody 3E2, there is also an increase in the frequency of mitotic aberrations associated with the presence of cells blocked in division for several hours and which do not break out, as well as the presence of cells that can not enter in division and which alternate repeated cycles of cellular attachment and detachment.
• mitotic aberrations can be attributed to death mechanisms involving adhesion, such as death by anoikis, as has already been demonstrated for an antibody targeting GD 2 ganglioside in small cell lung cancers.
• Anoikis is a phenomenon of death that was first identified in 1994 and characterizes the loss of contact of cells with their extracellular matrix.
• an anti-GD 2 antibody induces apoptosis via the ubiquitous cytoplasmic protein FAK (Focal Adhesion Kinase) found within the adhesion complex and which can be activated by integrins but also by different growth factors. cytokines or hormones.
• FAK Fluor Adhesion Kinase
• Gb3 it has already been shown for microvascular endothelial cells from patients with Fabry disease, that Gb3 present in large amounts was associated with an increased expression of molecules involved in cell adhesion such as ICAM proteins. -1, VCAM-1 and selectin E. Zemunic et al. (2004) compared the distribution of Gb3 with that of E-selectin in H FUVEC cells stimulated by T F- ⁇ .
• Gb3 may have a potential role in adhesion mechanisms within the endothelium (Zemunik et al., 2004).
• the migration of the cells requires the establishment of a finely regulated system of organization of the cytoskeleton and adhesion complexes.
• treatment with IFN- ⁇ increases the relative proportion of intracellular Gb4 that is associated with the cytoskeleton of the cell.
• the specific modulation of glycosphingolipids by IFN- ⁇ suggests that they may play a role in the adhesion mechanisms of activated endothelial cells.
• the most abundant glycolipids HUVEC, the Gb4 and GM 3 are located at the surface and intracellularly where they are associated with the intermediate filament vimentin cytoskeleton could play a role in the transport of glycosphingolipids.
• the strong membrane expression of an antigen can make it possible to obtain a high density of antigen-antibody complexes at the membrane, favoring the recruitment of the effectors of the antigen. immunity, and in particular complement.
• the classical pathway is activated by the antigen-antibody complex and among immunoglobulins, IgM are effective activators of complement-dependent cytotoxicity.
• the 3E2 antibody is capable of activating CDC as early as 0.5 ⁇ g / ml on HMEC-1 cells (20.6 +/- 3.2% of lysed cells) which have 1.7-10 6 Gb3 sites and 0.1 g / ml on Raji cells (15.7 ⁇ 5.2%) which have 2.0 June 10 sites.
• the CDC activity is observed as soon as 0.1 g / l on the HMEC-1 (22.2 ⁇ 6.9%) and Raji (25.8 ⁇ 3.9%) cells. Both antibodies are thus able to activate the complement pathway for weak antibody concentrations. This complement-dependent activity allows us to consider future preclinical studies in mice.
• 25C10, 11E10, 16G8) were obtained from a single somatic hybridization and a single cloning. They are all IgM isotype and all have a ⁇ light chain.
• the nucleotide sequences of the heavy chains of the seven anti-Gb3 antibodies are shown in FIG. 22.
• the seven Gp3 glycolipid-specific antibodies each use a different V gene with a germline homology degree of greater than 98% except for the 15C1 1 and 25C10 MABs which are respectively distinguishable. by 96.8% and 93, 1% homology.
• mAb 14C1 1 a homology of the V gene identical to the IGHV2-6-7 * 01 or 02 allele.
• mAb 15C11 which has 100% homology with the IGHJ3 allele.
• * 01 the other 6 mAbs all use the same gene J (IGHJ2 * 01) which combines with a D gene, different products VH sequences, different for each hybridoma.
• the length of the hypervariable region CDR3H is between 5 and 12 amino acids.
• the nucleotide sequences of the heavy chains of the seven anti-Gb35 antibodies are shown in FIG. 23.
• the genes used for the variable regions of the light chains are listed in the following table.
• the MAb 3E2 is encoded by the same J gene (IGKJ5 * 01) as the 14C 11 and 22F6 mAbs with the same degree of homology but with a different amino acid junction expression. one hybridoma to another.
• the seven anti-Gb3 antibodies were generated in a single somatic hybridization and the similarities observed in the heavy and light chains suggest that the antibodies are cloned.
• Our results indicate that Variety of V H and V L chains can encode for anti-Gb3 antibodies and some are subject to a maturation process revealed by several somatic mutations.
• the set of nucleotide data collected for the expression of the seven specific Gb3 mAbs confirms after cloning of the hybridomas the existence of distinct combinations of the VDJ and VJ genes for the expression of the mAbs without apparently any restriction.
• V H and V L regions of mAb are essential for designing and optimizing for human therapeutic purposes chimeric or even humanized antibodies failing to obtain human antibodies.
• This approach requires at least a three-dimensional modeling of the variable regions V H and V L of the antibody adapted to the specific recognition of Gb3 to substitute the hypervariable regions of a human antibody by those of the mAb 3E2 that we have defined as the leader of the seven antibodies that we have characterized with respect to glycolipid Gb3.
• EXAMPLE 7 In Vivo Confirmation of the Anti-Angiogenic Properties of the 3E2 Antibody in Two Tumor Models
• the therapeutic potential of the 3E2 antibody in the treatment of solid tumors was confirmed in vivo in two tumor models based on the murine neuroblastoma NXS2 line, and compared to that of an anti-GD2 antibody.
• NXS2 neuroblastoma
• the GD2 being another glycolipid which is, unlike Gb3, expressed by the NXS2 tumor cells and not expressed by the cells of the blood vessels.
• mice Male A / J mice (6-8 weeks old) were obtained from Harlan Sprague-Dawley (Sulzfeld, Germany). Animal studies were carried out in accordance with Directive 86/609 / EEC.
• mice were treated with intravenous injections of monoclonal antibodies at day 1, 2, 7 and 8 for the antibody 3E2 (IgM, 200 ⁇ g in 150 ⁇ l of PBS), or at days 1 and 2 for the antibody 14G2a ( IgG, 100 ⁇ g in 100 ⁇ l) or IgG isotype control antibody (100 ⁇ g in 100 ⁇ l), respectively.
• IgM antibody 3E2
• IgG antibody 14G2a
• IgG IgG isotype control antibody
• the molar amounts of antibody of each injection are similar for all antibodies, the larger mass used for the antibody 3E2 (IgM) relative to the other antibodies (IgG) being due to the higher molecular weight of IgM compared to IgG. .
• the new 3E2 antibody injections at days 7 and 8 are necessitated by the limited half-life of IgM in vivo (approximately 1 week), which is not the case for the other antibodies. isotype IgG, IgG having an in vivo half-life of about 3 weeks.
• mice received only PBS. The animals were sacrificed at day 28, their liver was weighed and the number of metastases evaluated.
• Frozen tumors were cut and fixed with cold acetone for 10 minutes.
• the samples were labeled for 90 minutes with the following monoclonal antibodies: a rat antibody against CD31 Murin (1: 50, Millipore, Molsheim, France) and a biotinylated antibody 3E2, 14G2a (anti-GD2) or its isotype control (Beckmann Coulter, Fullerton, CA, USA) (40 ⁇ g / ml).
• Antibody 3E2 and isotype control were biotinylated with an EZ-Link Sulfo-HS-LC-Biotinylation kit (Thermo Scientific, Courtaboeuf, France).
• the labeling was revealed by 90 minutes of incubation with a goat anti-rat antibody conjugated with AF 488 (1: 400, Invitrogen) for CD31 and with streptavidin conjugated with AF 568 (1: 200, Invitrogen) for the 3E2 antibody or isotype control thereof.
• the samples were finally coated with a ProLong gold antifade reagent with DAPI. Healthy animal organs have also been labeled with the 3E2 antibody or its isotype control using the same protocol. The markings were observed under a confocal fluorescence microscope (Nikon, Champigny sur Marne, France). Muscle sections were used as a normal tissue control.
• Tumor volumes ranged from 120 to 130 mm 3 and did not differ significantly upon injection of treatment (3E2, 14G2a, IgM control, or PB S).
• FIG. 24 The evolution of the tumor volume after treatment is shown in FIG. 24.
• the antitumor efficacy of the 3E2 antibody was then determined in the model of experimental liver metastases of mouse neuroblastoma NSX2 developed by Lode et al.
• the 3E2 antibody specifically targets the vessels within the tumor mass by histology and immunohistochemical staining.
• the results are shown in Table 21 below, and show that the antibody 3E2 generates an intense colocalized labeling with that of the anti-CD31 antibody (marker cells of the blood vessels), and no markings in the tumor mass.
• the muscle sections showed no Gb3 staining, only the endothelial cells within the tumor vessels being labeled with the 3E2 antibody.
• NXS2 tumor cells showed a high level of GD2.
• Table 21 Distribution of Gb3 within the tumor mass of NXS2 metastases.
• mice with 3E2 and 14G2a antibodies were highly effective in reducing hepatic neuroblastoma metastases, as indicated by decreased liver weight from 39 ⁇ 6.23 g (PBS-treated mice) to 17 ⁇ 5.03 g (3E2 antibody treated mice) and 6.75 ⁇ 2.06 g (14G2a antibody treated mice) (p> 0.05). These last two values are not significantly different from those found in healthy control animals (p> 0.1). The effect of treatment with monoclonal antibody 3E2 is not significantly different from treatment with monoclonal antibody 14G2a (p> 0.5). These data further confirm the specificity of 3E2 antibody therapy, since treatment with a control isotype antibody is completely ineffective.
• the 3E2 antibody is an IgM, whose distribution is limited to the intravascular compartment and because it recognizes blood vessels in solid tumors but not in healthy tissues, the results suggest that its therapeutic effects are related to its action. on these blood vessels.
• the 3E2 antibody injection is also effective for inhibiting subcutaneous NXS2 neuroblastoma tumors in the A / J mouse.

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