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WO2004020574A2 - Fragment scfv en rapport avec la cathepsine l humaine et leurs procedes d'utilisation - Google Patents

Fragment scfv en rapport avec la cathepsine l humaine et leurs procedes d'utilisation Download PDF

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WO2004020574A2
WO2004020574A2 PCT/IB2003/003581 IB0303581W WO2004020574A2 WO 2004020574 A2 WO2004020574 A2 WO 2004020574A2 IB 0303581 W IB0303581 W IB 0303581W WO 2004020574 A2 WO2004020574 A2 WO 2004020574A2
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seq
antibody
fragment
cathepsin
sequence
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Raymond Frade
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to the field of molecular biology, more precisely molecular immunology. More particularly, the invention relates to the cloning and characterization of a monoclonal antibody, named Mab 3D8, specifically recognizing active human pre-pro-cathepsin L, pro-cathepsin , and cathepsin .
  • Mab 3D8 specifically recognizing active human pre-pro-cathepsin L, pro-cathepsin , and cathepsin .
  • the inventors have determined the amino acid sequences of the H chain variable region and L chain variable region of Mab 3D8. Based on the determined amino acid sequences, recombinant antibodies against human Cathepsin and a method for producing the same can be envisionned.
  • Cathepsin L is a papain-type cysteine proteinase [Ishidoh et al . (1987) FEBS Lett. 223, 69-73 - Turk, B., Turk, D. and Turk, V. (2000) Biochem. Biophys . Acta 1477, 98-111], whose gene is localized on human chromosome 9q21 -22 [Chauhan et al . (1993) J. Biol. Chem. 268, 1039-1045].
  • Cathepsin L is successively translated as pre-pro-cathepsin L, transferred through the golgi apparatus as pro-cathepsin L and then stored in lysosomes as mature cathepsin L [Ishidoh et al . (1998) L.
  • Cathepsin L whose overexpression was demonstrated in transformed cells [Gottesmann et al .
  • Cathepsin L was also involved in tumor progression [Rozhin et al.(1989) Biochem. Biophys . Res. Commun. 164, 556-561 , Maciewicz et al . (1989) Int. J. Cancer 43, 478-486] .
  • scFV preferably chimerized or humanized, which recognizes with a high specificity and affinity pre-procathepsin L, pro- cathepsin L and/or cathepsin L.
  • mAb monoclonal antibody
  • This monoclonal antibody was used to generate a scFv, whose nucleotide and amino acid sequences were determined. The binding properties of this scFv was analyzed on recombinant and native cathepsin L. Then, this scFv was expressed in human melanoma. Indeed, the inventors demonstrate that expression of anti-cathepsin L scFv in human melanoma cells inhibits pro-cathepsin L secretion.
  • the present invention provides an isolated and/or purified antibody, or an isolated functional fragment thereof, that binds to cathepsin L and pro-cathepsin L, and eventually to pre-pro-cathepsin L, and that is optionally and preferably able to inhibit cellular pro-cathepsin L secretion, wherein said antibody, or fragment thereof comprises: a variable region of the light chain (VL) that comprises at least one CDR selected in the group consisting of the following amino acid sequences: o L-CDR 1 of sequence SER-SER-VAL-ASN-TYR (SEQ ID)
  • LEU (SEQ ID N°8) , or an amino acid sequences having at least 80% identity after optimal alignment with SEQ ID N°6, with sequence SER- THR-SER or SEQ ID N°8; and/or:
  • variable region of the heavy chain comprises at least one CDR selected in the group consisting of the following amino acid sequences: o H-CDR 1 of sequence GLY-TYR-THR-PHE-THR-SER-TYR-TRP (SEQ ID N°10) , o H-CDR 2 of sequence ILE-TYR-PRO-GLY-SER-GLY-ASN-ILE
  • the isolated antibody of the invention or a fragment thereof comprises : a variable region of the light chain (VL) that comprises at least one CDR selected in the group consisting of the following amino acid sequences: o L-CDR 1 of sequence SER-SER-VAL-ASn-TYR (SEQ ID N°6), o L-CDR 2 of sequence SER-THR-SER, o L-CDR 3 of sequence Gln-Gln-ARG-SER-SER-TYR-PRO-LEU (SEQ ID N°8) , or an amino acid sequences having at least 80% identity after optimal alignment with SEQ ID N°6, with sequence SER- THR-SER or SEQ ID N°8; and:
  • variable region of the heavy chain comprises at least one CDR selected in the group consisting of the following amino acid sequences: o H-CDR 1 of sequence GLY-TYR-THR-PHE-THR-SER-TYR-TRP
  • antibody refers to single chain, two- chain, and multi -chain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric, humanized and hetero immunoglobulins (monoclonal antibodies being preferred) ; it also includes synthetic and genetically engineered variants of these immunoglobulins .
  • the antibody of the invention preferably has the structure of a natural antibody or a fragment thereof. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond.
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL) .
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (Cnl, CH2 and CH3 , collectively referred to as CH) .
  • variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors.
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin consisting of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs) .
  • the CDR refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • FR refers to amino acid sequences interposed between CDRs.
  • the term antibody encompasses all the proteins comprising at least one of the sequence of the L-CDR1 (SEQ ID N° 6) , L-CDR2 (SER-THR-SER) , L- CDR3 (SEQ ID N°8), H-CDR1 (SEQ ID N°10), H-CDR2 (SEQ ID N°12), H-CDR3 (ARG) , the variable region of the light chain (VL) (SEQ ID N°2) and the variable region of the heavy chain (VH) (SEQ ID N°4) that are able to specifically binds to cathepsin L and/or pro-cathepsin L and/or pre-pro-cathepsin L.
  • the antibody of the invention as described above may be subjected to post-translational or post-synthetic modifications as a result of thermal treatment, chemical treatment (formaldehyde, glutaraldehyde etc.) or enzyme treatment
  • glycosylations include for example, glycosylations , acetylations, phosphorylations and the like.
  • glycosylation is often achieved when the polypeptide is expressed by a cell of a higher organism such as yeast or preferably a mammal. Glycosylation is normally found in connection with amino acid residues Asn, Ser, Thr or hydroxylysine .
  • modifications of the antibody of the invention may be useful for example to enhance the half-life of this polypeptide in an organism, or in cells, to enhance the solubility of this polypeptide, or to facilitate the purification of the protein fusion of the invention (e.g. tagging) .
  • antibody with substituted linkages for example covalent linkage of the heavy and the light chain of the immunoglobulin by a heterobifunctional linker
  • other modifications known in the art both naturally occurring and non-naturally occurring.
  • the antibody of the invention is a monoclonal antibody.
  • the term "monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as the below listed fragments. Monoclonal antibodies are generally prepared using the method of Kohler and Milstein (1975, Nature 1975; 256:495-497), or a modification thereof.
  • the invention also provides an isolated functional fragment of the antibody of the invention wherein said functional fragment is selected among Fv, Fab, (Fab') 2 , Fab', scFv, scFv- Fc fragments, diabodies, single domain antibodies, any antibody fragment with an increased half-live, and functional fragments which exhibit immunological binding properties of the parent antibody molecule.
  • the antibody fragments of the invention may be obtained by cleaving the antibody of the invention with proteases.
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab') 2 fragment which comprises both antigen-binding sites.
  • a "Fv" fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Alternatively, these antibody fragments may be constructed by using gene recombination techniques. Fv fragments are, more commonly derived using recombinant techniques known in the art.
  • the Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule.
  • the antibody fragments of the invention are capable of exhibiting immunological binding properties of the parent antibody molecule of the invention.
  • the antibody fragment of the invention is a scFv fragment.
  • a single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
  • the scFv fragment of the invention is able to refold into a 3 -dimensional conformation designed specifically to have affinity for human pro-cathepsin L, and/or cathepsin L and more generally to have affinity for cancer cells expressing pro-cathepsin L and/or cathepsin L such as melanoma cells.
  • the humanized scFv fragment of the invention includes CDRs previously described that are held in appropriate conformation by polypeptide segments analogous to the FRs of a Fv fragment derived from a native antibody molecule.
  • purified and “ isolated” it is meant, when referring to a polypeptide (i.e. the antibody of the invention) or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • the term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present.
  • nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • the isolated and/or purified antibody of the invention interacts with an epitope of the human pre-pro-cathepsin L (SEQ ID N°31) ; said epitope comprises or is of the sequence RLISLSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLD (SEQ ID N°32) , corresponding to the amino acids 157 to 197 of the human pre- pro-cathepsin L.
  • said epitope comprises a fragment of said sequence SEQ ID N°32 or a variant thereof.
  • the antibody of the invention and their fragments thereof are able to bind specifically and selectively to at least one of the following human proteins: the human cathepsin L, the human procathepsin L, and the human pre-pro-cathepsin L.
  • the antibody of the present invention may also be described or specified in terms of its cross-reactivity. Because of the sequence conservation among the species, the antibody of the invention, or a fragment thereof, is able to bind to homologous protein expresses by cells of others mammals, such as rodent, rat, rabbit, cattle, primate for example.
  • the antibody of the invention that bind to polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to human pre-pro-cathepsin L, human pro-cathepsin L and human cathepsin L are also included in the present invention.
  • binding affinities include those with a dissociation constant or Kd less than 10 "2 M, 10 "3 M, 10 "4 M, 10 "5 M, 10 "6 M, 10 "7 M, 10 “8 M, 10 "9 M, 5.10 "10 M, 10 "10 M, 5.10 "11 M, 10 "11 M, 5.10 "12 M, 10 “12 M, 5.10 “13 M, 10 “13 M, 5.10 "14 M, 10 “14 M, 5.10 "15 M, 10 "15 M.
  • the antibody of the invention or a fragment thereof are provided that inhibit proteinase activity of pre-pro-cathepsin L, and/or pro-cathepsin L, and/or cathepsin L by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50% of the activity in absence of the antibody.
  • the antibody of the invention is a murine isolated and/or purified antibody, or a fragment thereof, wherein the variable region of the light chain (VL) has the amino acid sequence SEQ ID N°2 or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99%, ou 99,5% identity after optimal alignment with SEQ ID N°2, and/or wherein the variable region of the heavy chain (VH) has the amino acid sequence SEQ ID N°4 or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99%, or 99.5% identity after optimal alignment with SEQ ID N°4.
  • VL variable region of the light chain
  • VH variable region of the heavy chain
  • percentage of identity between two amino acids sequences or two nucleic acids sequences, means the percentage of identical amino acids, respectively nucleotides, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the aminoacids or nucleic acids sequences.
  • best alignment or “optimal alignment” , means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences or nucleic acids are usually realised by comparing these sequences that have been previously align according to the best alignment. This comparison is realised on segments od comparison in order to identify and compared the local regions of similarity.
  • BLAST software with the BLOSUM 62 matrix, or the PAM or PAM 250 matrix.
  • the identity percentage between two sequences of amino acids or nucleic acids is determined by comparing these two sequences optimally aligned, the aminoacids or nucleic acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
  • amino acids sequence respectively nucleic acid sequence, having a percentage of identity of at least 80%, preferably, of at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99%, 99.5% after optimal alignment, means amino acids sequences respectively nucleic acid sequences having with regard to the reference sequence, modifications such as deletions, truncations, insertions, chimeric fusions, and/or substitutions, specially point mutations, the sequence of which presenting at least 80%, preferably, at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, 99%, 99.5% identity after optimal alignment with the sequence of reference.
  • the murine isolated antibody named 3D8 of the invention comprises a constant region of the heavy chain that comprises the amino acid sequence of a gamma-1 chain of a murine immunoglobulin, and comprises the constant region of the light chain that comprises the amino acid sequence of a kappa chain of a murine immunoglobulin.
  • constant region it is meant the portion of the antibody molecule which confers effector functions.
  • the antibody includes constant regions derived from murine immunoglobulins .
  • the antibody of the invention includes constant regions derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of five isotypes: alpha, delta, epsilon, gamma or mu. Heavy chains of various subclasses (such as the IgG subclass of heavy chains) are responsible for different effector functions. Thus, by choosing the desired heavy chain constant region, antibody with the desired effector function can be produced.
  • the light chain constant region can be of the kappa or lambda type, preferably the kappa type.
  • monoclonal antibodies in humans may be severely restricted when the monoclonal antibody is produced in a non- human animal.
  • Repeated injections in humans of a "foreign" antibody, such as a mouse antibody may lead to harmful hypersensitivity reactions, i. e., anti-mouse antibody (HAMA) or an anti-idiotypic response.
  • HAMA anti-mouse antibody
  • the HAMA response makes repeated administrations less effective due to an increased rate of clearance from the patient's serum and/or allergic reactions by the patient.
  • the invention provides a chimeric antibody or fragment thereof that binds to cathepsin L and/or pro-cathepsin L, and/or eventually to pre-procathepsin L, and that is optionally and preferably able to inhibit cellular pro-cathepsin L secretion.
  • chimeric antibody it is meant an antibody which includes sequences derived from two different antibodies, which typically are of different species. Most typically, chimeric antibodies include human and murine antibody fragments, generally human constant and murine variable regions.
  • chimeric antibodies of the invention can be formed by grafting non-human variable regions, such as mouse variable regions (SEQ ID N°l and 3) to human constant regions.
  • the invention provides a humanized antibody or fragment thereof that binds to cathepsin L and/or pro-cathepsin L, and/or eventually to pre-procathepsin L, and that is optionally and preferably able to inhibit cellular pro-cathepsin L secretion.
  • Human antibody can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences [See, U.S. Pat.
  • humanized monoclonal antibodies are formed by grafting all six (three light chain and three heavy chain) CDRs from a non-human antibody into Framework Regions (FRs) of a human antibody.
  • FRs Framework Regions
  • Such humanized antibody retains or substantially retains the antigen-binding properties of the parent antibody but is less immunogenic in humans than the parent antibody.
  • the CDRs are derived from a mouse monoclonal antibody 3D8 and the framework of the variable domains, and the constant domains, of the altered antibody is preferably derived from a human antibody.
  • a number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described (see for examples Winter et al .
  • the humanized isolated antibody of the invention comprises at least one of the frameworks (FR) of the light and/or heavy chain that comprises an amino acid sequence selected from common sequences (HMHCS: human most homologous consensus sequence) derived from human antibody subgroups (see, e.g., Chothia et al . , J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions).
  • FR frameworks
  • HHCS human most homologous consensus sequence
  • the invention provides antibody fragment that binds to cathepsin L and/or pro- cathepsin L, and/or optionally to pre-pro-cathepsin L, and that is optionally and preferably able to inhibit cellular pro-cathepsin L secretion.
  • Such fragments which are more preferably Fv antibodies or single chain Fv (scFv) antibodies, are usefull to reduce the risk of a HAMA response.
  • scFv single chain Fv
  • the antibody fragment of the invention is a scFv fragment of the antibody of the invention, more preferably of the antibody 3D8.
  • the chimeric antibodies, the humanized antibodies, and their fragment thereof and the antibody fragment of the isolated antibody of the invention more preferably Fv antibodies or single chain Fv (scFv) antibodies have substantially similar binding properties to human cathepsin L, and/or human pro-cathepsin L, and/or human pre-pro-cathepsin L, than the 3D8 monoclonal antibody of the invention.
  • substantially similar binding properties refers to a chimeric antibody, a humanized antibody or a fragment of antibody of the invention which retains the ability to specifically bind the antigen recognized by the parent antibody used to produce such engineered antibodies or fragments thereof.
  • the affinity of the humanized antibody is at least about 10% of the affinity of the parent antibody, more preferably at least about 25%, even more preferably at least about 50%.
  • the humanized antibody exhibits antigen-binding affinity that is at least about 75%, 85%, 95%, 100% of the affinity of the parent antibody.
  • the affinity of such engineered antibodies i.e. chimeric antibody, humanized antibody, Fv fragment, scFv fragment, chimeric Fv or scFv fragment, humanized Fv or scFv fragments
  • Methods for assaying antigen binding affinity are well known in the art and include half-maximal binding assays, competition assays, and Scatchard analysis. Methods for enhancing affinity are also well known to the man skilled in the art; point substitutions can be made in one or more CDRs using conventional cassette mutagenesis or other molecular biology techniques to enhance binding capabilities of an antigen-binding site. Modification of CDR- encoding sequences can be designed empirically under the invention based on sequence analysis of the Fv region of preexisting antibodies. Further, using a computer program such as, for example, COMPUGENE . RTM . , and known variable region DNA sequences, those of ordinary skill in the art can design and synthesize modified CDR-encoding sequences. Using transfection and gene delivery techniques as described above, modified CDR sequences can be readily expressed and the resultant antibody tested for binding and empirically refined by exchanging selected amino acids in CDRs sequences.
  • the invention also provides a hybridoma able to secrete an antibody of the invention.
  • the hybridoma is a murine hybridoma.
  • Hybridomas are produced according to methods well-known by a man skilled in the art
  • the fragment of the invention can be expressed in eukaryotic hybridoma cells.
  • a transgene molecule comprising at least one of the isolated DNA molecule of the invention (see below) and coding for a recombinant scFv
  • VH-linker-VL (VH-linker-VL, or VL-linker-VH) is inserted into an expression vector containing, for example, the immunoglobulin promoter, a secretion signal, immunoglobulin enhancers, and various introns .
  • the vector is then transfected into myeloma cells via established electroporation or protoplast fusion methods. Transfected host cells thus express scFv fragments.
  • the polypeptide product can then be purified by methods known in the art.
  • the invention also provides an isolated DNA molecule selected in the group coonsisting of molecule of SEQ ID N°5, SEQ ID N°7, SEQ ID N°9, SEQ ID N°ll, and molecule having at least 80%, or at least 85%, 90%, 95%, 99% identity after optimal alignment with SEQ ID N°5, SEQ ID N°7, SEQ ID N°9, SEQ ID N°ll.
  • the isolated DNA molecules of the invention may be expressed or coexpressed to provide monomeric or heterodimeric polypeptides, respectively.
  • the polypeptide are derived from VH and/or VL domains of the monoclonal antibody 3D8.
  • nucleotide sequences of the VH (SEQ ID N°3) and VL (SEQ ID N°l) domains of the 3D8 antibody molecule or of the 6 CDRs of the antibody molecule allows for the construction of synthetic nucleic acid molecules comprising nucleotide sequences which encode CDRs flanked by recombinantly engineered polypeptide regions.
  • a DNA molecule is provided which includes nucleotide sequences encoding either a heavy or light chain V domain having a CDR set derived from the VH or VL domain of the 3D8 antibody molecule, or the CDR sequence alone.
  • the present invention is also providing a vector comprising at least one DNA molecule of the invention.
  • a "vector” is a replicon in which another polynucleotide segment is attached, so as to bring the replication and/or expression to the attached segment.
  • vectors include plasmids, phages, cosmids, phagemid, yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , human artificial chromosome (HAC) , viral vector, such as adenoviral vector, retroviral vector, baculoviral vector and other DNA sequences which are able to replicate or to be replicated in vi tro or in a host cell, or to convey a desired DNA segment to a desired location within a host cell.
  • YAC yeast artificial chromosome
  • BAC bacterial artificial chromosome
  • HAC human artificial chromosome
  • viral vector such as adenoviral vector, retroviral vector, baculoviral vector and other DNA sequence
  • a vector can have one or more restriction endonuclease recognition sites at which the DNA sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment can be spliced in order to bring about its replication and cloning.
  • Vectors can further provide primer sites (e.g. for PCR), transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, selectable markers, etc.
  • the cloning vector can further contain a selectable marker suitable for use in the identification of cells transformed with the cloning vector.
  • the invention relates to a recombinant vector for cloning a DNA sequence according to the invention and/or for expressing an antibody according to the invention or a fragment thereof characterized in that it contains at least a DNA sequence selected among SEQ ID N°l, N°3, N°5, N°7, N°9, N°ll. More precisely, the invention provides an expression vector comprising at least one DNA molecule selected in the group consisting of a DNA molecule of the invention.
  • the vector according to the invention is characterized in that it includes components for the expression, possibly the secretion, of said sequences in a host cell. These vectors are useful for transforming host cells in order to clone or express DNA sequences of the invention.
  • Such expression vectors are typically replicable in a host organism either as episomes or as an integral part of the host chromosomal DNA.
  • the isolated DNA molecule or the vector of the invention may be introduced into a host cell either in vivo or in vi tro using known techniques, such as CaP0 4 precipitation, electroporation, cationic lipofection, use of artificial viral envelopes, direct injection (e.g., intravenous, intraperitoneal or intramuscular).
  • a host includes prokaryotic or eukaryotic organisms that can be genetically engineered. For examples of such hosts, see Sambrook et al .
  • the host cells are transformed under conditions allowing the antibody or a fragment thereof to be expressed.
  • the cell host may be selected from bacterial cells but also from yeast cells, as well as animal cells, in particular mammal cell cultures, but also insect cells wherein methods implementing baculoviruses for example may be used.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilus, Salmonella, Serratia and Pseudomonas .
  • Examples of eukaryotic hosts cells include yeast, including Saccharomyces, Sf9 insect cells, Sp2/0, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7 and MDCK cell lines.
  • yeast including Saccharomyces, Sf9 insect cells, Sp2/0, VERO and HeLa cells
  • Chinese hamster ovary (CHO) cell lines WI38, BHK, COS-7 and MDCK cell lines.
  • CHO cells mouse myeloma cells Sp2/0.
  • a clone exhibiting a high productivity can be selected using a chemical such as MTX (Immunol, Lett. 64, 139, 1998).
  • Such a strain exhibiting stable high productivity, if available, is useful in producing the recombinant anti-cathepsin L monoclonal antibody or a fragment thereof on an industrial scale.
  • the host cells of the invention may be part of a transgenic animal; thus the antibody expression is
  • a coexpression system can be established in a suitable host cell.
  • "Coexpression” as used herein refers to the expression of two or more polypeptides in a host cell.
  • one such system comprises DNA molecules (SEQ ID N°l and N°2) encoding respectively for monomeric VL (SEQ ID N°2) and VH (SEQ ID N°4) domain polypeptides which are harbored in a single vector, either under the control of the same regulatory elements or under the control of separate elements.
  • monomeric variable domain polypeptides expressed from the same vector but driven by separate regulatory elements would also be considered “coexpressed” .
  • a coexpression system is provided by the expression of two or more polypeptides from separate constructs.
  • the host cells can be transfected with two vectors; the first vector containing an expression cassette which includes nucleotide sequences (SEQ ID N°l) encoding the VL domain polypeptide (SEQ ID N°2) derived from a 3D8 antibody, and the second vector containing an expression cassette which includes nucleotide sequences (SEQ ID N°3) encoding the VH domain polypeptide (SEQ ID N°4) derived from a 3D8 antibody.
  • SEQ ID N°l nucleotide sequences
  • SEQ ID N°3 encoding the VH domain polypeptide
  • the vectors are identical --except in so far as the coding sequences and selectable markers are concerned- -so as to ensure that the VH and VL polypeptides are substantially equally expressed in the transfected host cell.
  • a non-covalent heterodimer which exhibits immunological binding properties of an immunoglobulin which binds to a human cathepsin L and/or pro-cathepsin L can be readily produced using a coexpression system.
  • a heterodimer can be formed by the coexpression of complementary VH and VL polypeptides in a transfected host cell.
  • the coexpressed polypeptides dimerize under the influence of non-covalent (e.g., electrostatic) interdomain contacts to form an antigen-binding site.
  • the dimeric molecule thus formed is substantially homologous to an Fv fragment isolated from a native immunoglobulin.
  • the heterodimer comprises six CDRs (a heavy chain CDR set and a light chain CDR set) disposed relative to each other in three dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is maintained by flanking FR residues to provide an antigen-binding site which retains much of the antigen recognition and binding capabilities of the parent antibody molecule.
  • the invention also relates to a method for producing a recombinant antibody against human pre-pro-cathepsin L, and/or human pro-cathepsin L and/or cathepsin L which comprises the steps of (i) transforming host cells by the DNA sequences or the expression vector of the invention, (ii) culturing the host cells under such conditions as to allow expression of the antibody, or a fragment thereof, and (iii) collecting the antibody or a fragment thereof thus produced by the host cells.
  • the present invention therefore relates to the recombinant polypeptide which may be obtained by the method shown above .
  • the gene products can be purified from lysats and cell extracts, from the supernatant of the culture medium, by methods either used individually or in combination, such as fractionation, ammonium sulfate precipitation, gel electrophoresis, chromatography methods, immuno-affinity techniques by means of specific mono- or polyclonal antibodies, etc.
  • Substantially pure immunoglobulins of at least about 90% to about 95% homogeneity are preferred, and 98% to 99% or more homogeneity most preferred for pharmaceutical uses.
  • the anti-cathepsin L monoclonal antibody of the invention secreted into the cell culture supernatant can be easily purified by a method using a resin binding protein A
  • a preferred alternative consists of producing a recombinant polypeptide fusioned to a "carrier" protein (chimeric protein) .
  • carrier chimeric protein
  • the advantage of this system is that it provides stabilization and a reduction in the proteolysis of the recombinant product, an increase in the solubility during renaturation in vi tro and/or a simplification of the purification when the fusion partner has an affinity for a specific ligand.
  • the value of the new antibody of the invention and their fragment thereof for therapeutic and diagnostic purposes is extraordinarily high.
  • the antibody of the invention, or a fragment thereof may be used therapeutically, or in developing and performing assays, in vivo or in vitro diagnostic procedures, and imaging.
  • the antibody of the invention, or fragments is particularly useful for the treatment of diseases such as cancer, in particular for treating or detecting melanoma cancer.
  • the antibody or a fragment thereof may be used in an in vitro diagnostic method of diseases characterized by an altered expression of pre-pro-cathepsin L and/or pro-cathepsin L and/or cathepsin L expression, such as melanoma cancer.
  • Such method comprises the step of: (i) contacting a sample of body fluid and/or body tissue collecting from a patient suspected of having such disease (e.g melanoma cancer), with the antibody of the invention or a fragment thereof; and (ii) detecting the binding of the antibody of the invention or a fragment thereof to an antigen or an anti-idiotypic antibody of said sample.
  • such method may be performed on tissue sections.
  • such diagnostic method constitutes an immunoassay.
  • immunoassays Numerous types of immunoassays are available and are well known to those of skill in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipition reactions, gel diffusion precipition reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • the antibody or a fragment thereof is linked to a solid support directly or indirectly via a spacer arm.
  • solid supports may include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Chemical attachment techniques of detectable moieties on to antibody or fragment thereof are well known in the art.
  • the invention provides a diagnostic agent for melanoma comprising the antibody of the invention or a fragment thereof, preferably the scFv fragment as an active ingredient.
  • the antibody or a fragment thereof may either be labeled or unlabeled.
  • Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody or a fragment of the invention.
  • the antibodies can be directly labeled.
  • labels can be employed, such as radionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens, biotin, avidine, streptavidine, digoxygenine ... ) , metal ions etc.
  • the antibody or a fragment thereof may be used for in vivo imaging and detection of cells characterized by an altered expression of pre-pro-cathepsin L and/or pro-cathepsin L and/or cathepsin L expression, such as melanoma cells.
  • the invention provides a diagnostic agent for melanoma that comprises an antibody of the invention or a fragment thereof, more preferably a scFv fragment as an active ingredient.
  • the invention provides a composition for such detection, localisation and imaging comprising the antibody or a fragment and a pharmaceutical acceptable carrier.
  • the antibody will be administered as a fragment such as scFv, F(ab) or F(ab')2 fragments because this provides superior results so that background substraction is unnecessary (see US 4 331 647) .
  • the subject molecules can be administered by intravenous or intramuscular injection. Mixtures of labeled antibody fragments may be used in a single preparation for tumor localization, detection or therapy. Fragments of different size may be used together. The mixture can use antibodies specific to antigens associated with the same tumor type to enhance localization and resolution. Alternatively, broad screening for various tumor types can be done using a mixture of antibodies with diverse tumor specificities. Antibody fragments may be labeled by any of several techniques known to the art.
  • the radioisotope is a metal ion, and the coupling is performed via a conjugated metal chelator (see, e.g., Khaw et al . , 1980 Science 209:295; US 4 427 200, US 4 472 509, US 4 479 930).
  • a conjugated metal chelator see, e.g., Khaw et al . , 1980 Science 209:295; US 4 427 200, US 4 472 509, US 4 479 930.
  • the foregoing are merely illustrative of the many methods of radiolabeling proteins known to the art.
  • gamma-emitters, positron-emitters, x-ray- emitters and fluorescence-emitters are suitable for localization and/or therapy, while beta-emitters and alpha- emitters may also be used for therapy.
  • Suitable radioisotopes for labeling antibodies include Iodine-131, Iodine-123, Iodine-126, Iodine-133, Bromine-77, Indium-Ill, Indium-113m, Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium- 103, Ruthenium- 105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105, Rhenium- 101, Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18.
  • the antibody can also be conjugated to a radio-opaque dye.
  • a radio-opaque dye for the in vivo imaging the antibody can also be conjugated to a radio-opaque dye.
  • the coupling of radioisotopes such as Iodine-131, Indium-Ill, and Technetium-99m to an antibody of the invention or a fragment thereof is preferred to perform tumor imaging because they are detectable with a gamma camera and have favorable half-lives for imagining in vivo .
  • the antibody or a fragment thereof, like the scFv fragment of the invention, can be labelled, for example, with radioactive atoms such as, but not limited to, Yttrium-90, Technetium-99m, or Indium-Ill via a conjugated metal chelator (see, e.g., Khaw et al . (1980) Science 209:295; U.S. Pat. No. 4,472,509 to Gansow et al . ; and U.S. Pat. No. 4,479,930 to Hnatowich), or by other standard means of isotope linkage to proteins known to those with skill in the art.
  • radioactive atoms such as, but not limited to, Yttrium-90, Technetium-99m, or Indium-Ill via a conjugated metal chelator (see, e.g., Khaw et al . (1980) Science 209:295; U.S. Pat. No. 4,472,509
  • kits for use with the subject antibody in the detection for the presence of pre-pro-cathepsin L, pro-cathepsin L, and/or cathepsin L may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional agents, such as secondary antibodies, labelled agents.
  • additional agents such as secondary antibodies, labelled agents.
  • the antibody which may be conjugated to a label or toxin, or unconjugated, are included in the kit with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use.
  • these materials will be present in less than about 5% wt . based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt . based again on the antibody concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% wt . of the total composition.
  • a second antibody capable of binding to the antibody, or a fragment thereof is employed in an assay, this will usually be present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above.
  • Antibodies of the present invention and their fragment thereof can further find a wide variety of utilities in vi tro .
  • the exemplary antibodies can be utilized for L-cathepsin producing cells typing or for isolating specific cathepsin L expressing cells over cathepsin L non- expressing cells for vaccine preparation, or the like.
  • Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al . , J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al . , J. Cell. Biol. 105:3087-3096 (1987).
  • the human anti-cathepsin L specific antibody of the invention typically find use in treating a cathepsin L overexpressing cell mediated disease state, such as melanoma cancer.
  • the invention aims at providing an antibody or a fragment thereof, more particulary a scFv fragment, as a drug.
  • such drug is useful for the preventive and curative treatment of melanoma, especially by inhibiting tumorigenic and metastatic phenotype of melanoma.
  • Human cathepsin plays an important role in regulation of humoral immune system.
  • Anti-cathepsin L antibody of the invention may therefore be immunosuppressive when administered in vivo .
  • Such antibodies may be useful as a result in the treatment of melanoma.
  • the invention encompasses antibodies that inhibit or reduce the proteinase activity of pro- cathepsin L and/or cathepsin L. Such inhibition can be assayed using techniques known in the art.
  • the antibody of the invention provides a convenient means for designing multi-functional molecules such as immunotoxins and the like.
  • Immunotoxins are characterized by two components and are particularly useful for killing selected cells in vi tro or in vivo .
  • One component is a cytotoxic agent which is usually fatal to a cell when attached or absorbed.
  • the second component known as the "delivery vehicle”, provides a means for delivering the toxic agent to a particular cell type, such as melanoma cells.
  • the two components are commonly chemically bonded together by any of a variety of well-known chemical procedures.
  • the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like.
  • heterobifunctional cross-linkers e.g., SPDP, carbodiimide, glutaraldehyde, or the like.
  • Production of various immunotoxins is well-known with the art, and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet", Thorpe et al . , Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982) .
  • a variety of cytotoxic agents are suitable for use in immunotoxins.
  • Cytotoxic agents can include radionuclides, such as Iodine-131, Yttrium-90, Rhenium-188, and Bismuth-212; a number of chemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, and cisplatin; and cytotoxic proteins such as ribosomal inhibiting proteins like pokeweed antiviral protein, Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, etc., or an agent active at the cell surface, such as the phospholipase enzymes (e.g., phospholipase C) .
  • radionuclides such as Iodine-131, Yttrium-90, Rhenium-188, and Bismuth-212
  • chemotherapeutic drugs such as vindesine, methotrexate, adriamycin, and cisplatin
  • cytotoxic proteins such as
  • the antibody of the present invention may display excellent cytotoxicity, in the absence of toxin and in the presence of effector cells, i.e. human monocytes.
  • the antibodies of the present invention may be used in combination with other molecules, such as antibodies, particularly human monoclonal antibodies reactive with other markers on melanoma cells or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7) , for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • antibodies particularly human monoclonal antibodies reactive with other markers on melanoma cells or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7) , for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents, antibiotics, and immunoglobulin) .
  • treatments e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents, antibiotics, and immunoglobulin
  • administration of products of a species origin or species reactivity in the case of antibodies
  • human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
  • the antibody can be used as separately administered compositions given in conjunction with chemotherapeutic or immunosuppressive agents.
  • the agents will include cyclosporin A or a purine analog (e.g., methotrexate, 6-mercaptopurine, or the like), but numerous additional agents (e.g., cyclophosphamide, prednisone, etc.) well-known to those skilled in the art may also be utilized.
  • the antibody of the invention, or a fragment thereof can be linked to a chemotherapeutic agent using immunoconjugate techniques well known in the art.
  • conjugation of a number of chemotherapeutic agents to immunoglobulin molecules have been described, including doxorubicin (Yang et al . (1988) Proc. Natl. Acad. Sci.
  • DNA molecules coding for part of the antibodies of the present invention (CDRs, or VH, VL domains), and their fragments thereof that can be used to develop gene targeting vectors (e.g retroviral vector, adenoviral vector,..) that target human cancer cells expressing pro-cathepsin L, such as melanoma cells, and are thus useful in the design and development of gene therapy strategies .
  • CDRs e.g retroviral vector, adenoviral vector,..
  • pro-cathepsin L such as melanoma cells
  • transgene e.g a DNA molecule
  • antibody/transgene conjugate will be useful to treat disorders involving cells expressing cathepsin L, such as melanoma cells.
  • the transgene will selectively transfected into specific melanoma tissues or cells.
  • Such transgene may encodes for any therapeutic protein, marker protein and the like.
  • the antibody of the invention of a fragment thereof can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the human pro-cathepsin L or human cathepsin L, using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1989), and Nissinoff, J. Immunol. 147 (8) :2429-2438 (1991)). Such anti-idiotype antibodies may be used as ingredients for a vaccine.
  • the antibody or a fragment thereof can be administered to a patient alone or in combination with a pharmaceutical formulation.
  • Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions.
  • the antibody or a fragment thereof must further be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms.
  • Sterile injectable solutions are prepared by incorporating at least the antibody, or a fragment thereof, more preferably the single chain polypeptide constructs of the invention scFv in the required amount in the appropriate solvent, such as sodium phosphate-buffered saline, followed by filter sterilization.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutially acceptable carrier, stabilizer or diluent and, as active ingredient, an antibody according to the invention, or a fragment thereof.
  • a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, antibacterial and antifungal agents that are non-toxic to humans, and the like. Examples of such carriers and stabilizers include human serum albumin and gelatin. The use of such media and agents for pharmaceutically active substances is well known in the art. The media or agent must be compatible with maintenance of proper conformation of the single polypeptide chains, and its use in the therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions.
  • the antibodies according to the invention can be administered to mammals, preferably human patients for therapy or diagnosis according to known procedures.
  • the antibody, or antibody fragments, or the composition of the invention will be injected parenterally, preferably intraperitoneally.
  • the antibody, antibody fragments, or the composition of the invention can also be administered intravenously or by others routes. Determination of appropriate titers of antibody, or the fragment thereof to administer is well within the skill of the art.
  • the dosage ranges for the administration of the antibody of the invention are those large enough to produce the desired tumor suppressing effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counter indications, immune tolerance or similar conditions.
  • Dosage can vary from 0.1 m g/kg to 70 mg/kg, preferably 0.1 mg/kg to 500 mg/kg/dose, in one or more doses administrations daily, for one or several days.
  • Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of the antibody (ies) of this invention sufficient to effectively treat the patient.
  • the antibody of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use.
  • This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.
  • FIGURE 1 Preparation of cathepsin L fusion proteins.
  • Part A Purified GST fusion proteins were separated by SDS- PAGE and stained with Comassie Blue. Lane 1: GST-Pro (3,3 ⁇ g) ; lane 2: GST-CTSL (1,8 ⁇ g) ; lane 3: GST (9 ⁇ g) . Part B: Purified His fusion proteins were separated by SDS-PAGE and stained with Comassie Blue. Lane 1: His-Pro; lane 2: His-CTSL; lane 3: His-ProCTSL.
  • Part C Culture supernatant from SF9 insect cells, after transfection without (lane 1) or with (lane 2) procathepsin L recombinant viral DNA, was submitted to SDS-PAGE and subsequently immunoblotted with the polyclonal anti-cathepsin L Ab. Purified ProCTSL-His (1 ⁇ g) was submitted to SDS-PAGE and stained with Comassie Blue (lane 3) .
  • FIGURE 2 Characterization of anti-cathepsin L 3D8 mAb.
  • Part A ELISA with purified 3D8 mAb using recombinant GST or GST-CTSL proteins.
  • Part B Immunoblot of purified fusion cathepsin L proteins (100 ng) using 3D8 mAb. Lane 1: ProCTSL- His; Lane 2: GST-Pro; Lane 3: GST-CTSL.
  • Part C Immunoprecipitation of ProCTSL-His by anti-v-src mAb (lane 1) or by anti-cathepsin L 3D8 mAb (lane 2) . Immunoprecipitated proteins were submitted to SDS-PAGE, then immunoblotted with polyclonal anti-CTSL Ab.
  • Part D Immunoblot using 3D8 mAb (lanes 1 and 2) or polyclonal anti-CTSL Ab (lanes 3 and 4) on conditioned media (lanes 1 and 3) or cell extracts (lanes 2 and 4) prepared from DM-4 cells.
  • Part E Immunoprecipitation from DM-4 cells conditioned media using anti-v-src mAb (lane 1) or by 3D8 anti-CTSL mAb (lane 2) . Immunoprecipitated proteins were submitted to SDS-PAGE, then revealed by polyclonal anti-CTSL Ab.
  • Part F Immunofluorescence staining of DM-4 melanoma cells without (Part 1) or with (Part 2) 3D8 mAb.
  • FIGURE 3 Localization of cathepsin L epitope recognized by 3D8 anti-CTSL mAb.
  • FIGURE 4 Preparation of 3D8 anti-CTSL ScFv.
  • Part A Ethidium-bromide-stained agarose gel separation of the purified DNA fragments corresponding to VL (lane 2) and VH
  • FIGURE 5 Nucleotide and deduced amino acid sequences of the VH and the VL regions of 3D8 ScFv.
  • Complementarity determining regions (CDR) 1, 2 and 3 are underlined.
  • the 3D8 ScFv nucleotide and amino acid deduced sequences have been assigned GenBank Accession Number AF488378.
  • FIGURE 6 Characterization of 3D8 anti-cathepsin L ScFv.
  • Part A ELISA using 3D8 ScFv on GST or GST-CTSL.
  • Part B Immunoblot analysis of purified fusion CTSL proteins using 3D8
  • Lane 1 100 ng GST; Lane 2: 100 ng GST-Pro; Lane 3: 100 ng GST-CTSL; Lane 4: 100 ng His-Pro; Lane 5: 200 ng His-CTSL.
  • FIGURE 7 Intracellular expression of 3D8 ScFv anti-cathespin L inhibits procathepsin L secretion.
  • Part A Immunoblot of extracts of A375SM cells transfected with 1 ⁇ g of pCMV/myc/ER (lane 1) or pCMV/myc/ER-3D8/ScFv (lane 2) with anti-myc mAb.
  • Part B Immunoblot of conditioned media of A375SM cells transfected with pCMV/myc/ER-3D8/ScFv alone (lane 1), pcDNA3. l/V5-His-ProCTSL alone (lane 2), and pcDNA3. l/V5-His-ProCTSL with increasing concentration of pCMV/myc/ER-3D8/ScFv (lanes 3 to 7) with anti-V5 mAb.
  • FIGURE 8 ScFv expression in stable transfected A375SM melanoma cells. cDNA (Panel A) and total cell extracts (Panel
  • FIGURE 9 Cathepsin L expression and secretion in transfected A375SM human melanoma cells.
  • cDNA Panel A
  • cell protein extracts Panel B
  • conditioned media Panel C
  • A375SM parental lane 1
  • A375SM neol lane 2
  • A375SM neo 2 lane 3
  • A375SM ScFvl lane 4
  • A375SM ScFv2 lane 5
  • Panel A Expression of cathepsin L cDNA was analyzed by RT-PCR.
  • GAPDH cDNA was also amplified from the same cDNA.
  • Cell extracts (Panel B) and conditioned media (Panel C) were submitted to SDS-PAGE and analyzed by western blot using anti- cathepsin L (clone 3D8) and anti-GAPDH moAbs .
  • FIGURE 10 Growth of parental and transfected A375 SM melanoma cells in nude mice.
  • FIGURE 11 Tumor MVD and apotosis (TUNEL) in parental, neo and anti-cathepsin L ScFv-transfected cells implanted into nude mice.
  • Parental (A) and neo-transfected (B) cells produced vascularized tumors (V-arrowed) with very few cells undergoing apotosis.
  • tumors produced by the anti-cathepsin L ScFv transfected cells (C) were significantly less vascularized.
  • the number of TUNEL-positive tumor cells was inversely correlated with MVD (A-arrowed) .
  • Example I Cloning and characterisation of anti-cathepsin L scFv fragment 1. Materials and methods
  • DM-4 cell line established from surgical specimen of lymph node metastasis
  • the P3X63Ag8.653 mouse myeloma cell line was obtained from American Type Culture Collection (ATCC, CRL- 1580) .
  • the melanoma and myeloma cell lines were grown in DMEM
  • Conditioned Media and Cell Extract Preparations were prepared as previously described [Jean et al . (1995) Biochem. J. 312, 961-969] . Briefly, DM-4 cells were incubated for 24 h in serum-free culture medium and conditioned media was concentrated 20 times on microcon 10
  • ELISA ELISA .
  • 96-well plates were coated with 100 ⁇ l GST or GST-CTSL solution at 5 ⁇ g/ml, at room temperature for 1 h.
  • plates were coated with 100 ⁇ l goat anti -mouse immunoglobins (Dako) at 5 ⁇ g/ml.
  • Dako goat anti -mouse immunoglobins
  • 100 ⁇ l supematants from hybridoma cells, purified 3D8 mAb or bacterial culture supernatant containing 3D8 ScFv were incubated in microtiter plates for 1 h at room temperature.
  • Plasmid pGHP3 corresponding to pGEM3 vector in which procathepsin L cDNA was cloned, was kindly provided by Dr M. M. Gottesman (National Institute of Health, Bethesda, ML) [Gal et al . (1988) Biochem. J. 253, 303-306] .
  • Glutathion-S- transferase (GST) fusion proteins vectors (ProCTSL: procathepsin L; CTSL: cathepsin L and Pro: proregion of cathepsin L) were generated by cloning cDNAs, amplified by PCR from pGHP3 plasmid, in the pGEX-4T3 vector (Amersham Biosciences) .
  • the 5' primers used were: 5'- GCGGATCCCATGACTCTAACATTTGATCAC-3' (SEQ ID N° 13.) for GST-Pro and GST-ProCTSL and 5 ' -GCGGATCCATGGCCCCCAGATCTGTGGAT-3 ' (SEQ ID N°14) for GST-CTSL.
  • the 3' primers used were: 5'- TATCTCGAGTCACACAGTGGGGTAGCTGG-3 ' (SEQ ID N°15) for GST-CTSL and GST-ProCTSL and 5 ' -TATCTCGAGTCACTCATAAAACAGAGGTTC-3 ' (SEQ ID N°16) for GST-Pro.
  • His fusion proteins were prepared either in a prokaryotic expression system (His-ProCTSL, His-CTSL and His-Pro) , by subcloning cathepsin L cDNAs, cloned in pGEX-4T3 vector, in pET-28a(+) vector (Novagen) or in the eukaryotic expression system: pre-procathepsin L cDNA was amplified by PCR from pGHP3 plasmid with primers 5 ' - GCGAAGCTTATGAATCCTACACTCATCCT-3 ' (SEQ ID N°17) and 5'- GCGGATCCGCACAGTGGGGTAGCTGGCTG-3 ' (SEQ ID N°18), and cloned in pTenHisCc (Qbiogen) , to generate pTenHisCc-ProCTSL.
  • pre-procathepsin L cDNA was amplified by PCR from pGHP3 plasmid
  • GST fusion proteins used to localize 3D8 mAb epitope were constructed by cloning PCR products, amplified from pTenHisCc-ProCTSL, in the pGEX-4T3 vector with 5' primers: 5'-
  • Mammalian expression vector encoding a fusion procathepsin L with a C-terminal V5 epitope was generated from pre-procathepsin L cDNA, amplified by PCR from pTenHisCc-ProCTSL vector with primers 5'- GCGAAGCTTATGAATCCTACACTCATCCT-3 ' (SEQ ID N°27) and 5'- GCGGATCCCACAGTGGGGTAGCTGGCTG-3 ' (SEQ ID N°28) and cloned in pcDNA3.
  • l/V5-HisB Invitrogen).
  • the 3D8 ScFv cDNA generated by PCR from pCANTAB5E-3D8/ScFv vector with primers 5'- ACGCGTCGACATGGCCCAGGTGAAGCTGCAGCAGTC-3 ' (SEQ ID N°29) and 5'- ATAAGAATGCGGCCGCCCGTTTGATTTCCAGATTGGTGCCAGCACC-3 ' (SEQ ID N°30) was then cloned in pCMV/myc/ER (Invitrogen) . The underlined restriction enzyme site in each primer was added to the 5'- end to facilitate subcloning.
  • GST fusion proteins (GST-Pro, GST-CTSL and GST-pepl to pep5) were produced in BL21 E. coli strain, following Amersham Biosciences method. GST-Pro and GST-CTSL were purified from soluble and insoluble fractions, respectively. His fusion proteins (His-ProCTSL, His-CTSL and His-Pro) were produced using the pET system (Novagen) , into BL21.DE3 E. coli strain and were purified using the His-bind resin kit (Novagen) from insoluble cell fractions. His fusion protein ProCTSL-His was generated using a baculovirus/insect expression system.
  • mice 25
  • 25 ⁇ g purified fusion procathepsin L (ProCTSL- His) 25 ⁇ g purified fusion procathepsin L (ProCTSL- His) .
  • mice were intraperitonealy immunized with 25 ⁇ g purified ProCTSL-His in incomplete Freund's adjuvant. This protocol was repeated five times, every 3 weeks.
  • titer of anti-CTSL antibody present in serum was higher than 1/30 000 and three days after a final boost with 50 ⁇ g purified ProCTSL-His, spleen cells were harvested for fusion, which was performed as previously described [Fiandino- Tirel et al .
  • Hybridoma cells surviving selection were analyzed by ELISA for antibody production and cathepsin L specificity.
  • Anti-cathepsin L antibody-producing cells were cloned by limiting dilution.
  • the immunoglobulin class and subclass of light chain type of the mAb was determined using the mouse-hybridoma subtyping kit (Roche) according to the manufacturer's instructions.
  • MAbs were purified using protein G-Sepharose affinity column (Sigma) and antibody concentration was determined using the BCA protein assay reagent (Pierce) .
  • 3D8 ScFv expression vector Total R.NA was isolated from 3D8 hybridoma cells with Trizol (Invitrogen) and submitted to DNAse I (Clontech) treatment, then mRNA was isolated using the mRNA purification kit (Amersham Biosciences) . First-strand cDNA synthesis was prepared with random primers using reverse transcription system kit (Promega) . PCR amplifications of the antibody heavy (VH) and light (VL) variable chains were performed using separate reaction mixtures with primers specific for each chain (Amersham Biosciences) . Purification of PCR products was performed by gel electrophoresis in agarose gel.
  • the DNA bands of 340 and 325 bp corresponding to VH and VL chains respectively were excised and the DNA purified by Sephaglas Bandprep Kit (Amersham Biosciences) .
  • the VH and VL chains were assembled with a linker by two rounds of PCR amplification as described in the Amersham Recombinant Phage Antibody System (RPAS) kit.
  • the 750 bp DNA band corresponding to the ScFv fragment was purified and cloned in pCANTAB5E phagemid vector (Amersham Biosciences) using -S-fi I and Not I restriction enzyme sites.
  • Microtiter plates were coated with 100 ⁇ l GST-CTSL solution at 10 ⁇ g/ml and recombinant phage antibodies were added. Phage binding was detected with a peroxidase-labelled sheep anti-M13 antibody (Amersham Biosciences) . Plates coated with GST were used as a negative control. Among 48 recombinant phage clones analyzed by this assay, 4 clones bound specifically to cathepsin L. The recombinant phage clone, which gave the strongest positive signal in ELISA, was used to infect E. coli HB2151 cells.
  • A375SM melanoma cells were transfected as previously described [Jean et al . (2002) Biochem. J. 361, 173-184] . Transfections were performed with the indicated amount of vector constructs and, in experiments with increasing amount of vector, molar equivalent of parent vector was added (without cDNA insert) to maintain an equal number of vector in each transfection. Twenty four hours after transfection, medium was replaced for 24 h by serum-free medium to generate conditionned media. Cell extracts were prepared 48 h after transfection.
  • cathepsin L cathepsin L
  • ProCTSL procathepsin L
  • Pro proregion alone of procathepsin L
  • Figure 1 different systems of fusion protein expression were used. GST (Part A) or His (Part B) fusion proteins were produced in prokaryotic cells. His fusion protein was also generated using a baculovirus/insect expression system (Part C) .
  • 3D8 mAb was an IgGl with a kappa light chain type. Characterization of 3D8 specificity was performed. As shown in Figure 2, (Part A) 3D8 mAb recognized GST-CTSL but not GST in ELISA. In addition (Part B) , 3D8 reacted with ProCTSL-His (lane 1) or GST-CTSL (lane 3) but not GST-Pro (lane 2) , in immunoblotting assay and (Part C) immunoprecipitated ProCTSL-His (lane 2) . An anti-v-src mAb, with the same IgGl isotype, was used as negative control (Part C, lane 1) .
  • 3D8 mAb was also analyzed on native forms of cathepsin L expressed by DM-4 human melanoma cells: i. e., the 41 kDa procathepsin L secreted by these cells and the two 34 kDa and 29 kDa mature forms present in cell extracts [Jean et al . (1997) . Immunol. Lett. 58, 107-112], et [Frade et al . (1998) Cancer Res. 58, 2733-2736] .
  • the 3D8 anti-cathepsin L mAb recognized (Part D) the 41 kDa procathepsin L in conditioned media (lane 1) , and in cell extracts (lane 2) , and the 29 kDa and 34 kDa mature forms of cathepsin L present only in cell extracts (lane 2) .
  • a polyclonal anti-cathepsin L Ab was used as positive control
  • 3D8 mAb specifically immunoprecipitated procathepsin L from DM-4 cells conditioned media (Part E, lane 2) .
  • 3D8 mAb also recognized in si tu cathepsin L, as demonstrated by specific positive immunofluorescent staining of permeabilized DM-4 melanoma cells (Part F) .
  • the inventor determined the domain of cathepsin L recognized by 3D8 mAb.
  • GST fusion proteins corresponding to several cathepsin L fragments were prepared. Specificities of 3D8 mAb were tested by immunoblot on the fusion peptide produced from each construct ( Figure 3) .
  • the 3D8 ScFv DNA fragment generated from these purified VH and VL DNA products linked into a single gene using a DNA linker fragment, was characterized by a length of approximately 750 bp (lane 4) .
  • This ScFv DNA fragment was cloned in the phagemid pCANTAB5E and transformed into E . coli TGI cells, which allowed production of phage-displayed recombinant ScFv, as described in Material and Methods. Phages, which gave a positive signal in ELISA for binding to cathepsin L via ScFv displayed on their tips, were used to infect E. coli HB2151 cells for production of soluble recombinant ScFv production.
  • 3D8 ScFv by HB2151 cells activated by IPTG was controlled by SDS-PAGE and immunoblot analysis using mAb directed against E tag peptide fused to the 3D8 ScFv in the phagemid pCANTAB5E (Part B) .
  • 3D8 ScFv characterized by a 28 kDa molecular weight, was expressed in the cell lysates (lane
  • ScFv secretion was estimated from the Western Blot with anti-TagE antibody using the public domain NIH Image program. ScFv secretion obtained after induction of bacteria cells (1 OD) with 1 mM IPTG in presence of glucose (0.1 %) at 30°C was set to 1.
  • the inventors analyzed these sequences using several databases.
  • the sequence analysis using mouse Ig database http: //vbase . dnaplot .de) revealed that the 3D8 VH belongs to the VHI family and the 3D8 VL belongs to the VK 4/5 Group IV subgroup and allowed to localize the complementary- determining regions (CDR) ( Figure 5) .
  • Genbank analysis using BLASTn sequence alignment software revealed that VH AND VL sequence share 97% homology with several previously described mouse immunoglobulin variable chain sequences (GenBank accession No. X59180 for VH, and L37409, U12579, M94023 for VL) .
  • A375SM cells were co- transfected with pCMV/myc/ER-3D8/ScFv and pcDNA3.
  • l/V5-His- ProCTSL vectors The pCMV/myc/ER-3D8/ScFv vector was prepared by subcloning cDNA of 3D8 ScFv into the pCMV/myc/ER vector. This vector targets and retains a protein in the endoplasmic reticulum, the subcellular compartment where procathepsin L is normally translated before being carried to lysosome or secreted [Ishidoh et al . (1998) Gene Biol. Chem. 379, 131- 135] .
  • the 3D8 ScFv was therefore expressed as a fusion protein with a N-terminal signal peptide, an endoplasmic reticulum retention signal (peptide SEKDEL) and the myc epitope in C- terminal, which added 2 kDa to the 3D8 ScFv.
  • the pcDNA3.l/V5- His-ProCTSL vector encodes for a fusion procathepsin L with a C-terminal V5 epitope (ProCTSL-V5) , which allowed protein detection with an anti-V5 mAb.
  • conditioned media and cell extracts were prepared from transfected A375SM cells and analyzed by immunoblot.
  • the highly tumorigenic and metastatic human melanoma A375SM cell line was established from nude mice lung metastases produced by the A375-P human cell line isolated from a lymph node metastasis (Kozlowski et al . (1984), J. Natl. Cancer Inst. 72, 913-917 - Li et al .
  • A375SM neo 1, A375SM neo 2, A375SM ScFvl and A375SM ScFv2 were grown in the same medium with G418 (Invitrogen) at 1.2 mg/ml .
  • the 3D8 ScFv cDNA was cloned in pCMV/myc/ER (Invitrogen) to generate pCMV/myc/ER- 3D8ScFv, as described above.
  • the 3D8 ScFv was fused with a N- terminal signal peptide, an endoplasmic reticulum retention signal (peptide SEKDEL) and the myc epitope in C-terminal, to allow expression and retention in the endoplasmic reticulum of melanoma cells.
  • peptide SEKDEL endoplasmic reticulum retention signal
  • A375SM melanoma cells were seeded in 6-well tissue culture plates, cultured for 24 h and transfected by using 5 ⁇ l of Lipofectin reagent (Invitrogen) and 1.5 ⁇ g of pCMV/myc/ER-3D8ScFv expression vector or control pCMV/myc/ER vector. Transfections were carried out according to the manufacturer's instructions. At 16 h following transfection, the medium was changed to complete culture medium. Cells were selected after 48 h with culture medium containing G418 at 1.2 mg/ml. G418-resistant colonies were isolated and established in culture.
  • Conditioned Media and Cell Extract Preparations. Conditioned media and cell extracts were prepared as described above, by incubating A375SM cells for 24 h in serum-free culture medium (Jean et al . (1995) Biochem. J. 312, 961-969). Concentration of solubilized proteins was determined by the bicinchoninic acid (BCA) protein assay reagent (Pierce) .
  • BCA bicinchoninic acid
  • cDNA was used for PCR amplification with specific primers: 5'- ACGCGTCGACATGGCCCAGGTGAAGCTGCAGCAGTC-3 ⁇ and 3 ' -ATAAGAATGCGGCC GCCCGTTTGATTTCCAGCTTGGTGCCAGCACC-5' for 3D8 ScFv, 5 ' -GCGGATCC ATGTATGAGGCCCCCAGATCT-3 ' and 3 ' -GCGGATCCGCACAGTGGGGTAGCTGGCTG- 5' for cathepsin L, 5 ' -GTCTTCACCACCATGGAGAAGGCT-3 ' and 5'- CATGCCAGTGAGCTTCCCGTTCA-3 ' for Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) .
  • GPDH Glyceraldehyde-3 -phosphate dehydrogenase
  • PCR reaction was carried out in a GeneAmp PCR system 9700 (Applied Biosystem) with an initial denaturation step (3 min at 94°C) , followed by 22 cycles of denaturation (30 s at 94°C) , annealing (30 s at 48°C) , and extension (30 s at 72°C) then followed by an extension time of 3 min at 72 °C.
  • PCR products were analyzed on a 3% agarose gel containing ethidium bromide.
  • Immunoblotting Analysis were performed as previously described in Barel et al . (1989) Proc. Natl. Acad. Sci. USA. 86, 10054-10058, using anti-cathepsin L moAb (clone 3D8) prepared as described above, anti -GAPDH moAb (Chemicon International, moAb374) or anti-myc tag moAb (Upstate Biotechnology, clone 9E10) . Bound antibodies were detected with peroxidase-linked goat anti-mouse immunoglobins antibody (Dako) and the enhanced chemiluminescence (ECL) system (Amersham Biosciences) .
  • Dako peroxidase-linked goat anti-mouse immunoglobins antibody
  • ECL enhanced chemiluminescence
  • mice were housed in laminar flow cabinets under specific-pathogen-free conditions and used at 8 weeks of age. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health.
  • TUNEL Tissue sections and the slides were prepared as previously described (Mills et al . (2002) Cancer Res. 62, 5106-5114) Slides were then stained with hematoxylin.
  • A375SM parental cells were used (lane 1) and the inventors also prepared and selected, using the same experimental conditions two other A375SM clones stably transfected with the empty vector and named A375SM neol and A375SM neo2 (lanes 2 and 3) .
  • GAPDH cDNA was amplified from the same cDNA (lanes 1 to 5) .
  • expression of the 3D8 ScFv protein was measured in total cell extracts by western blot using an anti- myc tag moAb ( Figure 8, Panel B) .
  • the 3D8 ScFv protein characterized as expected by a 30 kDa apparent molecular weight, was present at a 3 time higher level in cell extracts of A375SM ScFv2 (lane 5) , than in cell extracts of A375SM ScFvl (lane 4) and, as expected, totally absent in cell extracts of A375SM parental, neol and neo2 cells (lanes 1 to 3) .
  • GAPDH protein was constant, as measured by western blot using anti -GAPDH moAb (lanes 1 to 5) .
  • anti-myc tag moAb described above, we found that 3D8 ScFv protein was not secreted in medium conditioned from A375SM ScFvl and A375SM ScFv2 (data not shown) .
  • A375SM cells transfected with anti-cathepsin L 3D8 ScFv were stained with the anti-myc tag moAb to detect 3D8 ScFv or with anti-cathepsin L moAb.
  • A375SM ScFv transfected cells were stained with the anti-myc tag moAb to detect 3D8 ScFv or with anti-cathepsin L moAb.
  • the inventors observed colocalization of endogenous cathepsin L (green color) and anti-cathepsin L 3D8 ScFv (red color) : indeed, both chromophores totally superimposed in the same image, resulting in the yellow color.
  • A375SM neol or neo2 cells submitted to the same confocal analysis did not present any positive staining with anti-myc tag moAb (data not shown) .
  • Anti-cathepsin L ScFv expression inhibits procathepsin L secretion in stably transfected clones
  • the inventors next analyzed expression and secretion of procathepsin L in parental and in transfected A375SM melanoma cells ( Figure 9) .
  • Expression of cathepsin L mRNA was analyzed by RT-PCR using specific primers of cathepsin L.
  • GAPDH cDNA was amplified from the same cDNA and served as a control for DNA integrity and equal loading. Presence of the different forms of cathepsin L protein was analyzed in total cell extracts and in media conditioned from each A375SM cells, by western blot using anti-cathepsin L moAb .
  • cathepsin L mRNA level was expressed at similar level in parental and in empty vector or ScFv transfected A375SM cells (neol, neo2 , ScFvl or ScFv2) (Panel A); 2) in total cell extracts, cathepsin L protein forms were present in the mature forms characterized by Mr 34,000 and Mr 29,000 and in the proenzyme form characterized by Mr 41,000.
  • Tumorigenic, metastatic and invasiveness phenotype of anti- cathepsin L ScFv transfected melanoma cells Tumorigenic, metastatic and invasiveness phenotype of anti- cathepsin L ScFv transfected melanoma cells.
  • mice were injected i.v. with 10 ⁇ parental and neo or 3D8 ScFv transfected A375SM cells and 60 days later, the number of lung metastases was counted.
  • Table 2 Experimental Lung Metastasis of A375 SM Human Melanoma cells Parental or Transfected with empty vector (neol or neo2) or anti-cathepsin L ScFv gene (ScFvl or ScFv2) in nude mice
  • A373SM parental and A375SM neol or neo2 cells gave numbers of 888 +/-70, 940 +/-71, and 955+/-72, respectively, while under the same experimental conditions, A375SM ScFvl and A375SM ScFv2 only 25+/-5 and 11+/-4, migrated through the Matrigel -coated filters, respectively.
  • anti-cathepsin L transfection inhibited the ability of human melanoma cells to penetrate extracellular matrix, an important component in the process of tumor invasion and metastasis.
  • tumor-associated neovascularization as indicated by microvessel density (MVD) was examined by IHC using anti-CD31 Ab. As shown in Figure 11, it was found a significant reduction in tumor MVD/field in tumors produced by transfected cells. Moreover, the number of TUNEL-positive tumor cells was inversely correlated with MVD in the studied tumors ( Figure 11) •
  • the inventors herein clearly demonstrated that when stably transfected in human melanoma cells, the anti-cathepsin L ScFv also strongly inhibited melanoma tumor growth and metastasis in nude mice. This biological effect was related to the strong inhibition of procathepsin L secretion, while the amount and the ratio of intracellular forms of cathepsin L were not modified.

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Abstract

L'invention se rapporte au clonage et à la caractérisation d'un anticorps monoclonal, connu sous le nom de Mab 3D8, et à leurs fragments, notamment le fragment scFv, spécialement indiqué pour reconnaître la pré-pro-cathepsine L active, la pro-cathepsine L et la cathepsine L et pour inhiber la sécrétion cellulaire de pro-cathepsine L. L'invention se rapporte également à l'utilisation de cet anticorps monoclonal et de son fragment comme médicament pour le traitement de mélanomes. Les procédés correspondants de diagnostic et d'imagerie font également l'objet de cette invention ainsi qu'un procédé d'obtention dudit anticorps.
PCT/IB2003/003581 2002-08-30 2003-08-28 Fragment scfv en rapport avec la cathepsine l humaine et leurs procedes d'utilisation Ceased WO2004020574A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021101991A1 (fr) * 2019-11-18 2021-05-27 Board Of Regents, The University Of Texas System Anticorps monoclonal anti-b7-h3 et ses procédés d'utilisation
WO2022011270A3 (fr) * 2020-07-09 2022-02-17 Lankenau Institute For Medical Research Compositions comprenant des anticorps dirigés contre l'ido-2 humain
WO2024081146A1 (fr) * 2022-10-13 2024-04-18 The Feinstein Institute For Medical Research Utilisation d'anticorps monoclonaux neutralisant la procathepsine l pour traiter la septicémie, la polyarthrite rhumatoïde et d'autres maladies inflammatoires

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021101991A1 (fr) * 2019-11-18 2021-05-27 Board Of Regents, The University Of Texas System Anticorps monoclonal anti-b7-h3 et ses procédés d'utilisation
WO2022011270A3 (fr) * 2020-07-09 2022-02-17 Lankenau Institute For Medical Research Compositions comprenant des anticorps dirigés contre l'ido-2 humain
JP2023533755A (ja) * 2020-07-09 2023-08-04 ランケナウ・インスティテュート・フォー・メディカル・リサーチ ヒトｉｄｏ－２に対する抗体を含む組成物
WO2024081146A1 (fr) * 2022-10-13 2024-04-18 The Feinstein Institute For Medical Research Utilisation d'anticorps monoclonaux neutralisant la procathepsine l pour traiter la septicémie, la polyarthrite rhumatoïde et d'autres maladies inflammatoires

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