WO2010122181A1 - Protéines oligomériques et leurs applications - Google Patents

Protéines oligomériques et leurs applications Download PDF

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WO2010122181A1
WO2010122181A1 PCT/ES2009/070107 ES2009070107W WO2010122181A1 WO 2010122181 A1 WO2010122181 A1 WO 2010122181A1 ES 2009070107 W ES2009070107 W ES 2009070107W WO 2010122181 A1 WO2010122181 A1 WO 2010122181A1
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antibody
antigen
oligomeric protein
polypeptide
target
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PCT/ES2009/070107
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English (en)
Spanish (es)
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Ángel CUESTA MARTÍNEZ
David SÁNCHEZ-MARTÍN
Laura Sanz Alcober
Marta Compte Grau
Félix BONILLA VELASCO
Luis ÁLVAREZ-VALLINA
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Universidad Autónoma de Madrid
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Priority to PCT/ES2009/070107 priority Critical patent/WO2010122181A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the invention relates to an oligomeric protein comprising a plurality of fusion proteins, the same or different, and a label, wherein each fusion protein comprises a polypeptide comprising an antibody or a functionally equivalent fragment of said antibody, and a polypeptide comprising an oligomerization domain.
  • Said oligomeric proteins can be used to detect, visualize and locate targets of interest.
  • Imaging procedures in medicine refer to non-invasive techniques and processes used to create images of the internal appearance of an animal's body, for clinical purposes, for example, medical procedures to diagnose a disease or to locate said disease. In a broad sense, it includes techniques such as radiological sciences, endoscopy, medical thermography and microscopy. Traditionally, laboratory studies to evaluate the activity of anticancer therapies in mice have required the sacrifice of multiple animals at each stage to provide significant data. Imaging techniques, however, allow an accurate evaluation of tumor growth and in real time in laboratory animals, which considerably decreases the number of subjects required.
  • Positron Emission Tomography is a technique of a medical specialty called nuclear medicine and radiology, when combining CT images (examples of 3D images). It is a non-invasive diagnostic and imaging technique capable of measuring the metabolic activity of the different tissues of the human body, especially the central nervous system.
  • PET is based on detecting and analyzing the distribution that a radioisotope administered through an injection adopts inside the body. PET allows, for example, to locate the foci of abnormal cell growth throughout the organism, in a single study and regardless of the anatomical location where the neoplasm occurs (primary or metastatic), since The PET does not evaluate the morphology of the tissues, but their metabolism.
  • the fluorescence technique is an imaging technique that allows you to study the properties of organic or inorganic substances using the fluorescence phenomenon.
  • a component of interest in the sample is specifically labeled, with a fluorescent molecule called fluorophore.
  • the sample is illuminated with light of a certain wavelength / s, which is absorbed by the fluorinated forums, causing light emission at longer wavelengths (or at a different color than the absorbed light).
  • the amount and wavelength of the energy emitted depend on the fluorophore and the chemical environment of the fluorophore.
  • fluorescein isothiocyanate FITC
  • TRITC rhodamine derivatives
  • comarin and cyanine fluorophores "Alexa Fluor” and "DyLight Fluor”
  • fluorophores "Alexa Fluor” and "DyLight Fluor” the cyanine family (eg, Cy3 , Cy5 and Cy 7).
  • the in vivo fluorescence imaging technique is the same as fluorescence microscopy, it differs only in that it works at the macroscopic level.
  • the objects for fluorescence imaging are the whole body of (small) animals. Molecules that absorb in the near infrared region (NIR), 700-1,000 nm, can be used to visualize and investigate molecular targets in vivo, since most tissues generate little NIR fluorescence.
  • NIR near infrared region
  • the most common NIR organic fluorophores are polymethines (eg, pentametine and heptametin).
  • the probes used for in vivo fluorescence imaging can be directed or non-directed. Targeting has the advantage that they can specifically target a target tissue.
  • probes join their targets, while the unbound are removed from the circulation.
  • This approach is very useful for imaging tumors in vivo, since in cancer, certain surface receptors are normally overexpressed.
  • An example of targeted probes can be cyanine-conjugated antibody molecules or "quantum dots.”
  • Targeted smaller molecules can also be used, instead of antibodies, for example folic acid conjugated with an NIR fluorochrome that provides images of activated macrophages involved in inflammatory joint diseases. These molecules would be directed to folic acid receptor.
  • the antibodies have the advantage of their rapid isolation and that they have a high affinity for virtually any antigen.
  • An ideal antibody to locate tumors should be an intermediate-sized multivalent molecule that provides faster penetration into the tumor, greater retention in the target tissue and faster blood removal.
  • bivalent antibodies such as "diabodies” (60 kDa) may be more appropriate for imaging techniques [Williams et al., 2001; Biother cancer. Radiopharm .; 16: 25-35].
  • "Diabodies” are dimeric molecules not covalently bound, formed spontaneously in scFv format with short spacers that connect the variable region of genes. Due to their small size, they are rapidly removed through the kidneys, thus limiting their exposure to the bone marrow, which is often the dose-limiting organ with radiolabelled intact antibodies.
  • Bivalent larger molecules such as “minibodies” (scFv-CH3 dimers) and ScFv 2 -Fc can accumulate in tumors and the latter can be designed with a broad spectrum of serum half-lives, modulating the interaction with the FcRn receptor.
  • the "minibodies” can be ideal for locating tumors, since they penetrate better and have a rapid elimination, thus achieving better tumor-blood proportions than intact immunoglobulins (150 kDa) or Fab 2 fragments (110 kDa).
  • minibodies can be ideal for locating tumors, since they penetrate better and have a rapid elimination, thus achieving better tumor-blood proportions than intact immunoglobulins (150 kDa) or Fab 2 fragments (110 kDa).
  • the inventors have developed a new antibody format, in the form of an oligomeric protein comprising a plurality of fusion proteins, each fusion protein comprising a binding domain of an antibody or a functionally equivalent fragment thereof, and an oligomerization domain. , subsequently marked with a marker suitable for application in diagnostic imaging techniques. Said oligomeric proteins can be used to detect, visualize or locate a target of interest, such as an antigen associated with a pathological alteration.
  • said oligomeric proteins are effective agents for the location of tumor deposits in vivo.
  • the inventors have generated several trimeric proteins, sometimes identified as "trimerbodies", one specific against the NIP hapten, another against the human carcinoembryonic antigen (CEA), and another that recognizes an angiogenesis-associated laminin epitope , and have characterized them both in vitro and in vivo.
  • CCA human carcinoembryonic antigen
  • an angiogenesis-associated laminin epitope angiogenesis-associated laminin epitope
  • the invention relates to an oligomeric protein comprising a plurality of fusion proteins, the same or different, and a marker (M), wherein each fusion protein comprises:
  • polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; and (a) a polypeptide (B) comprising an oligomerization domain.
  • the invention relates to a process for obtaining said oligomeric protein.
  • the invention relates to the use of said oligomeric protein, in a method for the detection, visualization or localization of a target.
  • a method for the detection, visualization or localization of a target comprising the use of said oligomeric protein constitutes a further aspect of this invention.
  • the invention in another aspect, relates to a composition comprising said oligomeric protein and an appropriate medium.
  • the invention in another aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising said oligomeric protein and a pharmaceutically acceptable carrier.
  • the invention in another aspect, relates to a kit comprising said oligomeric protein.
  • kit comprising said oligomeric protein.
  • the use of said kit for the detection, visualization or location of a target constitutes an additional aspect of this invention.
  • Figure 1 describes the molecular characterization of purified trimerbodies.
  • A Elution profile of the gel filtration experiment of the L36 [L36 trimerbody (darker lines) and L36 scFv (lighter line)] constructs. The exclusion volumes (V 0 ) and total (V T ) are indicated. Elution volumes of selected molecular weight markers are indicated by arrows, as well as their corresponding molecular weights. To make it clear, the absorbance of both chromatograms has been scaled and moved in position in the figure.
  • trimerbodies were demonstrated by ELISA on plates upholstered with bovine serum albumin (BSA), murine laminin-1, BIP-conjugated NIP hapten (NIPio-BSA) and human CEA; and (C) by flow cytometry in CEA-negative and CEA-positive tumor cells. Isotype control (gray histogram), anti-CEA trimerbody (solid line) and anti-NIP trimerbody (dotted line) are shown.
  • D Analysis of the interaction of scFv B 1.8 and trimerbody B 1.8 to NIP 10 - BSA using BIAcore.
  • the curves show the data obtained after subtraction of the binding response to a reference surface upholstered with BSA (1900 UR), to eliminate the effects of non-specific binding. Representative sensograms are also shown corresponding to the adjusted affinity binding curves of the scFv Bl .8 trimerbodies and Bl .8 trimerbody (dil. 1200 and 800 nM) in HBS-EP buffer injected onto the same cells as above.
  • Figure 2 describes binding activities of mono-specific and bi-specific trimerbody molecules.
  • Supernatants of 293T cells transfected with pEGFP-Nl, pCR3.1-L36-NCl ES “ , or pCEP4-B1.8-NCl ES” are compared with supernatants of 293T cells transfected with pCR3.1-L36-NCl ES " and pCEP4 -B1.8-NCl ES " , using direct ELISA (A), using plates upholstered with BSA, conjugates NIPio-BSA and laminin-1, and by ELISA sandwich (B) using plates upholstered with laminin.
  • A direct ELISA
  • BSA conjugates NIPio-BSA and laminin-1
  • B ELISA sandwich
  • Figure 3 describes the stability of the trimerbody molecules in serum.
  • the purified L36 trimerbodies incubated in human or mouse serum at 37 0 C, as detailed in Example 1 (Materials and Methods) and functionality of the reaction mixtures were analyzed by ELISA.
  • Figure 4 describes the location of antibodies conjugated to a fluorophore (cyanine 5; Cy5) in a gastric cancer model (A-positive CE) Experimental in nude mice.
  • sc subcutaneous MKN45 gastric carcinoma
  • b bladder
  • t tumor.
  • Figure 5 describes the location of trimerbodies conjugated with a fluorophore (cyanine 5; Cy5) in several experimental human cancer models (CE A-negative) in nude mice.
  • CUA fluorophore
  • Figure 5 Near-infrared images of nude mice carrying human HT 1080 fibrosarcomas s.c. (A) or human cervix carcinomas (HeLa) s.c. (B). The images were taken 3, 24 and 48h after intravenous injection (i.v.) with trimerbodies labeled with Cy5.
  • Figure 6 describes a structural model of trimerbody. Side view (A) and top (B) of the molecular model of the L36 anti-laminin trimerbody.
  • the term "target” includes a part of a macromolecule that is recognized by an antibody; In a particular embodiment, said target is an antigenic epitope or determinant of an antigen.
  • An "antigen”, as used herein, refers to a substance that induces the generation of antibodies and can generate an immune response.
  • the antigens are usually proteins or polysaccharides, and include parts of cells, bacteria, viruses and other microorganisms.
  • an antigen can be (i) exogenous, that is, an antigen that has entered the body of a subject from the outside, for example, by inhalation, ingestion, injection, etc .; (ii) endogenous, that is, an antigen generated inside the cell as a result of normal cellular metabolism or due to an intracellular infection, eg, bacterial, viral, etc., including xenogeneic (heterologous), autologous and idiomatic or allogeneic antigens ( homologues); or (iii) autoantigen, that is, a normal protein (or a complex of proteins) that is recognized by the immune system of a subject suffering from an autoimmune disease.
  • exogenous that is, an antigen that has entered the body of a subject from the outside, for example, by inhalation, ingestion, injection, etc .
  • endogenous that is, an antigen generated inside the cell as a result of normal cellular metabolism or due to an intracellular infection, eg, bacterial, viral, etc
  • tumor antigens or neoantigens
  • MHC I complex molecules majority of histocompatibility type I
  • MHC II type II
  • TSA tumor-specific antigens
  • TAA tumor antigens that are presented by both tumor cells and normal cells
  • TAA tumor associated antigens
  • antibody refers to an immunoglobulin, or a fragment thereof, capable of binding to an antigen and includes all types of antibodies, eg, polyclonal antibodies, monoclonal antibodies, etc., as well as antibody fragments such as Fab, F (ab ') 2 , Fab' fragments, single chain Fv fragments (scFv), monodomain antibodies (V HH ), diabodies, etc.
  • said antibody is a fragment of an antibody, such as, for example, a Fab, F (ab ') 2 , Fab', scFv or V HH fragment, which can be produced directly by host cells recombinant
  • the antibody is an scFv.
  • An “Fv” fragment is a minimum antibody fragment that contains a complete antigen recognition and antigen binding site. This region is formed by a variable domain of a heavy chain (VH) and a variable domain of a light chain (VL) not covalently associated.
  • VH variable domain of a heavy chain
  • VL variable domain of a light chain
  • the "scFv” fragments comprise the VH and VL domains of an antibody, forming a single polypeptide chain (VH-VL).
  • the Fv polypeptide further comprises a linker polypeptide (linker) between the VH and VL domains (VH-linker-VL) that allows the scFv to form the desired structure for antigen binding.
  • linker linker polypeptide
  • Additional information on scFv can be found in "The Pharmacology of Monoclonal Antibodies", vol. 113, Rosenburg and Moore eds., Springer-Verlag, N. Y., p. 269-315 (1994).
  • diabodies refers to an antibody fragment with two antigen binding sites, comprising a VH connected to a VL in the same polypeptide chain (VH-linker-VL).
  • V HH refers to monodomain antibodies [Nguyen et al, Adv. Immunol (2001), 79: 261-96 .; Nguyen et al, Embo J. (2000), 19 (5): 921-30 .; Sheriff & Constantine, Nat. Struct. Biol. (1996), 3 (9): 733-6).
  • camelid species camels, dromedaries, llamas, etc.
  • a part of their antibodies lacks a light chain, the antigen recognition zone being constituted solely by the VH domain [Desmyter et al., J. Biol. Chem. (2002), 277 (26): 23645-50].
  • VH domain differs from the VH domains of other antibodies in that their antigen recognition zones are formed by larger loops.
  • the VH domains of camelid antibodies are called V HH .
  • This domain (V HH ) of approximately 15 kDa can be expressed in the periplasm of E. coli while maintaining its antigen binding capacity.
  • V HH are more stable molecules than scFv, and unlike they rarely add.
  • the V HH domains possess an intramolecular SS bond that stabilizes their tertiary structure and is necessary for proper folding [Desmyter et al, J. Biol. Chem. (2002), 277 (26 ): 23645-50].
  • the term "marker”, as used herein, refers to any type of molecule that indicates the existence of a chemical, physical or biological process, which can be introduced into an organism in order to examine some property.
  • the tides can be radioactive or non-radioactive. Radioactive or isotopic marking has been used more frequently, although it is progressively displaced by non-radioactive methods. Although there is no consensus on the ideal tide, in most cases it is done with 32 P. Since the short half-life of this isotope (14 days) creates difficulties in the preparation, commercialization and routine use of the tests, they are used also other alternatives ( 35 S, 3 H, 125 I, 14 C, etc.).
  • the detection is done by fluorometry or by adding a specific substrate whose enzymatic transformation by the marker enzyme can be detected by any of the following principles: (i) spectrophotometry: absorption measurement of light by reaction products, either soluble or insoluble, in ultraviolet or visible (colorimetry), (ii) fluorometry: measurement of the light emitted by reaction products that possess a molecule fluorescent, or (iii) chemiluminescence: measure of light emitted as a result of reactions with this characteristic.
  • spectrophotometry absorption measurement of light by reaction products, either soluble or insoluble, in ultraviolet or visible (colorimetry)
  • fluorometry measurement of the light emitted by reaction products that possess a molecule fluorescent
  • chemiluminescence measure of light emitted as a result of reactions with this characteristic.
  • fluorometry can be used for in vivo marking.
  • a component of interest can be specifically labeled in a sample, with a fluorescent molecule, called fluorophore.
  • fluorophore a fluorescent molecule
  • fluorescein isothiocyanate FITC
  • TRITC rhodamine derivatives
  • New generations of fluorophores include the "Alexa Fluor” and the "DyLight Fluor", more photostable, brighter and less sensitive to pH changes than other known markers.
  • Cy3 markers are yellow-orange (excitation at approximately 550 nm, emission at approximately 570 nm), while Cy5 are fluorescent in the red region (approximately 650/670 nm). They are normally synthesized with reactive groups in one or both side chains of nitrogen, so that they can be chemically coupled to both nucleic acids and proteins, being widely used in diagnostic imaging techniques.
  • organic fluorophores for near infrared (NIR) the most common are polymetins of general formula (I)
  • Ri and R 2 independently represent C 1 -C 10, sulphoalkyl
  • R 3 and R 4 independently represent sulfoalkyl C1 - C10, C1 - C10 haloalkyl, or C1 - C10 hydroxycarbonyl;
  • Y is C, O, S; and n is an integer between 1 and 10.
  • the invention relates to an oligomeric protein, hereinafter oligomeric protein of the invention, comprising a plurality of fusion proteins, the same or different, and a label (M), wherein each fusion protein comprises:
  • the marker (M) present in said oligomeric protein of the invention can be practically any marker; however, for its applications in diagnostic imaging techniques, said marker (M) is a suitable marker for said techniques, for example, (i) a fluorophore, such as a fluorophore for imaging by fluorescence in the near-infrared area , eg Cy5, Cy7 etc .; (ii) a radionuclide for radioimmunoscintigraphy [compounds that emit gamma particles: 99mTc, 111 In, 131 I, rhenium 186 ( 186 Re)] or for positron emission tomography (PET) (PET marker); etc.
  • a fluorophore such as a fluorophore for imaging by fluorescence in the near-infrared area , eg Cy5, Cy7 etc .
  • a radionuclide for radioimmunoscintigraphy compounds that emit gamma particles:
  • said marker (M) is a fluorophore, such as Cy5, Cy7, etc., or a PET marker, such as 64 Cu, 68 Ga, 18 F, 86 Y, 76 Br, 89 Zr, 124 I , etc.
  • a fluorophore such as Cy5, Cy7, etc.
  • a PET marker such as 64 Cu, 68 Ga, 18 F, 86 Y, 76 Br, 89 Zr, 124 I , etc.
  • markers type TEP markers type TEP
  • fluorophores eg, NIR fluorescent markers
  • the oligomeric protein of the invention comprises a plurality of fusion proteins, the same or different, and a marker (M), each of said fusion proteins comprising a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; and a polypeptide (B) comprising an oligomerization domain.
  • said oligomeric protein of the invention comprises a plurality of equal fusion proteins; in which case, each of said fusion proteins comprises the same polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; and the same polypeptide (B) comprising an oligomerization domain.
  • said oligomeric protein of the invention comprises a plurality of different fusion proteins, for example, two or more different fusion proteins.
  • one of said fusion proteins comprises a polypeptide (Al) comprising an antibody (1) or a functionally equivalent fragment of said antibody (1); and a polypeptide (B) comprising an oligomerization domain
  • another one of said fusion proteins comprises a polypeptide (A2) comprising an antibody (2) or a functionally equivalent fragment of said antibody (2); and a polypeptide (B) comprising an oligomerization domain, wherein said antibodies (1) and (2) are different.
  • the total number of fusion proteins present in the oligomeric protein of the invention will depend on the oligomerization domain, depending on the oligomerization domain being possible virtually any combination between said same or different fusion proteins, as long as the oligomerization domain is maintained.
  • the fact that the oligomeric protein of the invention comprises two or more different fusion proteins allows to recognize and interact with two or more different targets (eg, antigens).
  • the oligomeric protein of the invention may contain two different fusion proteins, as previously mentioned; or, alternatively, two fusion proteins equal to each other and a different fusion protein; or, alternatively, two fusion proteins equal to each other and two other fusion proteins different but equal to each other; or, alternatively, two fusion proteins equal to each other and two other fusion proteins different from each other and different from the two same fusion proteins; or, alternatively, three different fusion proteins from each other; etc.; in general, any combination of fusion proteins comprising a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody, and a polypeptide (B) comprising an oligomerization domain, in which said domain is maintained.
  • oligomerization comprising a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody, and a polypeptide (B) comprising an oligomerization domain, in which said domain is maintained.
  • the oligomeric protein of the invention comprises two or more fusion proteins, the same or different; therefore, it can recognize and interact with a single target (antigen), or, alternatively, with two or more different targets (eg, antigens), which makes it a very versatile monospecific reagent (that is, when it recognizes a single target or antigen), or multispecific (that is, when it recognizes more than one target or antigen, eg, bispecific, if it recognizes two different targets or antigens, triespecific, if it recognizes three different targets or antigens, etc.
  • the polypeptide (A) it comprises an antibody or a functionally equivalent fragment of said antibody, said antibody, or functionally equivalent fragment, recognizes a target, such as an antigenic epitope or determinant of an antigen; in a particular embodiment, said antigen is a tumor antigen, such as CEA (Begent RH et al. Nat Med. (1996) Sep; 2 (9): 979-84), or a non-tumor antigen that is expressed in areas of active angiogenesis in the peritumoral microenvironment, such as or the EDB domain of fibronectin (Ebbinghaus C et al. Curr Pharm Des. (2004); 10 (13): 1537-49).
  • a tumor antigen such as CEA (Begent RH et al. Nat Med. (1996) Sep; 2 (9): 979-84)
  • a non-tumor antigen that is expressed in areas of active angiogenesis in the peritumoral microenvironment, such as or the EDB domain of fibronectin (Ebbinghaus C
  • the polypeptide (A) comprises an antibody that recognizes a target or a functionally equivalent fragment of said antibody that recognizes said target.
  • Virtually any antibody, or functionally equivalent fragment thereof, that recognizes a target can be used in the present invention, for example, a monoclonal antibody (AcM) or a polyclonal antibody (AcP); alternatively, said polypeptide (A) may contain a functionally equivalent fragment of an antibody, such as an antibody fragment that maintains the ability to recognize the target recognized by the complete antibody from which it derives, eg, an scFv, a bispecific antibody or diabody that recognizes said target either in its complete recombinant format (Fab + Fc), or a monodomain antibody V HH preferably, a scFv (Example 1).
  • the polypeptide (A) comprises an antibody that recognizes a target or a functionally equivalent fragment of said antibody that recognizes said target.
  • Virtually any antibody, or functionally equivalent fragment thereof, that recognizes a target can be used in the present invention, eg, an AcM or polyclonal AcP monoclonal antibody; alternatively, said polypeptide (A) may contain a functionally equivalent fragment of an antibody, such as an antibody fragment that maintains the ability to recognize the target recognized by the complete antibody from which it derives, eg, an scFv, a bispecific antibody or diabody that recognizes said target or in its complete recombinant format (Fab + Fc), preferably, an scFv (Example 1).
  • said polypeptide (A) comprises a recombinant scFv derived from the anti-laminin AcM L36 (Example 1) [scFv L36] containing the heavy chain variable region (VH) of the fused L36 monoclonal antibody, through a spacer, such as a peptide comprising the sequence (Gly-Ser) 4 (or a sequence of the type Gly-Ser-Pro-Gly, or comprises the sequence Leu-Glu-Gly-Ala-Gly-Gly-Ser- Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser), to the variable region of the light chain (VL) of the AcM L36 and whose sequence has already been described [Sanz L et to the.
  • a spacer such as a peptide comprising the sequence (Gly-Ser) 4 (or a sequence of the type Gly-Ser-Pro-Gly,
  • said polypeptide (A) comprises a recombinant scFv derived from the hapten-specific NIP Bl .8 (Example 1) containing the heavy chain (VH) variable region of the fused AcM B 1.8, through a spacer, to the variable region of the light chain (VL) of the AcM Bl .8;
  • said spacer comprises a sequence of type Gly-Ser-Pro-GIy, or of type (Gly-Ser) 4, or it comprises the sequence Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly -Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser.
  • said polypeptide (A) comprises a recombinant scFv derived from the human embryonic carcinogen antigen (CEA) MMA23 (Example 1) containing the heavy chain (VH) variable region of the fused AcM MFE23, through from a spacer, to the variable region of the light chain (VL) of the AcM MFE23;
  • said spacer comprises a sequence of type Gly-Ser-Pro-Gly, or of type (Gly-Ser) 4 , or it comprises the sequence Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly -Gly-Ser-Ser-Gly-Ser-Asp- Gly-Ala-Ser-Gly-Ser.
  • Said polypeptide (A) can recognize and bind to a target, eg, present in a tumor antigen, and, as a result of that binding, locate the target (antigen) and allow its visualization by appropriate imaging techniques (eg, fluorescence, PET) , etc.) due to the presence of the marker (M) in the oligomeric protein of the invention.
  • a target eg, present in a tumor antigen
  • imaging techniques eg, fluorescence, PET
  • Polypeptide (B) comprises an oligomerization domain.
  • Said oligomerization domain can be practically any domain that allows the formation of oligomers, for example, dimers, trimers, tetramers, etc., of peptides or proteins, which can be expressed recombinantly and form a protein oligomer of the protein that He understands.
  • said oligomerization domain is a trimerization domain, such as the trimerization domain of the NCl domain of collagen XVIII or mammalian collagen XV.
  • said NC1 domain of collagen XVIII (and collagen XV) comprises a trimerization domain and the endostatin (ES) domain linked by hinge peptides.
  • the fusion protein present in the oligomeric protein of the invention comprises a polypeptide (B) comprising the NCl domain of collagen XV or the NCl domain of collagen XVIII which it contains the trimerization domain but to which the domain of ES (NC 1 ES " ) has been removed as well as all or part of the hinge peptides existing between said domains.
  • the invention provides an oligomeric protein of the invention comprising a marker (M) and a plurality of fusion proteins, the same or different, wherein each fusion protein comprises:
  • polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof;
  • polypeptide (B) comprising the NC1 ES " domain of mammalian collagen XVIII.
  • any other domain similar to the NC 1 domain of collagen XVIII that comprises an oligomerization domain can be used in the practice of the present invention, in order to generate the oligomeric protein of the invention.
  • the oligomeric protein of the invention may be constituted by 2 or more, for example, 3, 4, 5, or more fusion proteins. , the same or different from each other, giving rise to dimers, trimers, tetramers, pentamers, etc. of the fusion proteins provided by this invention.
  • said oligomeric protein of the invention is a trimer and comprises 3 fusion proteins provided by this invention; Since said fusion proteins incorporate an antibody or a functionally equivalent fragment thereof, said trimers have been referred to by the inventors under the generic name "trimerbody” (singular) or "trimerbodies” (plural).
  • said oligomeric protein of the invention is a trimer and comprises 3 equal fusion proteins provided by this invention; in another more concrete embodiment, said oligomeric protein of the invention is a trimer and comprises 3 fusion proteins provided by this invention, of which two are equal between yes and the other is different; and, in another more concrete embodiment, said oligomeric protein of the invention is a trimer and comprises 3 different fusion proteins provided by this invention.
  • the fusion protein provided by this invention may also contain, if desired, a third polypeptide (C) comprising the amino acid sequence of a flexible binding peptide between said polypeptides (A) and (B) and / or a peptide (D) to facilitate the isolation or purification of the fusion protein.
  • C third polypeptide
  • Said polypeptide (C) can comprise virtually any peptide sequence that defines a flexible binding peptide.
  • flexible binding peptides include sequences of the Gly-Ser-Pro-Gly type or the sequence (Gly-Ser) 4 .
  • said flexible binding peptide comprises the sequence Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser- Gly-
  • said fusion protein may contain, if desired, a peptide (D) capable of being used for the purpose of isolation or purification of the fusion protein, such as a peptide tag ("tag").
  • Said peptide (D) may be located at any position of the fusion protein that does not alter the functionality of any of the polypeptides (A) and (B); by way of illustration, not limitation, said peptide (D) may be located next to the polypeptide (B).
  • Virtually any peptide or peptide sequence that allows the isolation or purification of the fusion protein can be used, for example, polyhistidine sequences, peptide sequences capable of being recognized by antibodies that can be used to purify the resulting fusion protein by immunoaffinity chromatography , such as tag peptides, for example, epitopes derived from the hemagglutinin (HA) of influenza virus, C-myc, FLAG, V5, etc.
  • HA hemagglutinin
  • the oligomeric protein of the invention can be obtained by a method comprising contacting an oligomeric protein comprising a plurality of fusion proteins, wherein each fusion protein comprises:
  • polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof;
  • a po lip appointedti do (B) comprising an oligomerization domain, with a marker (M) under conditions that allow the binding of said marker (M) to the oligomeric protein.
  • Said process constitutes an additional aspect of this invention.
  • the oligomeric protein of the invention due to its own characteristics, can be used in numerous applications, for example, in the visualization of antigens by imaging techniques, both in vitro and in vivo (eg, fluorescence, PET, etc.), in the location of antigens, etc., in general, in the identification of pathological alterations associated with neo-expression or overexpression of antigens, eg, inflammatory pathologies (eg, integrins, etc.), vascular pathologies (eg, atheroma plaques, etc. .), tumor pathologies, etc.
  • said antigens are tumor antigens.
  • the invention relates to the use of an oligomeric protein of the invention, in a method for the detection, visualization or localization of a target, such as an antigen, by an appropriate technique, for example, a Image technique
  • a target such as an antigen
  • said target is an antigen that expresses de novo (i.e., that in normal conditions it is not expressed but that in a pathological alteration is expressed) or overexpressed (that is, that in normal conditions it is expressed at a baseline level and its expression increases in case of a pathological alteration), for example, in inflammatory, vascular, tumor pathologies, etc.
  • said target is a tumor antigen.
  • the invention relates to a method for the detection, visualization or localization of a target, such as an antigen, by an appropriate technique, for example, an imaging technique, comprising the use of an oligomeric protein. of the invention.
  • a target such as an antigen
  • said target is an antigen that expresses de novo or is overexpressed in a pathological alteration, for example, in inflammatory, vascular, tumor pathologies, etc.
  • said target is a tumor antigen.
  • the oligomeric protein of the invention will be in an appropriate and suitable medium for administration.
  • the invention relates to a composition
  • a composition comprising an oligomeric protein of the invention together with at least one appropriate medium, such as a medium that does not alter the stability of said oligomeric protein of the invention, for example, PBS, physiological saline, etc.
  • a medium constituted by a system for delivery and release of compounds, for example, a viral or non-viral vector (e.g., nanoparticles based on biocompatible polymers, liposomes, etc.).
  • a viral or non-viral vector e.g., nanoparticles based on biocompatible polymers, liposomes, etc.
  • Such vectors are, in general, known to those skilled in the art.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an oligomeric protein of the invention together with at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the invention comprises at least one oligomeric protein of the invention in an effective amount.
  • the term "effective amount" refers to the amount of oligomeric protein of the invention calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of the protein itself. oligomeric, the target (antigen) to visualize or locate, etc.
  • the pharmaceutical composition provided by this invention can be administered by any appropriate form of administration, for example, parenterally.
  • Vehicles that can be used in the preparation of the pharmaceutical composition provided by this invention will depend, among other things, on the manner of administration of said pharmaceutical composition.
  • a review of the various forms of administration of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faul ⁇ i Trillo, Luzán 5, S.A. of Editions, 1993.
  • the oligomeric proteins of the invention can be isolated and, if desired, easily purified, by conventional methods known to those skilled in the art, for example, by affinity chromatography.
  • the oligomeric protein of the invention is oligomeric in solution and possesses excellent stability and binding capacity to the target (antigen), recognizing with high efficiency both purified antigens immobilized on a plaque as antigens expressed or overexpressed in pathological alterations (eg, on the surface of a tumor cell).
  • the oligomeric proteins of the invention possess a greater binding signal than the monomeric antibody (which constitutes a structural unit thereof) and, apparently, a smaller dissociation, consistent with the multivalent binding to the antigen.
  • an oligomeric protein of the invention eg, the anti-NIP oligomeric protein - Example 1
  • NIP-BSA antigen-BSA
  • scFv that remain un interacting have an area of influence approximately 1 1 times, approximately, than other bivalent formats, such as diabodies and minibodies, increasing the likelihood of a second effective interaction.
  • the flexibility of the oligomeric proteins of the invention is also an advantage over other more compact or rigid structures of other formats (eg, collabody), since it increases the accessibility of scFv, which is a critical parameter for target location. in vivo
  • the multimerization of scFv constructs has numerous advantages for in vivo applications over other recombinant antibodies (diabodies and minibodies) that have shown their potential as in vivo localization agents.
  • the oligomeric proteins of the invention are intermediate sized multivalent molecules that exhibit high stability under physiological conditions.
  • the potential of said oligomeric proteins of the invention, in particular trimer (trimerbodies) for in vivo localization has been studied in experimental models of human cancer in nude mice (Example 1).
  • the invention relates to a kit comprising an oligomeric protein of the invention.
  • said target is an antigen that expresses de novo or is overexpressed in a pathological alteration, for example, in inflammatory, vascular, tumor pathologies, etc.
  • said target is a tumor antigen.
  • the kit of the invention is a product containing the different products (eg, oligomeric protein of the invention, additional reagents, etc.) forming the packaged composition so that it can be transported, stored and used.
  • the kits of the invention can thus contain one or more suspensions, syringes, etc., as well as means for reconstituting the oligomeric protein of the invention if it is in lyophilized form.
  • Other components that may be present in the kit of the invention is a package that allows to keep the formulations of the invention within certain limits. Suitable materials for preparing such containers include glass, plastic, polyethylene, polypropylene, polycarbonate and the like, bottles, vials, paper, sachets and the like.
  • kit of the invention may contain instructions for use. These instructions can be found in the form of printed material or in the form of electronic support that can store the instructions such that they can be read by a subject, such as electronic storage media (magnetic discs, tapes and the like), optical media (CD- ROM, DVD) and the like.
  • the media may additionally or alternatively contain websites on the Internet by providing such instructions.
  • the oligomeric protein of the invention comprises a plurality of fusion proteins (same or different), depending on the oligomerization domain present in the polypeptide (B), and each fusion protein comprises:
  • said fusion protein may include a polypeptide (C) comprising the amino acid sequence of a flexible binding peptide between said polypeptides (A) and (B) and / or a peptide (D) to facilitate the isolation or purification of the fusion protein.
  • the fusion protein provided by the invention can be obtained by conventional methods.
  • said fusion protein can be obtained by fusion of said polypeptides, obtained either by chemical peptide synthesis methods or by recombinant DNA technology.
  • said fusion protein provided by this invention can be obtained through the use of recombinant DNA technology, for which gene constructs, expression cassettes and vectors will be generated. corresponding.
  • the fusion proteins when the fusion proteins are the same, they can be obtained by incorporating the DNA encoding said fusion protein into an appropriate host cell (eg, bacteria, yeasts, animal cells, etc.).
  • the fusion proteins when they are different, they can be obtained, in general, by incorporating the DNAs encoding said fusion proteins into an appropriate host cell, where the different DNAs can be part of the same gene construct or gene constructs different (eg, by co-transformation or co-transfection of appropriate host cells).
  • the invention relates to a gene construct, hereinafter referred to as the gene construct of the invention, comprising at least: a) a first nucleic acid sequence (A '), which comprises the nucleotide sequence that encodes a polypeptide (A), wherein said polypeptide (A) comprises an antibody or a functionally equivalent fragment of said antibody; and b) a second nucleic acid sequence (B ') encoding a polypeptide (B) comprising an oligomerization domain, wherein the 3' end of said first nucleic acid sequence (A ') is attached to the 5' end of said second nucleic acid sequence (B '), or, alternatively, the 5' end of said first nucleic acid sequence (A ') is attached to the 3' end of said second nucleic acid sequence (B ').
  • a ' a first nucleic acid sequence
  • A comprises the nucleotide sequence that encodes a polypeptide (A)
  • said polypeptide (A) comprises an antibody
  • the nucleic acid sequence (A ') comprises the nucleotide sequence encoding a polypeptide (A) comprising an antibody that recognizes a target or a functionally equivalent fragment of said antibody that recognizes said target.
  • a target include an antigenic epitope or determinant of an antigen, for example, an antigen that is expressed de novo or overexpressed in a pathological alteration, for example, in inflammatory, vascular, tumor pathologies, etc.
  • said target is a tumor antigen.
  • the nucleic acid sequence (A ') encodes a polypeptide (A) comprising an antibody, or a functionally equivalent fragment thereof, that recognizes a specific target.
  • a polypeptide (A) comprising an antibody, or a functionally equivalent fragment thereof, that recognizes a specific target.
  • Virtually any antibody, or functionally equivalent fragment thereof, that recognizes a target can be used in the present invention, eg, an AcM, an AcP, a scFv, a bispecific antibody or diabody, a V HH , etc.
  • said nucleic acid sequence (A ') encodes a polypeptide (A) comprising a recombinant scFv derived from the anti-laminin L36 AcM [scFv L36], or a hapten-specific recombinant scMv derived from the AcM Bl.8 NIP, or a recombinant scFv derived from the ACM MFE23 specific to the human carcinoembryonic antigen (CEA) (Example 1).
  • a polypeptide (A) comprising a recombinant scFv derived from the anti-laminin L36 AcM [scFv L36], or a hapten-specific recombinant scMv derived from the AcM Bl.8 NIP, or a recombinant scFv derived from the ACM MFE23 specific to the human carcinoembryonic antigen (CEA) (Example 1).
  • the oligomeric protein of the invention comprises a plurality of different fusion proteins, for example, two or more different fusion proteins, each of them would be encoded by the corresponding DNA sequence, which could be in a single gene construct or in different gene constructs.
  • the nucleic acid sequence (B ') comprises the nucleotide sequence encoding a polypeptide (B) comprising an oligomerization domain.
  • an oligomerization domain allows the formation of oligomers (eg, dimers, trimers, tetramers, etc., of peptides or proteins).
  • said oligomerization domain is a trimerization domain, such as the trimerization domain of the NCl domain of collagen XVIII or collagen XV; in a specific embodiment, said oligomerization domain is the trimerization domain of the NCl domain of collagen XVIII or collagen XV to which all or part of the ES domain has been removed. Therefore, in a particular and preferred embodiment, the nucleic acid sequence (B ') comprises the nucleotide sequence encoding the mammalian collagen NCl domain XVIII or the mammalian collagen XV NCl domain, in which, optionally, all or part of the ES domain has been removed.
  • NCl domains of collagen XV and XVIII are known; by way of illustration, the sequence of the NC 1 domain of collagen XVIII has been previously described by Sasaki et al. [Sasaki et al. Structure, function and tissue forms of the C- globular terminal domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J. 1998 Aug 3; 17 (15): 4249-56].
  • the 3 'end of said nucleic acid sequence (A') is attached, in a particular embodiment, to the 5 'end of said nucleic acid sequence (B'); alternatively, in another particular embodiment, the 5 'end of said nucleic acid sequence (A') is attached to the 3 'end of said nucleic acid sequence (B').
  • the nucleic acid sequence (A ') is not fused directly to the nucleic acid sequence (B') but it is advantageous to introduce a flexible binding peptide (or spacer peptide) between the polypeptides encoded by said acid sequences nucleic (A ') and (B').
  • the gene construct of the invention may also contain, in addition, a third nucleic acid sequence (C) containing the nucleotide sequence encoding a flexible binding peptide located between said nucleic acid sequences ( A ') and (B').
  • the 5 'end of said nucleic acid sequence (C) is attached to the 3' end of said nucleic acid sequence (A ') and the 3' end of said nucleic acid sequence (C) is attached at the 5 'end of said nucleic acid sequence (B'); alternatively, in another particular embodiment, the 3 'end of said nucleic acid sequence (C) is attached to the 5' end of said nucleic acid sequence (A ') and the 5' end of said nucleic acid sequence (C) is attached to the 3 'end of said nucleic acid sequence (B').
  • said spacer peptide (C) is a peptide with structural flexibility. Virtually any peptide with structural flexibility can be used.
  • said flexible peptide may contain repetitions of amino acid residues, in particular of GIy and Ser residues or any other suitable amino acid residue repeat.
  • Virtually any peptide sequence that defines a flexible binding peptide can be used in the present invention.
  • Illustrative examples of flexible binding peptides include sequences of the Gly-Ser-Pro-Gly type (GSPG) or the sequence (Gly-Ser) 4 .
  • said flexible binding peptide comprises the sequence Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser- Gly-Ser Therefore, in a particular embodiment, the gene construct of the invention comprises, in addition to said nucleic acid sequences (A) and (B) a third nucleic acid sequence (C) comprising the nucleotide sequence encoding the Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser peptide.
  • the length and composition of the spacer peptide may vary; however, in a particular embodiment, it will be adjusted (greater or lesser length, greater or lesser rigidity) depending on the nature of the target (antigen) recognized by the antibody to achieve the best functional properties.
  • the gene construct of the invention may contain, if desired, a nucleic acid sequence encoding a peptide capable of being used for purposes. of isolation or purification of the fusion protein.
  • the gene construct of the invention includes, if desired, a nucleic acid sequence (D ') containing the nucleotide sequence encoding a peptide capable of being used for isolation or purification purposes, known as peptide tag ("tag").
  • Said nucleic acid sequence (D ') may be located in any position that does not alter the functionality of any of the polypeptides [(A) and (B)] expressed by said nucleic acid sequences (A') and (B ') .
  • said nucleic acid sequence (D ') may be located downstream of the 3' end of said nucleic acid sequence (B ').
  • any peptide or peptide sequence that allows the isolation or purification of the fusion protein can be used, for example, a histidine tail (eg, 6 His residues), a peptide sequence capable of being recognized by an antibody that can serve to purify the resulting fusion protein by immunoaffinity chromatography, such as tag peptides, etc., for example, epitopes derived from hemagglutinin (HA) from influenza virus, C-myc, FLAG, V5, etc.
  • HA hemagglutinin
  • the gene construct of the invention can be obtained by using techniques well known in the prior art [Sambrook et al, "Molecular Cloning, a Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989 VoI 1-3].
  • Said gene construct of the invention may, operably linked, incorporate a regulatory sequence of the expression of the nucleotide sequences encoding the polypeptides encoded by the nucleic acid sequences (A ') and (B'), thereby constituting a expression cassette
  • the term "operably linked” means that the polypeptides encoded by the nucleic acid sequences (A ') and (B'), and, where appropriate (C), are expressed in the correct reading frame under the control of the control or regulatory expression sequences.
  • the invention provides an expression cassette comprising the gene construct of the invention operably linked to an expression control sequence of the nucleotide sequence encoding the fusion protein provided by this invention comprising a polypeptide.
  • A comprising an antibody or a functionally equivalent fragment thereof and a polypeptide
  • B comprising an oligomerization domain.
  • Control sequences are sequences that control and regulate transcription and, where appropriate, translation of said fusion protein, and include promoter sequences, coding sequences for transcriptional regulators, ribosome binding sequences (RBS) and / or terminator sequences. of transcription.
  • said expression control sequence is functional in prokaryotic cells and organisms, for example, bacteria, etc.
  • said expression control sequence is functional in eukaryotic cells and organisms, for example. , insect cells, plant cells, mammalian cells, etc.
  • promoters include the human cytomegalovirus (hCMV) promoter, etc.
  • said expression cassette further comprises a marker or gene that codes for a motif or for a phenotype that allows the selection of the host cell transformed with said expression cassette.
  • markers that could be present in the expression cassette of the invention include antibiotic resistance genes, toxic compound resistance genes, and, in general, all those that allow genetically transformed plants to be selected.
  • the invention relates to a vector, such as an expression vector, comprising said gene construct of the invention or said expression cassette.
  • a vector such as an expression vector
  • the choice of the vector will depend on the host cell into which it will be subsequently introduced.
  • the vector where said nucleic acid sequence is introduced can be a plasmid or a vector which, when introduced into a host cell, whether or not it is integrated into the genome of that cell.
  • the obtaining of said vector can be carried out by conventional methods known to those skilled in the art [Sambrook et al., 1989, cited supra].
  • said recombinant vector is a vector useful for transforming animal cells.
  • Said vector can be used to transform, transfect or infect cells susceptible to being transformed, transfected or infected by said vector.
  • Said cells can be prokaryotic or eukaryotic. Therefore, in another aspect, the invention relates to a host cell transformed, transfected or infected with a vector provided by this invention.
  • Said transformed, transfected or infected cell thus comprises a gene construct of the invention, or said expression cassette or vector provided by this invention.
  • Transformed, transfected or infected cells can be obtained by conventional methods known to those skilled in the art [Sambrook et al., 1989, cited supra].
  • said host cell is an animal cell transformed, transfected or infected with an appropriate vector, said animal cell being transformed, transfected or infected capable of expressing the fusion protein provided by this invention, whereby said vectors can be used. for expression in animal cells of the fusion protein provided by this invention.
  • the gene construct of the invention can be used to produce said fusion proteins comprising a polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof and a polypeptide (B) comprising an oligomerization domain.
  • the invention relates to a method of producing said fusion protein provided by this invention comprising growing a cell or organism provided by this invention under conditions that allow the production of said fusion protein.
  • the conditions for optimizing the culture of said cell or organism will depend on the cell or organism used.
  • the method of producing a product of interest provided by this invention further includes isolation and purification of said fusion protein.
  • the oligomeric protein of the invention contains two or more different fusion proteins, said different fusion proteins may be expressed, if desired, in an appropriate host cell by co-transformation, co-transfection or co-infection using vectors containing the appropriate coding sequences.
  • AcMs Monoclonal antibodies used included the AcM 9E10 (Abcam, Cambridge, R. Kingdom) specific for human c-myc, and the AcM NCRC23 (AbD
  • AcPs bovine serum anti-albumin rabbit
  • HRP horseradish peroxidase
  • Fc Goat anti-mouse IgG Ac, specific to the constant domain of IgG (Fc), conjugated to HRP, all provided by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).
  • Laminin-1 purified from the EHS murine tumor was obtained from Becton Dickinson Labware (Bedford, MA, USA).
  • the human CEA and the BSA were provided by Sigma-Aldrich.
  • NIP 4-hydroxy-5-iodo-3-nitrophenyl
  • HEK-293 cells human embryonic kidney epithelial cells
  • HT-1080 cells human fibrosarcoma; CCL-121), MKN45 (adeno human gastric carcinoma; JCRB- 0254); and HeLa (human cervical carcinoma; CCL-2) were cultured in Dulbecco medium modified by Eagle (DMEM) supplemented with 10% (v / v) fetal bovine serum (SBF) heat-inactivated (all from Invitrogen, Carlsbad, CA ), hereinafter half DMEM complete (DCM).
  • DMEM Dulbecco medium modified by Eagle
  • SBF fetal bovine serum
  • HeLa CEA cells Compte, M. et al. 2007; Cancer Gene Ther. 14: 380-388 were grown in DCM supplemented with 750 ⁇ g / ml of G418 (Invitrogen).
  • PCR3 expression vectors 1-L36 and pCR3.1-L36-NCl ES " were constructed following known protocols [Sanz L et al. (2002). Gene Ther. 9: 1049-1053; Sanz L et al. (2001). Immunol Cancer. Immunother 50: 557-565].
  • the MFE-23 Ac expression cassette was digested with HindIII and Notl and cloned into the vector pCEP4.6xHis-myc [Sanz, L. et al. (2002). Gene Ther. 9: 1049-1053] to generate plasmid pCEP4-MFE-23.
  • Plasmid pCEP4-B1.8 containing the gene encoding Ac B 1.8 (anti-NIP) in scFv format and peptide tags (tags) of polyhistidine (6 His) and c-myc was constructed following a procedure already described [ Sanz, L. et al. (2002), cited supra].
  • HEK-293 cells and their 293T derived cells were transfected with Superfect according to the manufacturer's recommendations (QIAGEN GmbH, Hilden, Germany).
  • HEK-293 cells transfected with vectors pCR3.1-L36 and pCR3.1-L36-NCl ES " were selected in DCM supplemented with 0.5 mg / ml of neomycin (G418) (Promega)
  • HEK-293 cells transfected with pCEP4-MFE- vectors 23-NCl ES ⁇ and pCEP4-B1.8-NCl ES ⁇ were selected in DCM supplemented with 100 ⁇ g / ml hygromycin B (Invitrogen).
  • trimerbodies were studied by ELISA using the supernatant of 293T cells transfected either with a single vector (pCR3.1-L36-NCl ES " or pCEP4-B1.8-NCl ES ⁇ ) or with 2 vectors (pCR3 .1-L36-
  • CEA expression in HeLa and HeLa CEA tumor lines and recombinant antibody binding (trimerbodies) was analyzed following a protocol already described.
  • the BSA used as a negative control of the interaction, was immobilized at 1,900 UR over the reference flow cell. After each experiment, the surfaces were regenerated with 30 mM HCl, allowing the resonance signals to return to baseline levels. The analyzes were performed in duplicate.
  • NIPio-BSA approximately 100 UR
  • the individual samples consisting of scFv or purified trimerbodies, were passed on the surface of the chip at a flow rate of 20 ⁇ l / minute and the association / dissociation rates were measured. Changes in the refractive index were eliminated by subtracting the responses from the reference chips and the mean response of a negative control was subtracted from all the sensograms.
  • the kinetic results were obtained using the BIAevaluation v4.1 software, provided with the biosensor, and the kinetic data were adjusted to a 1: 1 Langmuir interaction model.
  • the structure of the binding domain of the L36 antibody in scFv format was modeled by comparative modeling using the 2GHW.B structure obtained from Protein Data Bank (PDB) as a template [Berman, HM et al. 2000; Nucleic Acids Res. 28: 235-42].
  • the structure of the NCl trimerization subdomain of the amino terminal end of murine collagen XVIII was obtained from ModBase [Pieper, U. et al. 2004, Nucleic Acids Res .; 32: D217-22]. Both domains are linked by a 21 amino acid spacer to form a monomer of the L36 antibody in trimerbody format.
  • the coordinates of the remaining monomers were obtained by applying a triple axis of rotation symmetry.
  • the L36 trimerbody model was formed by adding the coordinates of the three monomers.
  • the structure was optimized with GROMACS [Van Der Spoel, D. et al. 2005; J. Comput. Chem .; 26: 1701-18] and its energy evaluated with DFIRE [Zhou, H. et al. 2002; Prot. Sci., 11: 2714-26].
  • the energies were normalized by dividing them by the length of the sequence. Stability tests in the presence of serum
  • trimerbody maintained serum
  • 500 ng of purified L36 trimerbody incubated at 37 0 C for 72 hours, with 12.5% murine serum BALB / c mice (Harlan Iberica, Barcelona, Spain) .
  • Samples were removed for analysis at 3 h, 24 h and 72 h counted from the beginning of the incubation and frozen until the entire study was completed.
  • a second set of samples exposed to serum was immediately frozen to represent the "zero" time.
  • trimerbodies to retain their functional binding to murine laminin by ELISA was analyzed.
  • Cy5 The (molar) ratio of Cy5 to antibody (Cy5: antibody), calculated according to the procedure described by Birchler et al. (Birchler, M. et al. 1999; J. Immunol. Methods; 231: 239-48), was close to 1: 1.
  • the functionality of the Acs conjugated with Cy5 was verified by ELISA against specific antigens.
  • MKN45, HT 1080 or HeLa cells (1-) were implanted subcutaneously (s.c.)
  • trimerbodies with different specificities for the NIP hapten or human CEA.
  • the scFvs coding genes derived from the anti-NIP (B 1.8) and human anti-CEA (MFE23) Acs were assembled in a similar manner and expressed as soluble proteins in functionally active HEK-293 cells. Recombinant proteins were purified by affinity columns (IMAC) and their yield was greater than 95%, as verified in polyacrylamide gels (SDS-PAGE). Both Bl.8 and MFE-23 trimerbodies eluted from the column with single peaks (data not shown) comparable to those shown in the
  • trimerbodies The functionality of the purified trimerbodies was demonstrated by ELISA against the conjugated immobilized antigens NIP-BSA (NIPio-BSA), murine EHS laminin and human CEA ( Figure IB). Its ability to detect its antigen in a cellular context was investigated by immuno fluorescence mapping of human tumor cells expressing the CEA antigen on its cell surface. Fluorescence was observed after incubation with the MFE-23 trimerbody, followed by reaction with the anti-myc AcM and detection with the FITC conjugated mouse goat anti-IgG Ac.
  • trimerbodies not only recognize the immobilized antigen, but also recognize the native antigen expressed on the surface of tumor cells.
  • the SPR tests served to determine the influence of the scFv or trimerbody format on the functionality of Ac Bl.8.
  • the binding kinetics of each Ac format were compared using three different densities of the NIPio-BSA immobilized on the chip surface.
  • BSA coupled to dextran was used as nonspecificity reference.
  • To compare the binding responses during association-dissociation processes several concentrations of Ac B 1.8 (from 9 to 1,200 nM for the scFv and 6 to 800 nM for the trimerbody) were injected. Under these conditions, only the trimerbody reached the saturation of the antigen surface, while the scFv joined slowly and with a faster apparent dissociation. Sensograms indicate that the trimerbody has a greater binding capacity than its monovalent version (Figure ID).
  • trimerbody B 1.8 As the concentration of trimerbody B 1.8 increased, k a decreased from 4 x 10 5 M -1 S "1 (12.5 nM trimerbody) to 2 x 10 4 M -1 S “ 1 (800 nM trimerbody). These changes are compatibl it is with the changes in the proportion of antibody that binds to the ligand in a mono- or multivalent manner, since an approximation to surface saturation leads to a greater proportion of monovalent antibody binding.
  • the apparent ka has a narrower range, from 6 x 10 "4 (12.5 nM) to 9 x 10 " 4 (800 nM).
  • the functional affinity of the trimerbody calculated for that concentration could be K D (k d / k a ) of 1.5 x 10 "9 M or higher.
  • 293T cells were transfected with the L36, Bl .8 trimerbodies encoding plasmids or with both plasmids in a co-transfection.
  • Western blotting trials revealed that both trimerbodies were expressed in the cell supernatant and that they bound their respective antigens, and that the amount of trimerbody was higher in co-transfected 293T cells (with both plasmids) than in cells 293T monotransfected (data not shown).
  • conditioned medium of the monotransfected 293T cells bound to NIPiO-BSA or laminin, as well as the conditioned medium of the co-transfected 293T cells recognize both antigens Figure 2A.
  • conditioned medium of co-transfected 293T cells i.e., double transfected
  • laminin-linked trimerbodies were able to capture soluble NIPio-BSA ( Figure 2B), demonstrating its multivalence.
  • trimerbodies in the supernatant of 293T cells monotransfected with the plasmid encoding trimerbody L36 were unable to bind and capture soluble NIP 10 -BSA.
  • trimerbody L36 maintains 80-90% of its binding capacity to its antigen after 72 hours of incubation.
  • trimerbodies showed rapid renal clearance after iv injection, with a maximum signal intensity at 3 hours, being undetectable at 48 hours post-injection ( Figures 4 A and 4C).
  • Trimerbody B 1.8 showed no detectable tumor location in any of the three types of tumors studied ( Figures 4B and 5).
  • a selective and strong accumulation was observed in tumors expressing CEA (CEA + ) of the trimerbody MFE-23.
  • the maximum recorded signal occurred at 3 hours, the signal decaying at 24 hours and remaining detectable at 48 hours ( Figure 4B).
  • the trimerbody anti-laminin showed specific tumor location in all models studied (whether or not tumors expressing CEA) ( Figures 4B and 5).
  • the signal strength kinetics was different from the MFE-23 trimerbody, the maximum signal being at 24 hours, somewhat less than the maximum obtained by the MFE-23 trimerbody.
  • the anti-laminin scFv also showed tumor specific location but at a much lower level than that of the anti-laminin trimerbody ( Figures 4B and 4D).
  • trimerbodies were isolated in an active form from conditioned medium obtained from transfected HEK293 cells and were easily purified using immobilized metal affinity chromatography.
  • Trimerbodies are trimeric in solution, and possess excellent stability and antigen binding capacity. Trimerbodies are very efficient in recognizing purified antigens immobilized on a plate, or expressed on the surface of a tumor cell. SPR analysis showed that the trimerbody had a greater binding signal than the monomeric antibody and apparently a smaller dissociation, consistent with multivalent antigen binding. The researchers calculated that the anti-NIP trimerbody has a functional affinity for the antigen (NIP-BSA conjugates) about 100 times higher, compared to the monovalent version. This result suggests that this affinity gain could be due to the avidity effect of a second combination site in the trimerbody molecule.
  • trimerbodies Stable anti-laminin and anti-NIP bifunctional trimerbodies were easily produced by co-expression of two different constructs of trimerbodies in human cells.
  • the affinity gain through avidity makes trimerbodies attractive for in vivo imaging techniques as alternative agents to dimeric antibodies. It could, therefore, be speculated that trimerbodies will be preferred over dimeric antibodies (diabodies and minibodies), although this property may depend on the structure and density of the antigen recognized by scFv.
  • the antigen binding sites must point in the same direction.
  • trimerbodies are multivalent molecules of intermediate size presenting high stability under physiological conditions.
  • the potential of trimerbodies for in vivo localization was studied in experimental models of human cancer in nude mice.
  • Anti-CEA trimerbodies quickly and specifically locate CEA-positive tumors. The maximum signal in the tumor was reached at 3 hours after the injection and was slowly eliminated over time. Fluorescence was still detectable in the tumor after 48 hours after injection with the trimerbody.
  • trimerbody anti-laminin L36 antibody is located in all tumors studied regardless of their histological lineage. The maximum absorption of the anti-laminin trimerbodies was 24 hours after administration. Although the L36 anti-laminin scFv had a specific accumulation in the tumor, the accumulation in the tumor was lower, probably due to its faster removal of blood (with a half-life of less than 15 minutes) and by its nature monovalent (which implies low retention times) [Adams GP et al. (nineteen ninety five). Immunol cancer. Immunother 40: 299-306].
  • the epitope recognized by the L36 antibody is located in the middle part of the long arm of the laminin, in a highly flexible area, corresponding to a protease susceptible site.
  • the inventors have postulated that this epitope is only accessible during the assembly of the basement membrane (MB) [Sanz L et al. (2003) EMBO J. 22: 1508-1517], where the intact laminin during the polymerization process acts as a support for the recruitment of other components of the MB [Sasaki T et al. (2004) J. CeIl. Biol. 164: 959-963].
  • trimerbodies offer promising therapeutic opportunities based on the selective release of bioactive molecules in target tissues.
  • tumor-specific antigen-specific trimerbodies e.g., the human epidemic growth factor receptor 2, prostate-specific antigen
  • targeting the tumor stroma e.g., fibroblast activation protein
  • tumor-specific antigen-specific trimerbodies include the development of fusion proteins with angiogenic inhibitors [Sánchez-Arevalo VJ et al. (2006) Int J Cancer 119: 455-462], cytokines, enzymes, or truncated receptors, and radionuclide conjugation [Sanz L et al. (2004) Trends Immunol 25: 85-91].

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Abstract

L'invention concerne des protéines oligomériques qui comprennent une pluralité de protéines hybrides et un marqueur, chaque protéine hybride comportant un polypeptide qui contient un anticorps ou un fragment fonctionnellement équivalent dudit anticorps, et un polypeptide qui contient un domaine d'oligomérisation. Lesdites protéines oligomériques peuvent être utilisées pour détecter, visualiser et localiser des cibles d'intérêt.
PCT/ES2009/070107 2009-04-20 2009-04-20 Protéines oligomériques et leurs applications WO2010122181A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140348826A1 (en) * 2010-08-20 2014-11-27 Leadartis, S.L. Engineering multifunctional and multivalent molecules with collagen xv trimerization domain

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WO1995031540A1 (fr) * 1994-05-16 1995-11-23 Medical Research Council Polypeptides formant des trimeres, leur preparation et leur utilisation
WO2006048252A1 (fr) * 2004-11-02 2006-05-11 Universidad Autónoma de Madrid Inhibiteurs de l'angiogenese multifonctionnels et polyvalents
EP1798240A1 (fr) * 2005-12-15 2007-06-20 Industrial Technology Research Institute Polypeptides recombinants à structure en triple helice

Patent Citations (3)

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WO1995031540A1 (fr) * 1994-05-16 1995-11-23 Medical Research Council Polypeptides formant des trimeres, leur preparation et leur utilisation
WO2006048252A1 (fr) * 2004-11-02 2006-05-11 Universidad Autónoma de Madrid Inhibiteurs de l'angiogenese multifonctionnels et polyvalents
EP1798240A1 (fr) * 2005-12-15 2007-06-20 Industrial Technology Research Institute Polypeptides recombinants à structure en triple helice

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HOLLIGER P. ET AL.: "Engineered antibody fragments and the rise of single domains.", NATURE BIOTECHNOLOGY, vol. 23, no. 9, 7 September 2005 (2005-09-07), pages 1126 - 1136, XP008076746, DOI: doi:10.1038/nbt1142 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140348826A1 (en) * 2010-08-20 2014-11-27 Leadartis, S.L. Engineering multifunctional and multivalent molecules with collagen xv trimerization domain

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