Nothing Special   »   [go: up one dir, main page]

WO2010122181A1 - Oligomeric proteins and the uses thereof - Google Patents

Oligomeric proteins and the uses thereof Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
oligomeric protein
polypeptide
target
Prior art date
Application number
PCT/ES2009/070107
Other languages
Spanish (es)
French (fr)
Inventor
Ángel CUESTA MARTÍNEZ
David SÁNCHEZ-MARTÍN
Laura Sanz Alcober
Marta Compte Grau
Félix BONILLA VELASCO
Luis ÁLVAREZ-VALLINA
Original Assignee
Universidad Autónoma de Madrid
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad Autónoma de Madrid filed Critical Universidad Autónoma de Madrid
Priority to PCT/ES2009/070107 priority Critical patent/WO2010122181A1/en
Publication of WO2010122181A1 publication Critical patent/WO2010122181A1/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62DNA 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].

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The oligomeric proteins comprise a plurality of fusion proteins and a marker, each fusion protein comprising a polypeptide that comprises an antibody or a functionally equivalent fragment of said antibody, and a polypeptide that comprises an oligomerization domain. Said oligomeric proteins may be used to detect, view and locate targets of interest.

Description

PROTEÍNAS OLIGOMERICAS Y SUS APLICACIONES OLIGOMERIC PROTEINS AND ITS APPLICATIONS
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La invención se relaciona con una proteína oligomérica que comprende una pluralidad de proteínas de fusión, iguales o diferentes, y un marcador, en la que cada proteína de fusión comprende un polipéptido que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo, y un polipéptido que comprende un dominio de oligomerización. Dichas proteínas oligoméricas pueden ser utilizadas para detectar, visualizar y localizar dianas de interés.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.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
Los procedimientos de imagen en medicina se refieren a técnicas y procesos no invasivos usados para crear imágenes del aspecto interno del cuerpo de un animal, para propósitos clínicos, por ejemplo, procedimientos médicos para diagnosticar una enfermedad o para localizar dicha enfermedad. En un sentido amplio, incluye técnicas tales como ciencias radiológicas, endoscopia, termografía médica y microscopía. Tradicionalmente, los estudios de laboratorio para evaluar la actividad de las terapias anticancerígenas en ratones han requerido el sacrificio de múltiples animales en cada etapa para proporcionar datos significativos. Las técnicas de imagen, sin embargo, permiten una evaluación precisa del crecimiento tumoral y a tiempo real en animales de laboratorio, lo que disminuye de forma considerable el número de sujetos requeridos.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.
La tomografía por emisión de positrones, TEP o PET (por las siglas en inglés de Positrón Emission Tomography) es una técnica propia de una especialidad médica llamada medicina nuclear y de la radiología, al combinar imágenes de TAC (ejemplos de imágenes 3D). Se trata de una técnica no invasiva de diagnóstico e investigación por imagen capaz de medir la actividad metabólica de los diferentes tejidos del cuerpo humano, especialmente del sistema nervioso central. Al igual que el resto de técnicas diagnósticas en Medicina Nuclear, la TEP se basa en detectar y analizar la distribución que adopta en el interior del cuerpo un radioisótopo administrado a través de una inyección. La TEP permite, por ejemplo, localizar los focos de crecimiento celular anormal en todo el organismo, en un solo estudio e independientemente de la localización anatómica donde se presente la neoplasia (primaria o metastásica), ya que Ia TEP no evalúa la morfología de los tejidos, sino su metabolismo. Por tanto, se ha implantado con mucha fuerza como técnica diagnóstica en oncología, además de otras áreas, como son la cardiología, la neurología y la psicobiología, dada la posibilidad de cuantificar el metabolismo tanto cardiaco como en el sistema nervioso central. La técnica de fluorescencia es una técnica de imagen que permite estudiar las propiedades de sustancias orgánicas o inorgánicas utilizando el fenómeno de fluorescencia. En la mayoría de los casos, se marca específicamente un componente de interés en la muestra, con una molécula fluorescente llamada fluoróforo. La muestra se ilumina con luz de una determinada/s longitud/es de onda, que es absorbida por los fluoró foros, originando una emisión de luz a longitudes de onda mayores (o a diferente color que la luz absorbida). La cantidad y la longitud de onda de la energía emitida dependen del fluoróforo y del entorno químico del fluoróforo. Entre los fluoró foros más comúnmente utilizados se encuentran la fluoresceína isotiocianato (FITC), los derivados de la rodamina (TRITC), comarina y cianina, los fluoróforos "Alexa Fluor" y "DyLight Fluor", la familia de la cianina (e.g., Cy3, Cy5 y Cy 7).Positron Emission Tomography (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. Like the rest of diagnostic techniques in Nuclear Medicine, 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. Therefore, it has been implanted with great force as a diagnostic technique in oncology, in addition to other areas, such as cardiology, neurology and psychobiology, given the possibility of quantifying both cardiac and central nervous system metabolism. The fluorescence technique is an imaging technique that allows you to study the properties of organic or inorganic substances using the fluorescence phenomenon. In most cases, 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. Among the most commonly used fluorinated forums are fluorescein isothiocyanate (FITC), rhodamine derivatives (TRITC), comarin and cyanine, fluorophores "Alexa Fluor" and "DyLight Fluor", the cyanine family (eg, Cy3 , Cy5 and Cy 7).
La técnica de imagen por fluorescencia in vivo es igual que la microscopía de fluorescencia, difiere únicamente en que trabaja a nivel macroscópico. Los objetos para imagen por fluorescencia son el cuerpo completo de (pequeños) animales. Moléculas que absorben en la región del infrarrojo cercano (NIR), 700-1.000 nm, pueden ser utilizadas para visualizar e investigar dianas moleculares in vivo, ya que la mayoría de los tejidos generan poca fluorescencia NIR. Los fluoróforos orgánicos NIR más comunes son las polimetinas (e.g., pentametina y heptametina). Las sondas utilizadas para imagen por fluorescencia in vivo pueden ser dirigidas o no dirigidas. Las dirigidas tienen la ventaja de que se pueden dirigir específicamente a un tejido diana. De esta manera, las sondas se unen a sus dianas, mientras que las no unidas se eliminan de la circulación. Esta aproximación es muy útil para realizar imágenes de tumores in vivo, ya que en cáncer, normalmente se sobreexpresan ciertos receptores de superficie. Un ejemplo de sondas dirigidas pueden ser moléculas de anticuerpos conjugadas con cianinas o "quantum dots". También se pueden utilizar moléculas más pequeñas dirigidas, en lugar de anticuerpos, por ejemplo ácido fólico conjugado con un fluorocromo NIR que proporciona imágenes de macrófagos activados implicados en enfermedades inflamatorias de articulaciones. Dichas moléculas irían dirigidas al receptor de ácido fólico. Dentro del grupo de las sondas dirigidas, los anticuerpos presentan la ventaja de su rápido aislamiento y que tienen una alta afinidad por virtualmente, cualquier antígeno.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. 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. In this way, the 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. Within the group of targeted probes, the antibodies have the advantage of their rapid isolation and that they have a high affinity for virtually any antigen.
Por otra parte, diferentes ensayos sistemáticos in vivo han proporcionado una confirmación de que el tamaño es un parámetro importante en fármaco cinética y biodistribución de moléculas de anticuerpos monoclonales. Así, parece ser que moléculas grandes de IgG (150 kDa) que reconocen antígenos de superficie expresados por las células tumorales penetran en los tumores sólidos muy lentamente, con una distribución no uniforme, y tienen una vida media sérica muy larga [Holliger et al. Nature Biotec. 2005; 23: 1126-36]. Por el contrario, pequeños fragmentos de anticuerpo de cadena única (scFv) monovalentes, con un tamaño de 25-30 kDa son mucho más eficientes en la penetración al tumor, pero son eliminados demasiado rápido y poseen además una pobre retención en el tumor, debido a sus propiedades de unión monovalente. Un anticuerpo ideal para localizar tumores debería ser una molécula multivalente de tamaño intermedio, que proporcione una penetración más rápida en el tumor, una mayor retención en el tejido diana y una eliminación sanguínea más rápida. Estudios recientes indican que anticuerpos bivalentes, tales como los "diabodies" (60 kDa), pueden ser más apropiados para las técnicas de imagen [Williams et al., 2001; Cáncer Biother. Radiopharm.; 16:25-35]. Los "diabodies" son moléculas diméricas no unidas covalentemente, formadas espontáneamente en formato scFv con espaciadores cortos que conectan la región variable de los genes. Debido a su pequeño tamaño, se eliminan rápidamente a través de los ríñones, limitando, por tanto, su exposición a la médula ósea, que es frecuentemente el órgano dosis-limitante con los anticuerpos intactos radiomarcados. Moléculas más grandes bivalentes, tales como los "minibodies" (dímeros scFv-CH3) y ScFv2-Fc se pueden acumular en tumores y estos últimos pueden diseñarse con un espectro amplio de vidas medias en suero, modulando la interacción con el receptor FcRn. Los "minibodies" pueden ser ideales para localizar tumores, ya que penetran mejor y tienen una eliminación rápida con lo que consiguen mejores proporciones tumor-sangre que las inmunoglobulinas intactas (150 kDa) o los fragmentos Fab2 (110 kDa). Sin embargo, a pesar de los buenos resultados obtenidos con estos formatos en varios modelos [Williams et al, 2001; Cáncer Biother. Radiopharm.; 16:25-35 y Adams et al, 1998; Br. J. Cáncer 77:1405-12], aún existen algunas limitaciones que necesitan ser solucionadas con el fin de obtener las mayores ventajas de la capacidad de localización de estos anticuerpos recombinantes. Una de las limitaciones es su limitada flexibilidad, y, otra, la necesidad de que el segundo antígeno esté orientado de manera precisa en un área estrictamente definida una vez que el anticuerpo se una al primer antígeno. De esta manera, los antígenos unidos deberían encontrarse casi opuestos al "diabody", y en el caso del "minibody", en una pequeña área circular, que, en algunos casos impide la unión del segundo antígeno. Esto implica que parte de la afinidad observada depende principalmente de la unión y de la capacidad de volverse a unir ("rebinding"), y no de la unión simultánea a diferentes moléculas del antígeno.On the other hand, different systematic tests in vivo have provided confirmation that size is an important parameter in kinetic drug and biodistribution of monoclonal antibody molecules. Thus, it seems that large IgG molecules (150 kDa) that recognize surface antigens expressed by tumor cells penetrate solid tumors very slowly, with a non-uniform distribution, and have a very long serum half-life [Holliger et al. Nature Biotec 2005; 23: 1126-36]. On the contrary, small monovalent single chain antibody (scFv) fragments, with a size of 25-30 kDa are much more efficient in penetrating the tumor, but are removed too quickly and also have poor retention in the tumor, due to to its monovalent binding properties. 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. Recent studies indicate that 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). However, despite the good results obtained with these formats in several models [Williams et al, 2001; Biother cancer. Radiopharm .; 16: 25-35 and Adams et al, 1998; Br. J. Cancer 77: 1405-12], there are still some limitations that need to be solved in order to obtain the greatest advantages of the ability to localize these recombinant antibodies. One of the limitations is its limited flexibility, and, another, the need for the second antigen to be oriented precisely in a strictly defined area once the antibody binds to the first antigen. In this way, the bound antigens should be almost opposite to the "diabody", and in the case of the "minibody", in a small circular area, which, in some cases prevents the binding of the second antigen. This implies that part of the affinity observed depends mainly on the binding and on the ability to rejoin ("rebinding"), and not on the simultaneous binding to different antigen molecules.
Existe, por tanto, la necesidad de desarrollar reactivos para diagnóstico por imagen, basados en anticuerpos, que permitan la detección, visualización o localización de una diana de interés, tal como un antígeno asociado con una alteración patológica, que superen la totalidad o parte de los inconvenientes previamente mencionados.There is, therefore, the need to develop reagents for diagnostic imaging, based on antibodies, that allow the detection, visualization or localization of a target of interest, such as an antigen associated with a pathological alteration, that exceed all or part of the previously mentioned inconveniences.
COMPENDIO DE LA INVENCIÓNSUMMARY OF THE INVENTION
Los inventores han desarrollado un nuevo formato de anticuerpo, en forma de proteína oligomérica que comprende una pluralidad de proteínas de fusión, comprendiendo cada proteína de fusión un dominio de unión de un anticuerpo o de un fragmento funcionalmente equivalente del mismo, y un dominio de oligomerización, marcada posteriormente con un marcador adecuado para su aplicación en técnicas de diagnóstico por imagen. Dichas proteínas oligoméricas pueden ser utilizadas para detectar, visualizar o localizar una diana de interés, tal como un antígeno asociado con una alteración patológica.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.
Efectivamente, tal y como se ilustra en el Ej emplo 1 , dichas proteínas oligoméricas son agentes efectivos para la localización de depósitos tumorales in vivo. En una realización particular, los inventores han generado varias proteínas triméricas, en ocasiones identificadas como "trimerbodies", una específica frente al hapteno NIP, otra frente al antígeno carcinoembrionario (CEA) humano, y otra que reconoce un epítopo de la laminina asociado a angiogénesis, y las han caracterizado tanto in vitro como in vivo. Se ha estudiado en detalle su estabilidad, su especificidad y afinidad y sus propiedades de localización tumoral, observándose que todas ellas eran multivalentes y poseían una excelente característica de unión al antígeno, lo que les proporciona una elevada afinidad funcional (avidez). Asimismo, los trimerbodies anti-CEA conjugados químicamente con un marcador fluorescente mostraron una eficiente localización del tumor en un modelo experimental de carcinoma colorrectal humano en ratones y los trimerbodies anti- laminina mostraron una localización excelente en varios tipos de cáncer humano, incluyendo fibrosarcomas y carcinomas. Estos resultados demuestran el potencial de este nuevo formato de anticuerpo para aplicaciones terapéuticas y diagnósticas. Por tanto, en un aspecto, la invención se relaciona con una proteína oligomérica que comprende una pluralidad de proteínas de fusión, iguales o diferentes, y un marcador (M), en la que cada proteína de fusión comprende:Indeed, as illustrated in Example 1, said oligomeric proteins are effective agents for the location of tumor deposits in vivo. In a particular embodiment, 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. Its stability, its specificity and affinity and its tumor localization properties have been studied in detail, observing that all of them were multivalent and possessed an excellent antigen binding characteristic, which gives them a high functional affinity (greediness). Likewise, chemically conjugated anti-CEA trimerbodies showed an efficient tumor localization in an experimental model of human colorectal carcinoma in mice and anti-laminin trimerbodies showed excellent localization in several types of human cancer, including fibrosarcomas and carcinomas . These results demonstrate the potential of this new antibody format for therapeutic and diagnostic applications. Thus, in one aspect, 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:
(a) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y (a) un polipéptido (B) que comprende un dominio de oligomerización.(a) a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; and (a) a polypeptide (B) comprising an oligomerization domain.
En otro aspecto, la invención se relaciona con un procedimiento para la obtención de dicha proteína oligomérica.In another aspect, the invention relates to a process for obtaining said oligomeric protein.
En otro aspecto, la invención se relaciona con el empleo de dicha proteína oligomérica, en un método para la detección, visualización o localización de una diana. Un método para la detección, visualización o localización de una diana que comprende el empleo de dicha proteína oligomérica constituye un aspecto adicional de esta invención.In another aspect, 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.
En otro aspecto, la invención se relaciona con una composición que comprende dicha proteína oligomérica y un medio apropiado.In another aspect, the invention relates to a composition comprising said oligomeric protein and an appropriate medium.
En otro aspecto, la invención se relaciona con una composición farmacéutica que comprende dicha proteína oligomérica y un vehículo farmacéuticamente aceptable.In another aspect, the invention relates to a pharmaceutical composition comprising said oligomeric protein and a pharmaceutically acceptable carrier.
En otro aspecto, la invención se relaciona con un kit que comprende dicha proteína oligomérica. El empleo de dicho kit para la detección, visualización o localización de una diana constituye un aspecto adicional de esta invención. BREVE DESCRIPCIÓN DE LAS FIGURASIn another aspect, the invention relates to a 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. BRIEF DESCRIPTION OF THE FIGURES
La Figura 1 describe la caracterización molecular de los trimerbodies purificados. (A) Perfil de elución del experimento de filtración en gel de las construcciones L36 [L36 trimerbody (líneas más oscura) y L36 scFv (línea más clara)]. Los volúmenes de exclusión (V0) y total (VT) están indicados. Los volúmenes de elución de marcadores de peso molecular seleccionados se indican con flechas, así como sus correspondientes pesos moleculares. Para que quede de manera clara, la absorbancia de ambos cromatogramas se ha escalado y movido de posición en la figura. (B) La funcionalidad de los trimerbodies purificados se demostró por ELISA en placas tapizadas con albúmina sérica bovina (BSA), laminina-1 murina, hapteno NIP conjugados con BSA (NIPio-BSA) y CEA humano; y (C) mediante citometría de flujo en células tumorales CEA-negativas y CEA-positivas. Se muestran control de isotipo (histograma gris), trimerbody anti-CEA (línea sólida) y trimerbody anti-NIP (línea de puntos). (D) Análisis de la interacción del scFv B 1.8 y del trimerbody B 1.8 a NIP10- BSA utilizando BIAcore. Las curvas muestran los datos obtenidos tras la sustracción de la respuesta de unión a una superficie de referencia tapizada con BSA (1900 UR), para eliminar los efectos de la unión no específica. También se muestran sensogramas representativos que corresponden con las curvas de unión de afinidad ajustadas de los trimerbodies scFv Bl .8 y trimerbody Bl .8 (dil. 1200 y 800 nM) en buffer HBS-EP inyectado sobre las mismas celdas que anteriormente.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. (B) The functionality of the purified trimerbodies was 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.
La Figura 2 describe actividades de unión de moléculas de trimerbody mono- específicas y bi-específicas. Se comparan sobrenadantes de células 293T transfectadas con pEGFP-Nl, pCR3.1-L36-NClES", ó pCEP4-B1.8-NClES" con sobrenadantes de células 293T transfectadas con pCR3.1-L36-NClES" y pCEP4-B1.8-NClES", utilizando ELISA directo (A), usando placas tapizadas con BSA, conjugados NIPio-BSA y laminina-1, y por ELISA sandwich (B) utilizando placas tapizadas con laminina.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.
La Figura 3 describe la estabilidad de las moléculas de trimerbody en suero. Los trimerbodies L36 purificados se incubaron en suero humano o de ratón a 37 0C, tal y como se detalla en el Ejemplo 1 (materiales y métodos) y la funcionalidad de las mezclas de reacción se analizaron por ELISA.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.
La Figura 4 describe la localización de anticuerpos conjugados con un fluoróforo (cianina 5 ; Cy5) en un modelo de cáncer gástrico (C E A-positivo) experimental en ratones desnudos. Imágenes de infrarrojo cercano en ratones desnudos portadores de un carcinoma gástrico MKN45 subcutáneos (s.c). Imágenes ventral (A, C) y dorsal (B, D) tomadas 3, 24 y 48h tras la inyección intravenosa (i.v.) con trimerbodies (Bl.8, MFE-23 yL36) marcados con Cy5 (A, B), o scFv L36 marcados con Cy5 (C, D). b: bladder, t: tumor.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. Near-infrared images in nude mice carrying a subcutaneous MKN45 gastric carcinoma (sc). Ventral (A, C) and dorsal (B, D) images taken 3, 24 and 48h after intravenous (iv) injection with trimerbodies (Bl.8, MFE-23 and L36) marked with Cy5 (A, B), or scFv L36 marked with Cy5 (C, D). b: bladder, t: tumor.
La Figura 5 describe la localización de trimerbodies conjugados con un fluoróforo (cianina 5; Cy5) en varios modelos de cáncer humano (CE A-negativo) experimental en ratones desnudos. Imágenes de infrarrojo cercano de ratones desnudos portadores de fibrosarcomas HT 1080 humanos s.c. (A) o carcinomas de cervix (HeLa) humanos s.c. (B). Las imágenes fueron tomadas 3, 24 y 48h tras la inyección intravenosa (i.v.) con trimerbodies marcados con Cy5.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. 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.
La Figura 6 describe un modelo estructural de trimerbody. Vista lateral (A) y superior (B) del modelo molecular del trimerbody de anti-laminina L36.Figure 6 describes a structural model of trimerbody. Side view (A) and top (B) of the molecular model of the L36 anti-laminin trimerbody.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DefinicionesDETAILED DESCRIPTION OF THE INVENTION Definitions
Tal como aquí se utiliza, el término "diana" incluye una parte de una macromolécula que es reconocida por un anticuerpo; en una realización particular, dicha diana es un epítopo o determinante antigénico de un antígeno. Un "antígeno", tal como aquí se utiliza, se refiere a una sustancia que induce la generación de anticuerpos y puede generar una respuesta inmune. Los antígenos, habitualmente, son proteínas o polisacáridos, e incluyen partes de células, bacterias, virus y otros microorganismos. Atendiendo a su origen, un antígeno puede ser (i) exógeno, es decir, un antígeno que ha entrado en el cuerpo de un sujeto desde el exterior, por ejemplo, mediante inhalación, ingestión, inyección, etc.; (ii) endógeno, es decir, un antígeno generado en el interior celular como resultado del metabolismo celular normal o debido a una infección intracelular, e.g., bacteriana, vírica, etc., incluyendo antígenos xenogénicos (heterólogos), autólogos e idiomáticos o alogénicos (homólogos); o (iii) autoantígeno, es decir, una proteína (o un complejo de proteínas) normal que es reconocida por el sistema inmune de un sujeto que padece una enfermedad autoinmune. Una clase especialmente importante de antígenos tumorales (o neoantígenos), es decir, antígenos que son presentados por moléculas del complejo mayoritario de histocompatibilidad de tipo I (MHC I) o de tipo II (MHC II) sobre la superficie de células tumorales; cuando dichos antígenos tumorales son presentados únicamente por células tumorales pero no por células normales se denominan "antígenos específicos de tumor" (TSA, del inglés "tumor-specific antigen") y, en general, proceden de una mutación específica del tumor; alternativamente, los antígenos tumorales que son presentados tanto por células tumorales como por células normales se denominan "antígenos asociados a tumor" (TAA, del inglés "tumor-associated antigen") y, en general, proceden de una mutación específica del tumor.As used herein, 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. According to its origin, 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. An especially important class of tumor antigens (or neoantigens), that is, antigens that are presented by complex molecules majority of histocompatibility type I (MHC I) or type II (MHC II) on the surface of tumor cells; when these tumor antigens are presented only by tumor cells but not by normal cells, they are called "tumor-specific antigens" (TSA) and, in general, they come from a tumor-specific mutation; alternatively, tumor antigens that are presented by both tumor cells and normal cells are called "tumor associated antigens" (TAA) and, in general, come from a specific mutation of the tumor.
El término "anticuerpo", tal como aquí se utiliza, se refiere a una inmunoglobulina, o a un fragmento de la misma, con capacidad de unión a un antígeno e incluye todo tipo de anticuerpos, e.g., anticuerpos policlonales, anticuerpos monoclonales, etc., así como fragmentos de anticuerpos tales como fragmentos Fab, F(ab')2, Fab', fragmentos Fv de cadena única (scFv, del inglés single chain Fv), anticuerpos monodominio (VHH), diabodies, etc. En una realización particular de la invención, dicho anticuerpo es un fragmento de un anticuerpo, tal como, por ejemplo, un fragmento Fab, F(ab')2, Fab', scFv o VHH, los cuales pueden producirse directamente mediante células hospedadoras recombinantes. En una realización particular y preferida, el anticuerpo es un scFv.The term "antibody", as used herein, 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. In a particular embodiment of the invention, 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 In a particular and preferred embodiment, the antibody is an scFv.
Un fragmento "Fv" es un fragmento de anticuerpo mínimo que contiene un sitio completo de reconocimiento del antígeno y de unión al antígeno. Esta región está formada por un dominio variable de una cadena pesada (VH) y un dominio variable de una cadena ligera (VL) asociados no covalentemente.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.
Los fragmentos "scFv" comprenden los dominios VH y VL de un anticuerpo, formando una cadena polipeptídica única (VH-VL). Preferiblemente, el polipéptido Fv comprende adicionalmente un polipéptido conector (linker) entre los dominios VH y VL (VH- linker- VL) que permite al scFv formar la estructura deseada para la unión al antígeno. Información adicional sobre scFv puede encontrarse en "The Pharmacology of Monoclonal Antibodies", vol. 113, Rosenburg y Moore eds., Springer-Verlag, N. Y., págs. 269-315 (1994).The "scFv" fragments comprise the VH and VL domains of an antibody, forming a single polypeptide chain (VH-VL). Preferably, 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. 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).
El término "diabodies" se refiere a un fragmento de anticuerpo con dos sitios de unión al antígeno, comprendiendo una VH conectada a una VL en la misma cadena polipeptídica (VH- linker- VL). Mediante el uso de un conector que sea demasiado corto como para permitir el apareamiento entre los dos dominios en la misma cadena, los dominios son forzados a aparearse con los dominios complementarios de otra cadena, y crear dos sitios de unión al antígeno.The term "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). By using a connector that is too short to allow pairing between the two domains in the same chain, the domains are forced to mate with the complementary domains of another chain, and create two antigen binding sites.
El término "VHH", tal como aquí se utiliza se refiere anticuerpos monodominio [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). En especies de camélidos (camellos, dromedarios, llamas, etc.) una parte de sus anticuerpos carece de cadena ligera, estando la zona de reconocimiento de los antígenos constituida únicamente por el dominio VH [Desmyter et al., J. Biol. Chem. (2002), 277 (26):23645- 50]. Este dominio VH se diferencia de los dominios VH de otros anticuerpos en que sus zonas de reconocimiento del antígeno están formadas por bucles de mayor tamaño. Así, a los dominios VH de anticuerpos de camélidos se les denomina VHH. Este dominio (VHH) de aproximadamente 15 kDa puede expresarse en el periplasma de E. coli manteniendo la capacidad de unión a su antígeno. Los VHH son moléculas más estables que los scFv, y al contrario que éstos raramente agregan. Al igual que los dominios VL y VH de ratón, los dominios VHH poseen un enlace S-S intramolecular que estabiliza su estructura terciaria y es necesario para su correcto plegamiento [Desmyter et al, J. Biol. Chem. (2002), 277 (26):23645-50].The term "V HH ", as used herein 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). In 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]. This VH domain differs from the VH domains of other antibodies in that their antigen recognition zones are formed by larger loops. Thus, 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. Like the mouse VL and VH domains, 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].
El término "marcador", tal como aquí se utiliza, se refiere a cualquier tipo de molécula que indica la existencia de un proceso químico, físico o biológico, que se puede introducir en un organismo con el fin de examinar alguna propiedad. Los mareajes pueden ser radiactivos o no radiactivos. El mareaje radiactivo o isotópico se ha empleado con más frecuencia, aunque va siendo desplazado progresivamente por los métodos no radiactivos. Aunque no existe consenso acerca del mareaje ideal, en la mayoría de los casos se hace con 32P. Puesto que la corta vida media de este isótopo (14 días) crea dificultades en la preparación, comercialización y uso rutinario de los ensayos, se utilizan también otras alternativas (35S, 3H, 125I, 14C, etc.). En cuanto a los marcadores no radiactivos, en función del marcador empleado, la detección se hace por fluorometría o añadiendo un sustrato específico cuya transformación enzimática por la enzima marcadora puede detectarse por alguno de los siguientes principios: (i) espectrofotometría: medida de la absorción de luz por los productos de la reacción, bien solubles o insolubles, en el ultravioleta o el visible (colorimetría), (ii) fluorometría: medida de la luz emitida por productos de la reacción que poseen un molécula fluorescente, o (iii) quimioluminiscencia: medida de luz emitida como consecuencia de reacciones con esta característica. Estos y otros procedimientos se pueden aplicar a la detección de proteínas, antígenos o pequeños ligandos.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.). As for non-radioactive markers, depending on the marker used, 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. These and other procedures can be applied to the detection of proteins, antigens or small ligands.
En concreto, la fluorometría se puede utilizar para mareaje in vivo. De esta manera, se puede marcar específicamente un componente de interés en una muestra, con una molécula fluorescente, llamada fluoróforo. Uno de los fluoró foros más comúnmente utilizados es la fluoresceína isotiocianato (FITC), un reactivo derivado de la fluoresceína, que se acopla químicamente a otras moléculas para crear nuevas moléculas fluorescentes para una variedad de aplicaciones. Otros fluoróforos históricamente muy utilizados son los derivados de la rodamina (TRITC), comarina y cianina. Nuevas generaciones de fluoróforos incluyen el "Alexa Fluor" y los "DyLight Fluor", más fotoestables, más brillantes y menos sensibles a cambios de pH que otros marcadores conocidos. Dentro de la familia de la cianina, se incluyen los marcadores Cy3, Cy5, Cy5.5 y Cy7, que son reactivos fluorescentes solubles en agua. Los marcadores Cy3 son amarillo -naranja (excitación a 550 nm aproximadamente, emisión a aproximadamente 570 nm), mientras que los Cy5 son fluorescentes en la región del rojo (aproximadamente a 650/670 nm). Normalmente son sintetizados con grupos reactivos en una o en ambas cadenas laterales de nitrógeno, de manera que pueden ser acoplados químicamente, tanto a ácidos nucleicos como a proteínas, siendo ampliamente usados en técnicas de diagnóstico por imagen. Por otra parte, dentro de los fluoróforos orgánicos para el infrarrojo cercano (NIR), los más comunes son las polimetinas de fórmula general (I)Specifically, fluorometry can be used for in vivo marking. In this way, a component of interest can be specifically labeled in a sample, with a fluorescent molecule, called fluorophore. One of the most commonly used fluorinated forums is fluorescein isothiocyanate (FITC), a reagent derived from fluorescein, which chemically attaches to other molecules to create new fluorescent molecules for a variety of applications. Other historically widely used fluorophores are rhodamine derivatives (TRITC), comarin and cyanine. 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. Within the cyanine family, the Cy3, Cy5, Cy5.5 and Cy7 markers are included, which are water soluble fluorescent reagents. 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. On the other hand, within the organic fluorophores for near infrared (NIR), the most common are polymetins of general formula (I)
Figure imgf000011_0001
Figure imgf000011_0001
(I) donde(I) where
Ri y R2, independientemente entre sí, representan alquilo C1-C10, sulfoalquiloRi and R 2 independently represent C 1 -C 10, sulphoalkyl
C1-C10, cicloalquilo C3-C10, alcoxilo C1-C10, o arilo;C1-C10, cycloalkyl C 3 -C 10, alkoxyl C 1 -C 10 -alkyl, or aryl;
R3 y R4, independientemente entre sí, representan sulfoalquilo C1-C10, haloalquilo C1-C10, o hidroxicarbonilalquilo C1-C10;R 3 and R 4 independently represent sulfoalkyl C1 - C10, C1 - C10 haloalkyl, or C1 - C10 hydroxycarbonyl;
Y es C, O, S; y n es un número entero comprendido entre 1 y 10.Y is C, O, S; and n is an integer between 1 and 10.
Entre las polimetinas más útiles se encuentran las cianinas pentametina (n = 1) y heptametina (n= 2) que comprenden un grupo benzoxazol, benzotiazol, indolil, 2- quino lin ó 4-quinolin.Among the most useful polymethines are the cyanines pentametin (n = 1) and heptametin (n = 2) comprising a benzoxazole, benzothiazole, indolyl, 2- quino lin or 4-quinolin group.
Proteína oligomérica de la invenciónOligomeric protein of the invention
En otro aspecto, la invención se relaciona con una proteína oligomérica, en adelante proteína oligomérica de la invención, que comprende una pluralidad de proteínas de fusión, iguales o diferentes, y un marcador (M), en la que cada proteína de fusión comprende:In another aspect, 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:
(a) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y(a) a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y
(b) un polipéptido (B) que comprende un dominio de oligomerización. El marcador (M) presente en dicha proteína oligomérica de la invención puede ser prácticamente cualquier marcador; no obstante, para sus aplicaciones en técnicas de diagnóstico por imagen dicho marcador (M) es un marcador adecuado para dichas técnicas, por ejemplo, (i) un fluoróforo, tal como un fluoróforo para diagnóstico por imagen por fluorescencia en la zona cercana al infrarrojo, e.g. Cy5, Cy7 etc.; (ii) un radionucleido para radioinmunoescintigrafía [compuestos que emiten partículas gamma: 99mTc, 111In, 131I, renio 186 (186Re)] o para tomografía por emisión de positrones (TEP) (marcador TEP); etc. En una realización particular, dicho marcador (M) es un fluoróro, tal como Cy5, Cy7, etc., o un marcador TEP, tal como 64Cu, 68Ga, 18F, 86Y, 76Br, 89Zr, 124I, etc. En general, el uso en clínica humana de anticuerpos recombinantes con fines diagnóstico requiere el empleo de marcadores tipo TEP ("inmuno-TEP"), los cuales, por sus características, permitirán detectar lesiones pequeñas, incluyendo micrometástasis, localizadas en zonas profundas; alternativamente, para detectar lesiones más superficiales, se podrían utilizar fluoróforos, e.g., marcadores fluorescentes NIR, en localizaciones más accesibles, por ejemplo, piel, fondo de ojo, etc. [Wu & Olafsen. Cáncer J. (2008). May-Jun; 14(3):191-7; van Dongen GA et al. (2007). Oncologist. Dec; 12(12):1379-89.(b) a polypeptide (B) comprising an oligomerization domain. 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. In a particular embodiment, 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. In general, the use in clinical practice of recombinant antibodies for diagnostic purposes requires the use of markers type TEP ("immuno-TEP"), which, due to their characteristics, will allow to detect small lesions, including micrometastases, located in deep areas; alternatively, to detect more superficial lesions, fluorophores, eg, NIR fluorescent markers, could be used in more accessible locations, for example, skin, fundus, etc. [Wu & Olafsen. Cancer J. (2008). May-Jun; 14 (3): 191-7; van Dongen GA et al. (2007). Oncologist Dec; 12 (12): 1379-89.
La proteína oligomérica de la invención comprende una pluralidad de proteínas de fusión, iguales o diferentes, y un marcador (M), comprendiendo cada una de dichas proteínas de fusión comprende un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y un polipéptido (B) que comprende un dominio de oligomerización.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.
En una realización particular, dicha proteína oligomérica de la invención comprende una pluralidad de proteínas de fusión iguales; en cuyo caso, cada una de dichas proteínas de fusión comprende un mismo polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y un mismo polipéptido (B) que comprende un dominio de oligomerización.In a particular embodiment, 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.
En otra realización particular, dicha proteína oligomérica de la invención comprende una pluralidad de proteínas de fusión diferentes, por ejemplo, dos o más proteínas de fusión diferentes. En este caso, una de dichas proteínas de fusión comprende un polipéptido (Al) que comprende un anticuerpo (1) o un fragmento funcionalmente equivalente de dicho anticuerpo (1); y un polipéptido (B) que comprende un dominio de oligomerización, y otra de dichas proteínas de fusión comprende un polipéptido (A2) que comprende un anticuerpo (2) o un fragmento funcionalmente equivalente de dicho anticuerpo (2); y un polipéptido (B) que comprende un dominio de oligomerización, en donde dichos anticuerpos (1) y (2) son diferentes. El número total de proteínas de fusión presentes en la proteína oligomérica de la invención dependerá del dominio de oligomerización, dependiendo del dominio de oligomerización siendo posible prácticamente cualquier combinación entre dichas proteínas de fusión iguales o diferentes, siempre y cuando el dominio de oligomerización se mantenga. El hecho de que la proteína oligomérica de la invención comprenda dos o más proteínas de fusión diferentes permite reconocer e interaccionar con dos o más dianas (e.g., antígenos) diferentes. A modo ilustrativo, no limitativo, la proteína oligomérica de la invención puede contener dos proteínas de fusión diferentes entre sí, tal como se ha mencionado previamente; o, alternativamente, dos proteínas de fusión iguales entre sí y una proteína de fusión diferente; o, alternativamente, dos proteínas de fusión iguales entre sí y otros dos proteínas de fusión diferentes pero iguales entre sí; o, alternativamente, dos proteínas de fusión iguales entre sí y otros dos proteínas de fusión diferentes entre sí y diferentes a las dos proteínas de fusión iguales; o, alternativamente, tres proteínas de fusión diferentes entre sí; etc.; en general, cualquier combinación de proteínas de fusión que comprendan un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo, y un polipéptido (B) que comprende un dominio de oligomerización, en las que se mantenga dicho dominio de oligomerización.In another particular embodiment, said oligomeric protein of the invention comprises a plurality of different fusion proteins, for example, two or more different fusion proteins. In this case, 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, and 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). By way of illustration, not limitation, 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
La proteína oligomérica de la invención comprende dos o más proteínas de fusión, iguales o diferentes; por tanto, puede reconocer e interaccionar con una única diana (antígeno), o, alternativamente, con dos o más dianas (e.g., antígenos) diferentes, lo que la convierte en un reactivo muy versátil monoespecífico (es decir, cuando reconoce a una única diana o antígeno), o multiespecífico (es decir, cuando reconoce a más de una diana o antígeno, e.g., biespecífico, si reconoce dos dianas o antígenos diferentes, triespecíficos, si reconoce tres dianas o antígenos diferentes, etc. El polipéptido (A) comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo. Dicho anticuerpo, o fragmento funcionalmente equivalente, reconoce una diana, tal como un epítopo o determinante antigénico de un antígeno; en una realización particular, dicho antígeno es un antígeno tumoral, tal como CEA (Begent RH et al. Nat Med. (1996) Sep; 2(9):979-84), o un antígeno no tumoral que se expresa en áreas de angiogénesis activas en el microambiente peritumoral, tal como el dominio EDB de la fibronectina (Ebbinghaus C et al. Curr Pharm Des. (2004); 10(13):1537-49).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).
En una realización particular, el polipéptido (A) comprende un anticuerpo que reconoce una diana o un fragmento funcionalmente equivalente de dicho anticuerpo que reconoce dicha diana. Prácticamente cualquier anticuerpo, o fragmento funcionalmente equivalente del mismo, que reconozca una diana puede ser utilizado en la presente invención, por ejemplo, un anticuerpo monoclonal (AcM) o un anticuerpo policlonal (AcP); alternativamente, dicho polipéptido (A) puede contener un fragmento funcionalmente equivalente de un anticuerpo, tal como un fragmento de anticuerpo que mantiene la capacidad de reconocimiento de la diana reconocida por el anticuerpo completo del que deriva, e.g., un scFv, un anticuerpo biespecífico o diabody que reconoce dicha diana o bien en su formato recombinante completo (Fab + Fc), o un anticuerpo monodominio VHH preferentemente, un scFv (Ejemplo 1).In a particular embodiment, 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).
En una realización particular, el polipéptido (A) comprende un anticuerpo que reconoce una diana o un fragmento funcionalmente equivalente de dicho anticuerpo que reconoce dicha diana. Prácticamente cualquier anticuerpo, o fragmento funcionalmente equivalente del mismo, que reconozca una diana puede ser utilizado en la presente invención, por ej emplo, un anticuerpo monoclonal AcM o policlonal AcP; alternativamente, dicho polipéptido (A) puede contener un fragmento funcionalmente equivalente de un anticuerpo, tal como un fragmento de anticuerpo que mantiene la capacidad de reconocimiento de la diana reconocida por el anticuerpo completo del que deriva, e.g., un scFv, un anticuerpo biespecífico o diabody que reconoce dicha diana o bien en su formato recombinante completo (Fab + Fc), preferentemente, un scFv (Ejemplo 1).In a particular embodiment, 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).
En una realización particular, dicho polipéptido (A) comprende un scFv recombinante derivado del AcM L36 anti-laminina (Ejemplo 1) [scFv L36] que contiene la región variable de la cadena pesada (VH) del anticuerpo monoclonal L36 fusionada, a través de un espaciador, tal como un péptido que comprende la secuencia (Gly-Ser)4 (o una secuencia de tipo Gly-Ser-Pro-Gly, o bien comprende la secuencia Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser), a la región variable de la cadena ligera (VL) del AcM L36 y cuya secuencia ya ha sido descrita [Sanz L et al. Cáncer Immunology and Immunotherapy, 2001 Dec; 50(10)557- 65], en donde el extremo 3' de la secuencia codificante de VH está unido al extremo 5' de la secuencia codificante de dicho linker y el extremo 3 ' de la secuencia de nucleótidos codificante de dicho linker está unido al extremo 5 ' de la secuencia codificante de VL. El AcM L36 reconoce lamininas de diferentes especies de animales, por ejemplo, de ratón, rata, humanos, etc., ya que interacciona con una región que está muy conservada entre diferentes especies de animales [Sanz L et al. EMBO J 2003, VoI. 22(7):1508-1517]. En otra realización particular, dicho polipéptido (A) comprende un scFv recombinante derivado del AcM Bl .8 específico del hapteno NIP (Ejemplo 1) que contiene la región variable de la cadena pesada (VH) del AcM B 1.8 fusionada, a través de un espaciador, a la región variable de la cadena ligera (VL) del AcM Bl .8; en una realización particular, dicho espaciador comprende una secuencia de tipo Gly-Ser-Pro- GIy, o de tipo (Gly-Ser)4, o bien comprende la secuencia Leu-Glu-Gly-Ala-Gly-Gly- Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser.In a particular embodiment, 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. Cancer Immunology and Immunotherapy, 2001 Dec; 50 (10) 557-65], wherein the 3 'end of the VH coding sequence is attached to the 5' end of the linker coding sequence and the 3 'end of the linker coding nucleotide sequence is attached at the 5 'end of the VL coding sequence. The L36 AcM recognizes laminins of different animal species, for example, mouse, rat, human, etc., since it interacts with a region that is highly conserved between different animal species [Sanz L et al. EMBO J 2003, VoI. 22 (7): 1508-1517]. In another particular embodiment, 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; in a particular embodiment, 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.
En otra realización particular, dicho polipéptido (A) comprende un scFv recombinante derivado del AcM MFE23 específico del antígeno carcino embrionario (CEA) humano (Ejemplo 1) que contiene la región variable de la cadena pesada (VH) del AcM MFE23 fusionada, a través de un espaciador, a la región variable de la cadena ligera (VL) del AcM MFE23; en una realización particular, dicho espaciador comprende una secuencia de tipo Gly-Ser-Pro-Gly, o de tipo (Gly-Ser)4, o bien comprende la secuencia Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp- Gly-Ala-Ser-Gly-Ser.In another particular embodiment, 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; in a particular embodiment, 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.
Dicho polipéptido (A) puede reconocer y unirse a una diana, e.g., presente en un antígeno tumoral, y, como resultado de esa unión, localizar la diana (antígeno) y permitir su visualización mediante técnicas de imagen apropiadas (e.g., fluorescencia, TEP, etc.) debido a la presencia del marcador (M) en la proteína oligomérica de la invención.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.
El polipéptido (B) comprende un dominio de oligomerización. Dicho dominio de oligomerización puede ser prácticamente cualquier dominio que permite la formación de oligómeros, por ejemplo, dímeros, trímeros, tetrámeros, etc., de péptidos o proteínas, susceptible de ser expresado de forma recombinante y de formar un oligómero proteico de la proteína que lo comprende. No obstante, en una realización particular, dicho dominio de oligomerización es un dominio de trimerización, tal como el dominio de trimerización del dominio NCl de colágeno XVIII o del colágeno XV de mamífero. Como es conocido, dicho dominio NC l del colágeno XVIII (y del colágeno XV) comprende un dominio de trimerización y el dominio endostatina (ES) unidos por unos péptidos bisagra. Por tanto, en una realización particular, la proteína de fusión presente en la proteína oligomérica de la invención comprende un polipéptido (B) que comprende el dominio NCl del colágeno XV o el dominio NCl del colágeno XVIII que contiene el dominio de trimerización pero al que se le ha eliminado el dominio de ES (NC 1ES") así como la totalidad o parte de los péptidos bisagras existentes entre dichos dominios. Las secuencias de dichos dominios NCl de colágeno XV y de colágeno XVIII son conocidas; a modo ilustrativo, la secuencia del dominio NCl del colágeno XVIII ha sido descrita previamente por Sasaki et al. [Sasaki et al. Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J. 1998 Aug 3;17(15):4249-56].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. However, in a particular embodiment, said oligomerization domain is a trimerization domain, such as the trimerization domain of the NCl domain of collagen XVIII or mammalian collagen XV. As is known, said NC1 domain of collagen XVIII (and collagen XV) comprises a trimerization domain and the endostatin (ES) domain linked by hinge peptides. Therefore, in a particular embodiment, 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 sequences of said NCl domains of collagen XV and collagen XVIII they are known, by way of illustration, the sequence of the NCl domain of collagen XVIII has been previously described by Sasaki et al. [Sasaki et al. Structure, function and tissue forms of the globular C-terminal domain of collagen XVIII containing the angiogenesis inhibitor endostatin EMBO J. 1998 Aug 3; 17 (15): 4249-56].
Por tanto, a modo ilustrativo, en una realización particular, la invención proporciona una proteína oligomérica de la invención que comprende un marcador (M) y una pluralidad de proteínas de fusión, iguales o diferentes, en donde cada proteína de fusión comprende:Thus, by way of illustration, in a particular embodiment, 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:
(i) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente del mismo; y(i) a polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof; Y
(ii) un polipéptido (B) que comprende el dominio NC1ES" de colágeno XVIII de mamífero.(ii) a polypeptide (B) comprising the NC1 ES " domain of mammalian collagen XVIII.
No obstante, cualquier otro dominio similar al dominio NC 1 de colágeno XVIII que comprenda un dominio de oligomerización puede ser utilizado en la puesta en práctica de la presente invención, con el fin de generar la proteína oligomérica de la invención.However, 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.
Debido a la presencia del dominio de oligomerización en la proteína de fusión presente en la proteína oligomérica de la invención, la proteína oligomérica de la invención puede estar constituida por 2 ó más, por ejemplo, 3, 4, 5, o más proteínas de fusión, iguales o diferentes entre sí, dando lugar a dímeros, trímeros, tetrámeros, pentámeros, etc. de las proteínas de fusión proporcionadas por esta invención. En una realización particular, dicha proteína oligomérica de la invención es un trímero y comprende 3 proteínas de fusión proporcionadas por esta invención; dado que dichas proteínas de fusión incorporan un anticuerpo o un fragmento funcionalmente equivalente del mismo, dichos trímeros han sido denominados por los inventores con el nombre genérico de "trimerbody" (singular) o "trimerbodies" (plural). En una realización más concreta, dicha proteína oligomérica de la invención es un trímero y comprende 3 proteínas de fusión iguales proporcionadas por esta invención; en otra realización más concreta, dicha proteína oligomérica de la invención es un trímero y comprende 3 proteínas de fusión proporcionadas por esta invención, de las cuales dos son iguales entre sí y la otra es diferente; y, en otra realización más concreta, dicha proteína oligomérica de la invención es un trímero y comprende 3 proteínas de fusión diferentes proporcionadas por esta invención.Due to the presence of the oligomerization domain in the fusion protein present in 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. In a particular embodiment, 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). In a more concrete embodiment, 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.
La proteína de fusión proporcionada por esta invención puede contener, además, si se desea, un tercer polipéptido (C) que comprende la secuencia de aminoácidos de un péptido de unión flexible entre dichos polipéptidos (A) y (B) y/o un péptido (D) para facilitar el aislamiento o purificación de la proteína de fusión.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.
Dicho polipéptido (C) puede comprender prácticamente cualquier secuencia peptídica que defina un péptido de unión flexible. Ejemplos ilustrativos de péptidos de unión flexibles incluyen secuencias de tipo Gly-Ser-Pro-Gly o la secuencia (Gly-Ser)4. No obstante, en una realización particular, dicho péptido de unión flexible comprende la secuencia Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Said polypeptide (C) can comprise virtually any peptide sequence that defines a flexible binding peptide. Illustrative examples of flexible binding peptides include sequences of the Gly-Ser-Pro-Gly type or the sequence (Gly-Ser) 4 . However, in a particular embodiment, 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. Asimismo, con el fin de facilitar el aislamiento y purificación de la proteína de fusión de la invención, dicha proteína de fusión puede contener, si se desea, un péptido (D) susceptible de ser utilizado con fines de aislamiento o purificación de la proteína de fusión, tal como un péptido etiqueta ("tag"). Dicho péptido (D) puede estar situado en cualquier posición de la proteína de fusión que no altere la funcionalidad de ninguno de los polipéptidos (A) y (B); a modo ilustrativo, no limitativo, dicho péptido (D) puede estar situado a continuación del polipéptido (B). Prácticamente cualquier péptido o secuencia peptídica que permita el aislamiento o purificación de la proteína de fusión puede ser utilizado, por ejemplo, secuencias de polihistidina, secuencias peptídicas susceptibles de ser reconocidas por anticuerpos que pueden servir para purificar la proteína de fusión resultante por cromatografía de inmunoafinidad, tales como péptidos etiqueta, por ejemplo, epítopos derivados de la hemaglutinina (HA) del virus de la gripe, C-myc, FLAG, V5, etc.Ser. Likewise, in order to facilitate the isolation and purification of the fusion protein of the invention, 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.
La proteína oligomérica de la invención puede obtenerse mediante un procedimiento que comprende poner en contacto una proteína oligomérica que comprende una pluralidad de proteínas de fusión, en la que cada proteína de fusión comprende: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:
(i) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente del mismo; y(i) a polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof; Y
(ii) un po lip épti do (B ) que comprende un dominio de oligomerización, con un marcador (M) bajo condiciones que permiten la unión de dicho marcador (M) a la proteína oligomérica. Dicho procedimiento constituye un aspecto adicional de esta invención.(ii) a po lip épti 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.
Las características de dichos polipéptidos (A) y (B) así como las del marcador (M) ya han sido definidas previamente. Las condiciones que permiten la unión de dicho marcador (M) a dicha proteína oligomérica dependen del marcador (M) elegido y de la proteína oligomérica y, en general, son conocidas por los técnicos en la materia.The characteristics of said polypeptides (A) and (B) as well as those of the marker (M) have been previously defined. The conditions that allow the binding of said marker (M) to said oligomeric protein depend on the marker (M) chosen and the oligomeric protein and, in general, are known to those skilled in the art.
La proteína oligomérica de la invención, debido a sus características propias, puede ser utilizada en numerosas aplicaciones, por ejemplo, en la visualización de antígenos mediante técnicas de imagen, tanto in vitro como in vivo (e.g., fluorescencia, TEP, etc.), en la localización de antígenos, etc., en general, en la identificación de alteraciones patológicas asociadas con la neoexpresión o sobreexpresión de antígenos, e.g., patologías inflamatorias (e.g., integrinas, etc.), patologías vasculares (e.g., placas de ateroma, etc.), patologías tumorales, etc. En una realización particular, dichos antígenos son antígenos tumorales.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. In a particular embodiment, said antigens are tumor antigens.
Por tanto, en otro aspecto, la invención se relaciona con el empleo de una proteína oligomérica de la invención, en un método para la detección, visualización o localización de una diana, tal como un antígeno, mediante una técnica apropiada, por ejemplo, una técnica de imagen. En una realización particular dicha diana es un antígeno que se expresa de novo (es decir, que en condiciones normales no se expresa pero que en una alteración patológica se expresa) o sobreexpresa (es decir, que en condiciones normales se expresa en un nivel basal y su expresión aumenta en caso de una alteración patológica), por ejemplo, en patologías inflamatorias, vasculares, tumorales, etc. En una realización particular, dicha diana es un antígeno tumoral. Alternativamente, en otro aspecto, la invención se relaciona con un método para la detección, visualización o localización de una diana, tal como un antígeno, mediante una técnica apropiada, por ejemplo, una técnica de imagen, que comprende el empleo de una proteína oligomérica de la invención. En una realización particular dicha diana es un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica, por ejemplo, en patologías inflamatorias, vasculares, tumorales, etc. En una realización particular, dicha diana es un antígeno tumoral. Para su empleo en dichas aplicaciones, la proteína oligomérica de la invención se encontrará en un medio apropiado y adecuado para su administración.Therefore, in another aspect, 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 In a particular embodiment 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. In a particular embodiment, said target is a tumor antigen. Alternatively, in another aspect, 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. In a particular embodiment said target is an antigen that expresses de novo or is overexpressed in a pathological alteration, for example, in inflammatory, vascular, tumor pathologies, etc. In a particular embodiment, said target is a tumor antigen. For use in such applications, the oligomeric protein of the invention will be in an appropriate and suitable medium for administration.
Por tanto, en otro aspecto, la invención se relaciona con una composición que comprende una proteína oligomérica de la invención junto con, al menos, un medio apropiado, tal como un medio que no altera a la estabilidad de dicha proteína oligomérica de la invención, por ejemplo, PBS, solución salina fisiológica, etc. Alternativamente, dicha proteína oligomérica de la invención puede encontrarse en un medio constituido por un sistema de suministro y liberación de compuestos, por ejemplo, un vector viral o no viral (e.g., nanopartículas a base de polímeros biocompatibles, liposomas, etc.). Dichos vectores son, en general, conocidos por los técnicos en la materia.Therefore, in another aspect, the invention relates to 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. Alternatively, said oligomeric protein of the invention can be found in 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.). Such vectors are, in general, known to those skilled in the art.
Para sus aplicaciones in vivo, dicho medio debe ser farmacéuticamente aceptable. Por tanto, en otro aspecto, la invención se relaciona con una composición farmacéutica que comprende una proteína oligomérica de la invención junto con, al menos, un vehículo farmacéuticamente aceptable. En una realización particular, la composición farmacéutica de la invención comprende, al menos, una proteína oligomérica de la invención en una cantidad eficaz. En el sentido utilizado en esta descripción, la expresión "cantidad eficaz" se refiere a la cantidad de proteína oligomérica de la invención calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de la proteína oligomérica, la diana (antígeno) a visualizar o localizar, etc. La composición farmacéutica proporcionada por esta invención puede ser administrada por cualquier forma de administración apropiada, por ejemplo, por vía parenteral. Los vehículos que pueden utilizarse en la elaboración de la composición farmacéutica proporcionada por esta invención dependerán, entre otras cosas, de la forma de administración de dicha composición farmacéutica. Una revisión de las distintas formas de administración de principios activos, de los excipientes a utilizar y de sus procedimientos de fabricación puede encontrarse en el Tratado de Farmacia Galénica, C. Faulí i Trillo, Luzán 5, S.A. de Ediciones, 1993.For its applications in vivo, said medium must be pharmaceutically acceptable. Therefore, in another aspect, the invention relates to a pharmaceutical composition comprising an oligomeric protein of the invention together with at least one pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition of the invention comprises at least one oligomeric protein of the invention in an effective amount. In the sense used in this description, 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. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993.
Las proteínas oligoméricas de la invención pueden aislarse y, si se desea, purificarse, fácilmente, por métodos convencionales conocidos por los técnicos en la materia, por ejemplo, mediante cromatografía de afinidad. En general, la proteína oligomérica de la invención es oligomérica en solución y posee una excelente estabilidad y capacidad de unión a la diana (antígeno), reconociendo con alta eficiencia tanto antígenos purificados inmovilizados sobre una placa como antígenos expresados o sobreexpresados en alteraciones patológicas (e.g., en la superficie de una célula tumoral). Las proteínas oligoméricas de la invención poseen una señal de unión mayor que el anticuerpo monomérico (que constituye una unidad estructural de la misma) y, aparentemente, una disociación menor, consistente con la unión multivalente al antígeno. De hecho, se ha calculado que una proteína oligomérica de la invención (e.g., la proteína oligomérica anti-NIP - Ejemplo 1) tiene una afinidad funcional por el antígeno muy superior (NIP-BSA) unas 100 veces mayor que su versión monovalente. Este resultado sugiere, aunque no se desea estar vinculado por ninguna teoría, que esta ganancia de afinidad podría ser debida al efecto de avidez de un segundo sitio de combinación en la molécula de la proteína oligomérica de la invención. La presencia de al menos dos sitios de unión funcionales en una sola molécula de una proteína oligomérica de la invención ha sido posteriormente demostrada mediante la generación de proteínas de oligoméricas de la invención biespecíficas (Ejemplos 1); efectivamente, proteínas oligoméricas (trimerbodies) biespecíficas anti-laminina y anti-NIP estables fueron fácilmente producidos por co-expresión de dos construcciones diferentes de proteínas oligoméricas de la invención en células humanas.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. In general, 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. In fact, it has been calculated that an oligomeric protein of the invention (eg, the anti-NIP oligomeric protein - Example 1) has a much higher functional affinity for the antigen (NIP-BSA) about 100 times greater than its monovalent version. This result suggests, although it is not desired to be bound by any theory, that this affinity gain could be due to the avidity effect of a second combination site in the oligomeric protein molecule of the invention. The presence of at least two functional binding sites in a single molecule of an oligomeric protein of the invention has subsequently been demonstrated by the generation of bispecific oligomeric proteins of the invention (Examples 1); indeed, stable anti-laminin and anti-NIP bispecific oligomeric proteins (trimerbodies) were easily produced by co-expression of two different oligomeric protein constructs of the invention in human cells.
Esta ganancia en afinidad a través de la avidez convierte a las proteínas oligoméricas de la invención en unos reactivos muy atractivos para técnicas de imagen in vzvo como agentes alternativos, y preferidos, a los anticuerpos diméricos (diabodies y minibodies). Como es conocido, para una completa avidez en anticuerpos multivalentes dirigidos a moléculas unidas a la superficie, los sitios de unión del antígeno deben apuntar hacia la misma dirección; si la unión múltiple simultánea no es posible estéricamente, entonces la ganancia aparente en afinidad funcional es probable que sea menor y debida únicamente al efecto de unión aumentado, que es dependiente en tasas de difusión y la concentración del antígeno de superficie. El análisis del modelo de las proteínas oligoméricas de la invención, en particular en su formato trimérico (trimerbody), sugiere una estructura con forma de trípode con los dominios scFv orientados hacia fuera. La flexibilidad entre los sitios de unión al antígeno es otro aspecto importante en el diseño de anticuerpos multivalentes, requeridos para el entrecruzamiento de receptores de superficie, bien en la misma célula o en adyacentes. En este sentido, el espaciador flexible presente en algunas realizaciones particulares de esta invención permite numerosas geometrías de unión. Cuando una interacción antígeno-anticuerpo ocurre, la posibilidad de establecer una segunda interacción depende de la valencia, orientación y flexibilidad del sitio de unión del antígeno. Según los cálculos de los inventores, en una molécula de una proteína oligomérica de la invención, tal como un trímero (trimerbody), los scFv que permanecen sin interaccionar tienen un área de influencia unas 1 1 veces mayor, aproximadamente, que otros formatos bivalentes, tales como los diabodies y los minibodies, aumentando la probabilidad de una segunda interacción efectiva. La flexibilidad de las proteínas oligoméricas de la invención constituye también una ventaja frente a otras estructuras más compactas o rígidas de otros formatos (e.g., collabody), ya que aumenta la accesibilidad de los scFv, que es un parámetro crítico para la localización de las dianas in vivo.This affinity gain through avidity makes the oligomeric proteins of the invention very attractive reagents for in vzvo imaging techniques as alternative, and preferred, agents to dimeric antibodies (diabodies and minibodies). As is known, for a complete avidity in multivalent antibodies directed to surface bound molecules, the antigen binding sites must point in the same direction; If simultaneous multiple binding is not sterically possible, then the apparent gain in functional affinity is likely to be less and due solely to the increased binding effect, which is dependent on diffusion rates and the concentration of the surface antigen. The analysis of the oligomeric protein model of the invention, particularly in its trimeric format, suggests a tripod-shaped structure with the scFv domains oriented outward. Flexibility between antigen binding sites is another important aspect in the design of multivalent antibodies, required for cross-linking of surface receptors, either in the same cell or in adjacent ones. In this sense, the flexible spacer present in some particular embodiments of This invention allows numerous joining geometries. When an antigen-antibody interaction occurs, the possibility of establishing a second interaction depends on the valence, orientation and flexibility of the antigen binding site. According to the calculations of the inventors, in a molecule of an oligomeric protein of the invention, such as a trimer (trimerbody), 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
Por tanto, la multimerización de las construcciones scFv presenta numerosas ventajas para las aplicaciones in vivo frente a otros anticuerpos recombinantes (diabodies y minibodies) que han mostrado su potencial como agentes de localización in vivo. Las proteínas oligoméricas de la invención son moléculas multivalentes de tamaño intermedio que presentan una alta estabilidad en condiciones fisiológicas. El potencial de dichas proteínas oligoméricas de la invención, en particular de unos trímeros (trimerbodies) para la localización in vivo se ha estudiado en modelos experimentales de cáncer humano en ratones desnudos (Ejemplo 1). En otro aspecto, la invención se relaciona con un kit que comprende una proteína oligomérica de la invención. El empleo de dicho kit para la detección, visualización o localización de una diana, e.g., un antígeno, mediante una técnica apropiada, por ejemplo, una técnica de imagen. En una realización particular dicha diana es un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica, por ejemplo, en patologías inflamatorias, vasculares, tumorales, etc. En una realización particular, dicha diana es un antígeno tumoral.Therefore, 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). In another aspect, the invention relates to a kit comprising an oligomeric protein of the invention. The use of said kit for the detection, visualization or localization of a target, e.g., an antigen, by an appropriate technique, for example, an imaging technique. In a particular embodiment said target is an antigen that expresses de novo or is overexpressed in a pathological alteration, for example, in inflammatory, vascular, tumor pathologies, etc. In a particular embodiment, said target is a tumor antigen.
El kit de la invención es un producto que contiene los diferentes productos (e.g., proteína oligomérica de la invención, reactivos adicionales, etc.) formando la composición empaquetada de modo que permita su transporte, almacenamiento y su empleo. Los kits de la invención pueden contener de este modo una o más suspensiones, jeringuillas, etc., así como medios para reconstituir la proteína oligomérica de la invención en caso de que esta estuviera en forma liofilizada. Otros componentes que pueden estar presentes en el kit de la invención es un envase que permite mantener las formulaciones de la invención dentro de determinados límites. Los materiales adecuados para preparar tales envases incluyen vidrio, plástico, polietileno, polipropileno, policarbonato y similares, botellas, viales, papel, bolsitas y similares. Adicionalmente, el kit de la invención puede contener instrucciones para su empleo. Dichas instrucciones se pueden encontrar en forma de material impreso o en forma de soporte electrónico que puede almacenar las instrucciones tal que puedan ser leídas por un sujeto, tal como medios de almacenamiento electrónico (discos magnéticos, cintas y similares), medios ópticos (CD-ROM, DVD) y similares. Los medios pueden adicional o alternativamente contener sitios Web en Internet proporcionando dichas instrucciones.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. Additionally, the 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.
Proteína de fusión [polipéptido ( A)-polipéptido (B)IFusion protein [polypeptide (A) -polypeptide (B) I
La proteína oligomérica de la invención comprende una pluralidad de proteínas de fusión (iguales o diferentes), dependiendo del dominio de oligomerización presente en el polipéptido (B), y cada proteína de fusión comprende: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:
(a) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y(a) a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y
(b) un polipéptido (B) que comprende un dominio de oligomerización. Adicionalmente, si se desea, dicha proteína de fusión puede incluir un polipéptido (C) que comprende la secuencia de aminoácidos de un péptido de unión flexible entre dichos polipéptidos (A) y (B) y/o un péptido (D) para facilitar el aislamiento o purificación de la proteína de fusión.(b) a polypeptide (B) comprising an oligomerization domain. Additionally, if desired, 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.
Las características de dichos polipéptidos (A), (B), (C) y (D) ya han sido descritas previamente, al igual que la posibilidad de que dichas proteínas de fusión sean iguales o diferentes.The characteristics of said polypeptides (A), (B), (C) and (D) have already been described previously, as well as the possibility that said fusion proteins are the same or different.
La proteína de fusión proporcionada por la invención puede ser obtenida por métodos convencionales. A modo ilustrativo, dicha pro teína de fusión puede obtenerse mediante la fusión de dichos polipéptidos, obtenidos bien por métodos de síntesis química de péptidos o bien mediante la tecnología del ADN recombinante. Alternativamente, dicha proteína de fusión proporcionada por esta invención puede obtenerse por mediante el empleo de la tecnología del ADN recombinante, para lo cual se generarán las construcciones génicas, cassettes de expresión y vectores correspondientes. En general, cuando las proteínas de fusión son iguales, pueden obtenerse mediante incorporación del ADN que codifica dicha proteína de fusión en una célula huésped apropiada (e.g., bacterias, levaduras, células animales, etc.). Alternativamente, cuando las proteínas de fusión son diferentes, pueden obtenerse, en general, mediante incorporación de los ADN que codifican dichas proteínas de fusión en una célula huésped apropiada, en donde los distintos ADN pueden formar parte de la misma construcción génica o de construcciones génicas diferentes (e.g., mediante co- transformación o co-transfección de células huésped apropiadas).The fusion protein provided by the invention can be obtained by conventional methods. By way of illustration, said fusion protein can be obtained by fusion of said polypeptides, obtained either by chemical peptide synthesis methods or by recombinant DNA technology. Alternatively, 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. In general, 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.). Alternatively, when the fusion proteins 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).
Por tanto, en otro aspecto, la invención se relaciona con una construcción génica, en adelante construcción génica de la invención, que comprende, al menos: a) una primera secuencia de ácido nucleico (A'), que comprende la secuencia de nucleótidos que codifica un polipéptido (A), en donde dicho polipéptido (A) comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y b) una segunda secuencia de ácido nucleico (B') que codifica un polipéptido (B) que comprende un dominio de oligomerización, en donde el extremo 3' de dicha primera secuencia de ácido nucleico (A') está unido al extremo 5' de dicha segunda secuencia de ácido nucleico (B'), o, alternativamente, el extremo 5 ' de dicha primera secuencia de ácido nucleico (A') está unido al extremo 3 ' de dicha segunda secuencia de ácido nucleico (B').Therefore, in another aspect, 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 ').
La secuencia de ácido nucleico (A') comprende la secuencia de nucleótidos que codifica un polipéptido (A) que comprende un anticuerpo que reconoce una diana o un fragmento funcionalmente equivalente de dicho anticuerpo que reconoce dicha diana. Ejemplos ilustrativos de dicha diana incluyen un epítopo o determinante antigénico de un antígeno, por ejemplo, un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica, por ejemplo, en patologías inflamatorias, vasculares, tumorales, etc. En una realización particular, dicha diana es un antígeno tumoral.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. Illustrative examples of said 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. In a particular embodiment, said target is a tumor antigen.
Por tanto, en una realización particular, la secuencia de ácido nucleico (A') codifica un polipéptido (A) que comprende un anticuerpo, o un fragmento funcionalmente equivalente del mismo, que reconoce una diana concreta. Prácticamente cualquier anticuerpo, o fragmento funcionalmente equivalente del mismo, que reconozca una diana puede ser utilizado en la presente invención, e.g., un AcM, un AcP, un scFv, un anticuerpo biespecífico o diabody, un VHH, etc. En una realización particular, dicha secuencia de ácido nucleico (A') codifica un polipéptido (A) que comprende un scFv recombinante derivado del AcM L36 anti-laminina [scFv L36], o un scFv recombinante derivado del AcM Bl.8 específico del hapteno NIP, o un scFv recombinante derivado del AcM MFE23 específico del antígeno carcinoembrionario (CEA) humano (Ejemplo 1).Thus, in a particular embodiment, the nucleic acid sequence (A ') encodes 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. In a particular embodiment, 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).
En caso de que la proteína oligomérica de la invención comprenda una pluralidad de proteínas de fusión diferentes, por ejemplo, dos o más proteínas de fusión diferentes, cada una de ellas estaría codificada por la correspondiente secuencia de ADN, las cuales podrían estar en una única construcción génica o en construcciones génicas diferentes.In case 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.
La secuencia de ácido nucleico (B') comprende la secuencia de nucleótidos que codifica un polipéptido (B) que comprende un dominio de oligomerización. Como se ha mencionado previamente, un dominio de oligomerización permite la formación de oligómeros (e.g., dímeros, trímeros, tetrámeros, etc., de péptidos o proteínas). Prácticamente cualquier dominio de oligomerización, por ejemplo, un dominio de dimerización, trimerización, tetramerización, etc., presente en distintas proteínas, tanto de origen eucariótico como procariótico, susceptible de ser expresado de forma recombinante y de formar un oligómero proteico de la proteína que lo comprende puede ser utilizado en la presente invención. En una realización particular, dicho dominio de oligomerización es un dominio de trimerización, tal como el dominio de trimerización del dominio NCl de colágeno XVIII o del colágeno XV; en una realización concreta, dicho dominio de oligomerización es el dominio de trimerización del dominio NCl de colágeno XVIII o del colágeno XV al que se la ha eliminado la totalidad o parte del dominio de ES. Por tanto, en una realización particular y preferida, la secuencia de ácido nucleico (B') comprende la secuencia de nucleótidos que codifica el dominio NCl de colágeno XVIII de mamífero o el dominio NCl del colágeno XV de mamífero, en el que, opcionalmente, se ha eliminado la totalidad o parte del dominio de ES. Las secuencias de dichos dominios NCl de colágeno XV y XVIII son conocidas; a modo ilustrativo, la secuencia del dominio NC l del colágeno XVIII ha sido descrita previamente por Sasaki et al. [Sasaki et al. Structure, function and tissue forms of the C- terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J. 1998 Aug 3;17(15):4249-56].The nucleic acid sequence (B ') comprises the nucleotide sequence encoding a polypeptide (B) comprising an oligomerization domain. As previously mentioned, an oligomerization domain allows the formation of oligomers (eg, dimers, trimers, tetramers, etc., of peptides or proteins). Virtually any oligomerization domain, for example, a dimerization, trimerization, tetramerization, etc. domain, present in different proteins, both eukaryotic and prokaryotic, capable of being recombinantly expressed and forming a protein oligomer of the protein that It comprises it can be used in the present invention. In a particular embodiment, 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. The sequences of said 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].
En la construcción génica de la invención, el extremo 3 ' de dicha secuencia de ácido nucleico (A') está unido, en una realización particular, al extremo 5 ' de dicha secuencia de ácido nucleico (B'); alternativamente, en otra realización particular, el extremo 5 ' de dicha secuencia de ácido nucleico (A') está unido al extremo 3 ' de dicha secuencia de ácido nucleico (B').In the gene construct of the invention, 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').
En general, la secuencia de ácido nucleico (A') no se fusiona directamente a la secuencia de ácido nucleico (B') sino que resulta ventajoso introducir un péptido de unión flexible (o péptido espaciador) entre los polipéptidos codificados por dichas secuencias de ácido nucleico (A') y (B'). Por tanto, si se desea, la construcción génica de la invención también puede contener, además, una tercera secuencia de ácido nucleico (C) que contiene la secuencia de nucleótidos que codifica para un péptido de unión flexible situada entre dichas secuencias de ácido nucleico (A') y (B'). En una realización particular, el extremo 5' de dicha secuencia de ácido nucleico (C) está unido al extremo 3' de dicha secuencia de ácido nucleico (A') y el extremo 3' de dicha secuencia de ácido nucleico (C) está unido al extremo 5' de dicha secuencia de ácido nucleico (B'); alternativamente, en otra realización particular, el extremo 3' de dicha secuencia de ácido nucleico (C) está unido al extremo 5 ' de dicha secuencia de ácido nucleico (A') y el extremo 5 ' de dicha secuencia de ácido nucleico (C) está unido al extremo 3' de dicha secuencia de ácido nucleico (B'). Ventajosamente, dicho péptido espaciador (C) es un péptido con flexibilidad estructural. Prácticamente, cualquier péptido con flexibilidad estructural puede ser utilizado. A modo ilustrativo, dicho péptido flexible puede contener repeticiones de restos de aminoácidos, en particular de restos de GIy y Ser o cualquier otra repetición de restos de aminoácidos adecuada. Prácticamente cualquier secuencia peptídica que defina un péptido de unión flexible puede ser utilizada en la presente invención. Ejemplos ilustrativos de péptidos de unión flexibles incluyen secuencias de tipo Gly-Ser-Pro-Gly (GSPG) o la secuencia (Gly-Ser)4. No obstante, en una realización particular, dicho péptido de unión flexible comprende la secuencia Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly- Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser. Por tanto, en una realización particular, la construcción génica de la invención comprende, además de dichas secuencias de ácido nucleico (A) y (B) una tercera secuencia de ácido nucleico (C) que comprende la secuencia de nucleótidos que codifica para el péptido Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly- Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser. La longitud y composición del péptido espaciador puede variar; no obstante, en una realización particular, se ajustará (mayor o menor longitud, mayor o menor rigidez) en función de la naturaleza de la diana (antígeno) reconocida por el anticuerpo para conseguir las mejores propiedades funcionales.In general, 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'). Thus, if desired, 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'). In a particular embodiment, 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'). Advantageously, said spacer peptide (C) is a peptide with structural flexibility. Virtually any peptide with structural flexibility can be used. By way of illustration, 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 . However, in a particular embodiment, 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.
Asimismo, con el fin de facilitar el aislamiento y purificación de la proteína de fusión obtenida mediante la presente invención, la construcción génica de la invención puede contener, si se desea, una secuencia de ácido nucleico que codifica un péptido susceptible de ser utilizado con fines de aislamiento o purificación de la proteína de fusión. Por tanto, en una realización particular, la construcción génica de la invención incluye, si se desea, una secuencia de ácido nucleico (D') que contiene la secuencia de nucleótidos que codifica un péptido susceptible de ser utilizado con fines de aislamiento o purificación, conocido como péptido etiqueta ("tag"). Dicha secuencia de ácido nucleico (D') puede estar situada en cualquier posición que no altere la funcionalidad de ninguno de los polipéptidos [(A) y (B)] expresados por dichas secuencias de ácidos nucleicos (A') y (B'). A modo simplemente ilustrativo, no limitativo, dicha secuencia de ácido nucleico (D') puede estar situada aguas abajo del extremo 3' de dicha secuencia de ácido nucleico (B'). Prácticamente cualquier péptido o secuencia peptídica que permita el aislamiento o purificación de la proteína de fusión puede ser utilizado, por ejemplo, una cola de histidinas (e.g., 6 restos de His), una secuencias peptídica susceptible de ser reconocida por un anticuerpo que pueden servir para purificar la proteína de fusión resultante por cromatografía de inmunoafinidad, tales como péptidos etiqueta, etc., por ejemplo, epítopos derivados de la hemaglutinina (HA) del virus de la gripe, C-myc, FLAG, V5, etc.Also, in order to facilitate the isolation and purification of the fusion protein obtained by the present invention, 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. Thus, in a particular embodiment, 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 ') . By way of illustration, but not limitation, said nucleic acid sequence (D ') may be located downstream of the 3' end of said nucleic acid sequence (B '). Virtually 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.
La construcción génica de la invención puede obtenerse mediante el empleo de técnicas ampliamente conocidas en el estado de la técnica [Sambrook et al, "Molecular cloning, a Laboratory Manual", 2nd ed., CoId Spring Harbor Laboratory Press, N.Y., 1989 VoI 1-3]. Dicha construcción génica de la invención puede incorporar, operativamente unida, una secuencia reguladora de la expresión de las secuencias de nucleótidos que codifican para los polipéptidos codificados por las secuencias de ácido nucleico (A') y (B'), constituyendo de este modo un cassette de expresión. Tal como se utiliza en esta descripción, la expresión "operativamente unida" significa que los polipéptidos codificados por las secuencias de ácido nucleico (A') y (B'), y, en su caso (C), son expresados en el marco de lectura correcto bajo el control de las secuencias de control o reguladoras de expresión.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 As used in this description, 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.
Por tanto, en otro aspecto, la invención proporciona un cassette de expresión que comprende la construcción génica de la invención operativamente unida a una secuencia de control de expresión de la secuencia de nucleótidos que codifica la proteína de fusión proporcionada por esta invención que comprende un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente del mismo y un polipéptido (B) que comprende un dominio de oligomerización. Las secuencias de control son secuencias que controlan y regulan la transcripción y, en su caso, la traducción de dicha proteína de fusión, e incluyen secuencias promotoras, secuencias codificantes para reguladores transcripcionales, secuencias de unión a ribosomas (RBS) y/o secuencias terminadoras de transcripción. En una realización particular, dicha secuencia de control de expresión es funcional en células y organismos procariotas, por ejemplo, bacterias, etc., mientras que en otra realización particular, dicha secuencia de control de expresión es funcional en células y organismos eucariotas, por ejemplo, células de insecto, células vegetales, células de mamífero, etc. Ejemplos ilustrativos de promotores que pueden estar presentes en el cassette de expresión proporcionado por esta invención incluyen el promotor de citomegalovirus humano (hCMV), etc.Thus, in another aspect, 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. In a particular embodiment, said expression control sequence is functional in prokaryotic cells and organisms, for example, bacteria, etc., while in another particular embodiment, said expression control sequence is functional in eukaryotic cells and organisms, for example. , insect cells, plant cells, mammalian cells, etc. Illustrative examples of promoters that may be present in the expression cassette provided by this invention include the human cytomegalovirus (hCMV) promoter, etc.
Ventajosamente, dicho cassette de expresión comprende, además, un marcador o gen que codifica para un motivo o para un fenotipo que permita la selección de la célula hospedadora transformada con dicho cassette de expresión. Ejemplos ilustrativos de dichos marcadores que podrían estar presentes en el cassette de expresión de la invención incluyen genes de resistencia a antibióticos, genes de resistencia a compuestos tóxicos, y, en general, todos aquellos que permitan seleccionar a las plantas transformadas genéticamente.Advantageously, 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. Illustrative examples of said 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.
La construcción génica de la invención, o el cassette de expresión proporcionado por esta invención, pueden ser insertados en un vector apropiado. Por tanto, en otro aspecto, la invención se relaciona con un vector, tal como un vector de expresión, que comprende dicha construcción de génica de la invención o dicho cassette de expresión. La elección del vector dependerá de la célula hospedadora en la que se va a introducir posteriormente. A modo ilustrativo, el vector donde se introduce dicha secuencia de ácido nucleico puede ser un plásmido o un vector que, cuando se introduce en una célula hospedadora, se integra o no en el genoma de dicha célula. La obtención de dicho vector puede realizarse por métodos convencionales conocidos por los técnicos en la materia [Sambrook et al., 1989, citado supra]. En una realización particular, dicho vector recombinante es un vector útil para transformar células animales. Dicho vector puede ser utilizado para transformar, transfectar o infectar células susceptibles de ser transformadas, transfectadas o infectadas por dicho vector. Dichas células pueden ser procariotas o eucariotas. Por tanto, en otro aspecto, la invención se relaciona con una célula hospedadora transformada, transfectada o infectada con un vector proporcionado por esta invención. Dicha célula transformada, transfectada o infectada comprende, por tanto, una construcción génica de la invención, o bien dicho cassette de expresión o vector proporcionado por esta invención. Células transformadas, transfectadas o infectadas pueden ser obtenidas por métodos convencionales conocidos por los técnicos en la materia [Sambrook et al., 1989, citado supra]. En una realización particular, dicha célula hospedadora es una célula animal transformada, transfectada o infectada con un vector apropiado, siendo dicha célula animal transformada, transfectada o infectada capaz de expresar la proteína de fusión proporcionada por esta invención, por lo que dichos vectores pueden utilizarse para la expresión en células animales de la proteína de fusión proporcionada por esta invención.The gene construct of the invention, or the expression cassette provided by this invention, can be inserted into an appropriate vector. Therefore, in another aspect, the invention relates to a vector, such as an expression vector, comprising said gene construct of the invention or said expression cassette. The choice of the vector will depend on the host cell into which it will be subsequently introduced. By way of illustration, 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]. In a particular embodiment, 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]. In a particular embodiment, 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.
La construcción génica de la invención puede ser utilizada para producir dichas proteínas de fusión que comprenden un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente del mismo y un polipéptido (B) que comprende un dominio de oligomerización.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.
Por tanto, en otro aspecto, la invención se relaciona con un método para producir dicha proteína de fusión proporcionada por esta invención que comprende crecer una célula u organismo proporcionado por esta invención bajo condiciones que permiten la producción de dicha proteína de fusión. Las condiciones para optimizar el cultivo de dicha célula u organismo dependerán de la célula u organismo utilizado. Si se desea, el método para producir un producto de interés proporcionado por esta invención incluye, además, el aislamiento y purificación de dicha proteína de fusión. El experto en la materia entenderá que, si la proteína oligomérica de la invención contiene dos o más proteínas de fusión diferentes, dichas proteínas de fusión diferentes pueden expresarse, si se desea, en una célula huésped apropiada mediante co- transformación, co-transfección o co-infección utilizando los vectores que contienen las secuencias codificantes apropiadas.Therefore, in another aspect, 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. If desired, the method of producing a product of interest provided by this invention further includes isolation and purification of said fusion protein. The person skilled in the art will understand that if 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.
El siguiente Ejemplo sirve para ilustrar la invención y no debe ser considerado como limitativo del alcance de la misma.The following Example serves to illustrate the invention and should not be considered as limiting its scope.
EJEMPLO 1EXAMPLE 1
Localización y visualización in vivo de tumores mediante proteínas oligoméricas que comprenden fragmentos de anticuerpos y secuencias derivadas de colágenoIn vivo localization and visualization of tumors by oligomeric proteins comprising antibody fragments and collagen derived sequences
I. MATERIALES Y MÉTODOSI. MATERIALS AND METHODS
Anticuerpos y reactivosAntibodies and reagents
Los anticuerpos monoclonales (AcMs) utilizados incluyeron el AcM 9E10 (Abcam, Cambridge, R. Unido) específico de c-myc humano, y el AcM NCRC23 (AbDMonoclonal antibodies (AcMs) used included the AcM 9E10 (Abcam, Cambridge, R. Kingdom) specific for human c-myc, and the AcM NCRC23 (AbD
Serotec, Kidlington, R. Unido) específico del antígeno carcinoembrionario (CEA) humano. Los anticuerpos policlonales (AcPs) utilizados incluyeron un anticuerpo (Ac) de conejo anti-albúmina de suero bovina (BSA); un Ac de cabra anti-IgG de conejo conjugado con peroxidasa de rábano picante (HRP); y un Ac de cabra anti-IgG de ratón, específico del dominio constante de la IgG (Fc), conjugado con HRP, todos ellos proporcionados por Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, EEUU). La laminina-1 purificada a partir del tumor murino EHS (Engelbreth-Holm-Swarm) se obtuvo de Becton Dickinson Labware (Bedford, MA, USA). El CEA humano y la BSA fueron proporcionados por Sigma-Aldrich. Para la generación de conjugados de BSA con el hapteno 4-hidroxi-5-iodo-3-nitrofenil (NIP) (Sigma-Aldrich) en una relación molar 10: 1 (NIPio:BSA) se siguió un protocolo anteriormente descrito [Reth, M. et al.Serotec, Kidlington, United Kingdom) specific to the human carcinoembryonic antigen (CEA). The polyclonal antibodies (AcPs) used included a bovine serum anti-albumin rabbit (BSA) antibody (Ac); a goat anti-rabbit IgG Ac conjugated to horseradish peroxidase (HRP); and a 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 (Engelbreth-Holm-Swarm) was obtained from Becton Dickinson Labware (Bedford, MA, USA). The human CEA and the BSA were provided by Sigma-Aldrich. For the generation of BSA conjugates with the hapten 4-hydroxy-5-iodo-3-nitrophenyl (NIP) (Sigma-Aldrich) in a 10: 1 molar ratio (NIPio: BSA) a previously described protocol was followed [Reth, M. et al.
(1979). Eur. J. Immunol. 9:1004-1013].(1979). Eur. J. Immunol. 9: 1004-1013].
Células y condiciones de cultivo Las células HEK-293 (células epiteliales de riñon embrionario humano; CRL-Cells and culture conditions HEK-293 cells (human embryonic kidney epithelial cells; CRL-
1573), y sus células derivadas 293T (CRL-11268), así como las células HT-1080 (fibrosarcoma humano; CCL- 121), MKN45 (adeno carcinoma gástrico humano; JCRB- 0254); y HeLa (carcinoma de cérvix humano; CCL-2) fueron cultivadas en medio Dulbecco modificado por Eagle (DMEM) suplementado con 10% (v/v) de suero bovino fetal (SBF) inactivado por calor (todos de Invitrogen, Carlsbad, CA), en adelante medio DMEM completo (DCM). Las células HeLaCEA (Compte, M. et al. 2007; Cáncer Gene Ther. 14:380-388) se cultivaron en DCM suplementado con 750 μg/ml de G418 (Invitrogen).1573), and its 293T derived cells (CRL-11268), as well as 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). HeLa CEA cells (Compte, M. et al. 2007; Cancer Gene Ther. 14: 380-388) were grown in DCM supplemented with 750 μg / ml of G418 (Invitrogen).
Construcción de los vectores de expresiónConstruction of expression vectors
Los vectores de expresión pCR3. 1-L36 y pCR3.1-L36-NClES" fueron construidos siguiendo protocolos conocidos [Sanz L et al. (2002). Gene Ther. 9:1049- 1053; Sanz L et al. (2001). Cáncer Immunol. Immunother. 50:557-565].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].
El plásmido pVOMl .C23 que contiene el gen que codifica el Ac MFE-23 (anti- CEA humano) en formato de fragmento variable de cadena única (scFv), fue proporcionado por el Dr. R. E. Hawkins (University of Manchester, UK). El cassette de expresión del Ac MFE-23 se digirió con HindIII y Notl y se clonó en el vector pCEP4.6xHis-myc [Sanz, L. et al. (2002). Gene Ther. 9:1049-1053] para generar el plásmido pCEP4-MFE-23.Plasmid pVOMl .C23 containing the gene encoding Ac MFE-23 (human anti-CEA) in single chain variable fragment format (scFv), was provided by Dr. R. E. Hawkins (University of Manchester, UK). 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.
El plásmido pCEP4-B1.8 que contiene el gen que codifica el Ac B 1.8 (anti-NIP) en formato scFv y las etiquetas (tags) peptídicas de polihistidina (6 His) y c-myc fue construido siguiendo un procedimiento ya descrito [Sanz, L. et al. (2002), citado supra]. Para generar los plásmidos pCEP4-MFE-23-NClES~ y pCEP4-B1.8-NClES~, el fragmento de 252 pares de bases (pb) derivado del plásmido pCR3.1-L36-NClES", obtenido por digestión con Notl, se clonó en los plásmidos pCEP4-MFE-23 o pCEP4- B 1.8 respectivamente.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]. To generate plasmids pCEP4-MFE-23-NCl ES ~ and pCEP4-B1.8-NCl ES ~ , the 252 base pair fragment (bp) derived from plasmid pCR3.1-L36-NCl ES " , obtained by digestion with Notl, it was cloned into plasmids pCEP4-MFE-23 or pCEP4-B 1.8 respectively.
Transfecciones celulares y purificación de anticuerpos recombinantesCellular transfections and purification of recombinant antibodies
Las células HEK-293 y sus células derivadas 293T fueron transfectadas con Superfect según las recomendaciones del fabricante (QIAGEN GmbH, Hilden, Germany). Para la obtención de líneas celulares (transfectantes) estables, las células HEK-293 transfectadas con los vectores pCR3.1-L36 y pCR3.1-L36-NClES", respectivamente, se seleccionaron en DCM suplementado con 0,5 mg/ml de neomicina (G418) (Promega). Las células HEK-293 transfectadas con los vectores pCEP4-MFE- 23-NClES~ y pCEP4-B1.8-NClES~, respectivamente, se seleccionaron en DCM suplementado con 100 μg/ml de higromicina B (Invitrogen). Los sobrenadantes de las poblaciones celulares transfectadas se analizaron para determinar la expresión de proteínas por ELISA, SDS-PAGE y transferencia Western utilizando el AcM anti-myc. Los transfectantes estables de las células HEK-293 se utilizaron para obtener medio condicionado libre de suero (MCLS) (aproximadamente 1 litro) que fue concentrado (xlO) con un filtro de 10.000 MWCO Vivaflow 50 (Vivascience AG, Hannover, Germany), dializado frente a tampón fosfato salino (PBS) (pH 7,4) y cargado en una columna HisTrap HP de 1 mi utilizando el sistema ÁKTA Prime plus (GE Healthcare, Uppsala, Suecia). Las proteínas (anticuerpos) recombinantes purificadas se dializaron frente a PBS, se analizaron mediante SDS-PAGE bajo condiciones reductoras y no- reductoras, y se almacenaron a -2O0C hasta su uso.HEK-293 cells and their 293T derived cells were transfected with Superfect according to the manufacturer's recommendations (QIAGEN GmbH, Hilden, Germany). To obtain stable cell lines (transfectants), HEK-293 cells transfected with vectors pCR3.1-L36 and pCR3.1-L36-NCl ES " , respectively, 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 ~ , respectively, were selected in DCM supplemented with 100 μg / ml hygromycin B (Invitrogen). Supernatants from transfected cell populations were analyzed for protein expression by ELISA, SDS-PAGE and Western blotting using the anti-myc AcM. Stable transfectants of HEK-293 cells were used to obtain serum-free conditioned medium (MCLS) (approximately 1 liter) that was concentrated (xlO) with a 10,000 MWCO Vivaflow 50 filter (Vivascience AG, Hannover, Germany), dialyzed versus phosphate buffered saline (PBS) (pH 7.4) and loaded onto a 1 ml HisTrap HP column using the AKTA Prime plus system (GE Healthcare, Uppsala, Sweden). The purified recombinant proteins (antibodies) were dialyzed against PBS, analyzed by SDS-PAGE under reducing and non-reducing conditions, and stored at -2O 0 C until use.
Cromatografía analítica de filtración en gel Los análisis se realizaron utilizando columnas Superdex 200 10/300GL (GEAnalytical gel filtration chromatography The analyzes were performed using Superdex 200 10 / 300GL columns (GE
Healthcare) equilibradas con PBS (pH 7,4) en un equipo ÁKTA FPLC (GE Healthcare). Las muestras (100 μl) de proteínas recombinantes purificadas, en una concentración comprendida ente 0,5 y 1,0 mg/ml, se inyectaron en la columna y se eluyeron con una velocidad de flujo de 0,5 ml/minuto. La columna se calibró con marcadores de peso molecular que comprendían desde 16 kDa hasta 655 kDa (GE Healthcare). El azul dextrano se utilizó como volumen de exclusión.Healthcare) balanced with PBS (pH 7.4) in an ÁKTA FPLC (GE Healthcare) device. Samples (100 μl) of purified recombinant proteins, in a concentration between 0.5 and 1.0 mg / ml, were injected into the column and eluted at a flow rate of 0.5 ml / minute. The column was calibrated with molecular weight markers ranging from 16 kDa to 655 kDa (GE Healthcare). Dextran blue was used as exclusion volume.
ELISAELISA
La capacidad de reconocimiento y unión de las proteínas recombinantes (trímeros de anticuerpos (scFv) - en ocasiones identificadas en esta descripción comoThe ability to recognize and bind recombinant proteins (antibody trimers (scFv) - sometimes identified in this description as
"trimerbodies") purificadas [L36, MFE23 o Bl.8] a laminina-1 murina, CEA humano o conjugados NIPI0-BSA, fue estudiada mediante ELISA [Sanz, L. et al. 2003; EMBO J.,Purified "trimerbodies" [L36, MFE23 or Bl.8] to murine laminin-1, human CEA or NIP I0- BSA conjugates, was studied by ELISA [Sanz, L. et al. 2003; EMBO J.,
22:1508-17]. La multivalencia de los trimerbodies se estudió mediante ELISA utilizando el sobrenadante de las células 293T transfectadas bien con un sólo vector (pCR3.1-L36-NClES" o pCEP4-B1.8-NClES~) o bien con 2 vectores (pCR3.1-L36-22: 1508-17]. The multivalence of the trimerbodies was 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-
NC1ES" y pCEP4-B1.8-NClES"). Placas de 96 pocilios Maxisorp (NUNC BrandNC1 ES " and pCEP4-B1.8-NCl ES" ). 96-well plates Maxisorp (NUNC Brand
Products, Roskilde, Dinamarca) fueron recubiertas con laminina (0,5 μg/pocillo) mediante una incubación de 16 horas a 40C en PBS (10 μg/ml); posteriormente, y, después de lavar las placas con PBS y se bloquearlas con 200 μl de PBS-5% de leche en polvo desnatada, y se añadieron 100 μl de sobrenadante procedente de células 293T transfectadas bien con un sólo vector (pCR3.1-L36-NClES" o pCEP4-B1.8-NClES~) o bien con 2 vectores (pCR3.1-L36-NClES" y pCEP4-B1.8-NClES~) durante 1 hora a temperatura ambiente. Al cabo de 3 lavados, se añadieron 100 μl de NIPio-BSA (10 μg/ml), incubándose durante 1 hora a temperatura ambiente. Posteriormente, tras 3 lavados, se añadieron 100 μl del Ac de conejo anti-BSA (1 :1.000) en 0,05% Tween-20- PBS. Tras tres nuevos lavados, se añadieron 100 μl del Ac de cabra anti-IgG de conejo conjugado con HRP durante 1 hora a temperatura ambiente, tras lo cual, las placas se lavaron y revelaron.Products, Roskilde, Denmark) were coated with laminin (0.5 μg / well) by a 16 hour incubation at 4 0 C in PBS (10 μg / ml); subsequently, and, after washing the plates with PBS and blocking them with 200 μl of PBS-5% skimmed milk powder, and 100 μl of supernatant from 293T cells transfected well with a single vector was added (pCR3.1- L36-NCl ES " or pCEP4-B1.8-NCl ES ~ ) or with 2 vectors (pCR3.1-L36-NCl ES" and pCEP4-B1.8-NCl ES ~ ) for 1 hour at room temperature. After 3 washes, 100 µl of NIPio-BSA (10 µg / ml) was added, incubating for 1 hour at room temperature. Subsequently, after 3 washes, 100 µl of the anti-BSA rabbit Ac (1: 1,000) in 0.05% Tween-20-PBS was added. After three new washes, 100 µl of the goat anti-rabbit IgG conjugated with HRP was added for 1 hour at room temperature, after which the plates were washed and revealed.
Citometría de flujo, detección de antígenos en la superficie celularFlow cytometry, detection of cell surface antigens
La expresión de CEA en las líneas tumorales HeLa y HeLaCEA y la unión de los anticuerpos recombinantes (trimerbodies) se analizó siguiendo un protocolo ya descritoCEA expression in HeLa and HeLa CEA tumor lines and recombinant antibody binding (trimerbodies) was analyzed following a protocol already described.
[Blanco B et al, (2003). J. Immunol. 171 :1070-1077] mediante inmuno fluorescencia indirecta. Brevemente, las células se incubaron con AcM anti-CEA humano (5 μg/ml), o con los trimerbodies purificados (anti-NIP o anti-CEA, 10 μg/ml) y el AcM 9E10 (4 μg/ml) en 100 μl durante 45 minutos. Tras lavado, las células fueron tratadas con las diluciones apropiadas de Ac de cabra anti-IgG de ratón conjugado con FITC (Sigma-[Blanco B et al, (2003). J. Immunol. 171: 1070-1077] by indirect immuno fluorescence. Briefly, cells were incubated with human anti-CEA mAb (5 μg / ml), or with purified trimerbodies (anti-NIP or anti-CEA, 10 μg / ml) and AcM 9E10 (4 μg / ml) in 100 μl for 45 minutes. After washing, the cells were treated with the appropriate dilutions of goat anti-mouse IgG conjugated to FITC (Sigma-
Aldrich). Los estudios se realizaron en un citómetro EPICS XL (Coulter Electronics).Aldrich). The studies were performed on an EPICS XL cytometer (Coulter Electronics).
Estudios de afinidad mediante resonancia de plasmen superficieAffinity studies by surface plasmon resonance
Todos los estudios de afinidad mediante resonancia de plasmón de superficie (SPR, del inglés "Surface Plasmon Resonance") se realizaron a temperatura ambiente utilizando un sistema Biacore 3000 (GE Healthcare). Se utilizaron chips sensores de dextrano carboximetilado (CM5) (GE Healthcare) y tampón HBS-EP (HEPES 0,01 M; pH 7,4; NaCl 0,15 M; EDTA 3 mM; Surfactante P20 0,005%) previamente filtrado a través de un filtro de 0,22 μm y desgasificado antes de su uso. Las proteínas recombinantes (trimerbodies) se disolvieron en acetato sódico 10 mM (pH 4,5). El conjugado NIPio-BSA se inmovilizó directamente sobre la superficie del chip sensor siguiendo las instrucciones del fabricante en celdas de flujo independiente a, aproximadamente, 100, 1.800 y 8.500 unidades de resonancia (US). La BSA, utilizada como control negativo de la interacción, se inmovilizó a 1.900 UR sobre la celda de flujo de referencia. Después de cada experimento, las superficies se regeneraron con HCl 30 mM, permitiendo la vuelta de las señales de resonancia a niveles básales. Los análisis se realizaron por duplicado.All affinity studies using surface plasmon resonance (SPR) were performed at room temperature using a Biacore 3000 (GE Healthcare) system. Carboxymethylated dextran (CM5) sensor chips (GE Healthcare) and HBS-EP buffer (0.01 M HEPES; pH 7.4; 0.15 M NaCl; 3 mM EDTA; Surfactant P 20 0.005%) previously filtered at through a 0.22 μm filter and degassed before use. Recombinant proteins (trimerbodies) were dissolved in 10 mM sodium acetate (pH 4.5). The NIPio-BSA conjugate was immobilized directly on the surface of the sensor chip following the manufacturer's instructions in independent flow cells a, approximately 100, 1,800 and 8,500 resonance units (US). 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.
Para los análisis cinéticos se utilizó una celda de flujo con cantidades pequeñas de NIPio-BSA (aproximadamente 100 UR) con el fin de minimizar los efectos de transporte de masa y re-unión. Las muestras individuales, constituidas por scFv o trimerbodies purificados, se pasaron sobre la superficie del chip a una velocidad de flujo de 20 μl/minuto y se midieron las velocidades de asociación/disociación. Los cambios en el índice de refracción se eliminaron mediante la sustracción de las respuestas de los chips de referencia y la respuesta media de un control negativo se restó a todos los sensogramas. Los resultados cinéticos se obtuvieron utilizando el software BIAevaluation v4.1, proporcionado con el biosensor, y los datos cinéticos se ajustaron a un modelo de interacción de Langmuir 1 :1.For the kinetic analyzes a flow cell with small amounts of NIPio-BSA (approximately 100 UR) was used in order to minimize the effects of mass transport and re-binding. The individual samples, consisting of scFv or purified trimerbodies, were passed on the surface of the chip at a flow rate of 20 μl / 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.
Modelado comparativo de proteínasComparative protein modeling
La estructura del dominio de unión del anticuerpo L36 en formato scFv (scFv L36) fue modelada mediante modelado comparativo utilizando como molde la estructura 2GHW.B obtenida del Protein Data Bank (PDB) [Berman, H. M. et al. 2000; Nucleic Acids Res. 28:235-42]. La estructura del subdominio de trimerización NCl del extremo amino terminal del colágeno XVIII murino fue obtenido a partir de ModBase [Pieper, U. et a l. 2004, Nucleic Acids Res.; 32:D217-22]. Ambos dominios se encuentran unidos por un espaciador de 21 aminoácidos para formar un monómero del anticuerpo L36 en formato trimerbody. Las coordenadas de los monómeros restantes se obtuvieron mediante la aplicación de un eje triple de simetría de rotación. El modelo del trimerbody L36 se formó mediante la suma de las coordenadas de los tres monómeros. La estructura fue optimizada con GROMACS [Van Der Spoel, D. et al. 2005; J. Comput. Chem.; 26:1701-18] y su energía evaluada con DFIRE [Zhou, H. et al. 2002; Prot. Sci., 11 : 2714-26]. Con el objetivo de comparar los valores de DFIRE entre el monómero y el trímero, las energías fueron normalizadas dividiéndolas por la longitud de la secuencia. Ensayos de estabilidad en presencia de sueroThe structure of the binding domain of the L36 antibody in scFv format (scFv L36) 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]. In order to compare DFIRE values between the monomer and the trimer, the energies were normalized by dividing them by the length of the sequence. Stability tests in the presence of serum
Para determinar si el trimerbody mantenía su capacidad funcional en suero, 500 ng de trimerbody L36 purificado se incubaron a 370C, durante 72 horas, con 12,5% de suero murino de ratones BALB/c (Harían Ibérica, Barcelona, España). Se retiraron muestras para su análisis a las 3 h, 24 h y 72 h contadas desde el comienzo de la incubación y se congelaron hasta que se completó la totalidad del estudio. Como control, se congeló inmediatamente un segundo conjunto de muestras expuestas al suero para representar el tiempo "cero". A continuación, se analizó la capacidad de los trimerbodies de retener su unión funcional a laminina murina por ELISA.To determine if the functional capacity 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. As a control, a second set of samples exposed to serum was immediately frozen to represent the "zero" time. Next, the ability of trimerbodies to retain their functional binding to murine laminin by ELISA was analyzed.
Conjugación de anticuerpos recombinantes con dañina 5 scFv L36 y los trimerbodies purificados se marcaron con el fluorocromo Cianina 5 (Cy5) NHS esteres siguiendo las instrucciones del fabricante (GE Healthcare). La reacción de mareaje se realizó a temperatura ambiente durante 30 minutos, añadiendo 200 μl de una solución de Cy5 (2 mg/ml) en dimetilsulfóxido (DMSO). Las moléculas de Cy5 no unidas se eliminaron mediante cromatografía de exclusión en Sephadex G25- M (columnas PD-IO, GE Healthcare) y se concentraron en un filtro 10.000 MWCO Vivaspin 500 (Vivascience) a aproximadamente 1 mg/ml. La relación (molar) de Cy5 a anticuerpo (Cy5: anticuerpo), calculada según el procedimiento descrito por Birchler y col. (Birchler, M. et al. 1999; J. Immunol. Methods; 231 :239-48), era próxima a 1 :1. La funcionalidad de los Acs conjugados con Cy5 fue verificada mediante ELISA frente a los antígenos específicos.Conjugation of recombinant antibodies with harmful 5 scFv L36 and purified trimerbodies were labeled with the fluorochrome Cyanine 5 (Cy5) NHS esters following the manufacturer's instructions (GE Healthcare). The tidal reaction was carried out at room temperature for 30 minutes, adding 200 μl of a solution of Cy5 (2 mg / ml) in dimethylsulfoxide (DMSO). Unbound Cy5 molecules were removed by Sephadex G25-M exclusion chromatography (PD-IO columns, GE Healthcare) and concentrated on a 10,000 MWCO Vivaspin 500 (Vivascience) filter at approximately 1 mg / ml. 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.
Ensayos de localización de tumores en ratones portadores con anticuerpos recombinantes mediante imagen molecular - Inmunofotodetección infrarroja en ratones portadores de tumoresTumor localization assays in mice carrying recombinant antibodies by molecular imaging - Infrared immunophotodetection in tumor bearing mice
Se implantaron subcutáneamente (s.c.) células MKN45, HT 1080 o HeLa (1-MKN45, HT 1080 or HeLa cells (1-) were implanted subcutaneously (s.c.)
2x106) en la región dorsal de ratones hembra atímicos desnudos nu/nu de 6 semanas Hsd (ratones atímicos Nude-Foxi"" - Harían Ibérica). Las dimensiones de los nodulos se utilizaron para calcular el volumen tumoral utilizando la fórmula: (anchura)2 x2x10 6 ) in the dorsal region of nude nude female mice nu / nu of 6 weeks Hsd (Nude-Foxi athletic mice "" - Harían Ibérica). The nodule dimensions were used to calculate the tumor volume using the formula: (width) 2 x
(longitud) x 0,52. Cuando los tumores alcanzaron un tamaño apropiado (0,2-0,4 cm ), se administraron por vía intravenosa (i.v.), en la vena de la cola, 100 μl de una solución de anticuerpo recombinante purificado en formato trimerbody marcado con Cy5 en PBS (5 mg/kg). Las imágenes de fluorescencia in vivo se tomaron a diversos tiempos (3, 24 y 48 horas) después de la administración i.v. del anticuerpo marcado, con un sistema Hamamatsu dotado de una cámara digital de alta resolución con dispositivos de cargas acopladas (CCD) ORCA-2BT (Hamamatsu Photonics). Para el análisis y procesamiento de las imágenes se empleó el software Wasabi (Hamamatsu Photonics). Los protocolos utilizados para la manipulación de animales que a continuación se detallan, han sido aprobados por el por el Comité de Ética Animal del Hospital Universitario Puerta de Hierro.(length) x 0.52. When the tumors reached an appropriate size (0.2-0.4 cm), they administered intravenously (iv), in the tail vein, 100 µl of a solution of purified recombinant antibody in trimerbody format labeled with Cy5 in PBS (5 mg / kg). Fluorescence images in vivo were taken at various times (3, 24 and 48 hours) after iv administration of the labeled antibody, with a Hamamatsu system equipped with a high resolution digital camera with ORCA- coupled charge devices (CCD). 2BT (Hamamatsu Photonics). Wasabi software (Hamamatsu Photonics) was used to analyze and process the images. The protocols used for the handling of animals that are detailed below, have been approved by the Animal Ethics Committee of the Puerta de Hierro University Hospital.
II. RESULTADOSII. RESULTS
Diseño y expresión de las construcciones triméricas Los análisis estructurales del dominio NCl del colágeno XVIII murino sugieren que consiste en tres segmentos: un dominio de trimerización amino terminal implicado en el autoensamblaje de los homotrímeros; una región flexible sensible a proteasas; y un dominio compacto carboxiterminal de endostatina (ES) [Sasaki, T. et al. 1998; EMBO J. 17:4249-56]. Los investigadores han demostrado previamente que un Ac recombinante conteniendo el scFv L36 anti-laminina fusionado al dominio amino terminal NC l del colágeno XVIII murino era producido y secretado en forma funcionalmente activa por células HEK-293 [Sánchez- Aré valo, LV et al. (2006). Int. J. Cáncer 119:455-462]. Además, para proporcionar la suficiente flexibilidad espacial al scFv situado en la región amino terminal, se insertó un conector artificial de 21 aminoácidos [Sánchez-Arévalo, LV et al. (2006), citado supra].Design and expression of trimeric constructs Structural analyzes of the NCl domain of murine collagen XVIII suggest that it consists of three segments: an amino terminal trimerization domain involved in the self-assembly of homotymers; a flexible protease sensitive region; and a compact carboxy-terminal domain of endostatin (ES) [Sasaki, T. et al. 1998; EMBO J. 17: 4249-56]. Researchers have previously shown that a recombinant Ac containing the anti-laminin scFv L36 fused to the amino terminal NC domain of murine collagen XVIII was produced and functionally secreted by HEK-293 cells [Sánchez-Aré valo, LV et al. (2006). Int. J. Cancer 119: 455-462]. In addition, to provide sufficient spatial flexibility to the scFv located in the amino terminal region, an artificial 21 amino acid connector was inserted [Sánchez-Arévalo, LV et al. (2006), cited supra].
La naturaleza trimérica de la proteína de fusión scFv-NCl fue demostrada por ultracentrifugación [Sánchez-Arévalo, LV et al. (2006), citado supra] y cromatografía analítica de filtración en gel (Figura IA). La elución del scFv L36 mediante cromatografía analítica de filtración en gel pone de manifiesto que es un monómero. La calibración de la columna según los marcadores utilizados proporciona un peso molecular de 24,2 kDa, acorde con el teórico (26,9 kDa). Por el contrario, la elución de la fusión scFv L36-NC1 se corresponde con un peso molecular de 109,3 kDa, indicando su naturaleza trimérica (el peso teórico del trímero es de 111,4 kDa), acorde con los datos anteriores de ultracentrifugación [Sánchez-Arévalo, LV et al. (2006), citado supra]. Este formato de anticuerpo trimérico ha sido designado como "trimerbody".The trimeric nature of the scFv-NCl fusion protein was demonstrated by ultracentrifugation [Sánchez-Arévalo, LV et al. (2006), cited above] and analytical gel filtration chromatography (Figure IA). Elution of sc36 L36 by analytical gel filtration chromatography shows that it is a monomer. The calibration of the column according to the markers used provides a molecular weight of 24.2 kDa, in accordance with the theoretical one (26.9 kDa). In contrast, elution of the scFv L36-NC1 fusion corresponds to a molecular weight of 109.3 kDa, indicating its trimeric nature (the theoretical weight of the trimer is 111.4 kDa), in accordance with the previous ultracentrifugation data [Sánchez-Arévalo, LV et al. (2006), cited supra]. This trimeric antibody format has been designated as "trimerbody".
En la presente invención, se ha ampliado el concepto mediante el diseño de trimerbodies con especificidades diferentes para el hapteno NIP o el CEA humano. Los genes codificantes de los scFvs derivados de los Acs anti-NIP (B 1.8) y anti-CEA humano (MFE23) se ensamblaron de forma similar y se expresaron como proteínas solubles en células HEK-293 en forma funcionalmente activa. Las proteínas recombinantes fueron purificadas mediante columnas de afinidad (IMAC) y el rendimiento de las mismas fue superior al 95%, según se verificó en geles de poliacrilamida (SDS-PAGE). Tanto los trimerbodies Bl.8 como MFE-23 eluyeron de la columna con picos únicos (datos no mostrados) comparables a los mostrados en laIn the present invention, the concept has been extended by the design of 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
Figura IA.Figure IA
Estudios de unión a su antígenoAntigen binding studies
La funcionalidad de los trimerbodies purificados se demostró mediante ELISA frente a los antígenos inmovilizados conjugados NIP-BSA (NIPio-BSA), laminina EHS murina y CEA humano (Figura IB). Su capacidad para detectar a su antígeno en un contexto celular fue investigada mediante el mareaje por inmuno fluorescencia de células tumorales humanas que expresan el antígeno CEA en su superficie celular. La fluorescencia se observó tras la incubación con el trimerbody MFE-23, seguido de reacción con el AcM anti-myc y detección con el Ac de cabra anti-IgG de ratón conjugado con FITC. Por el contrario, la incubación de células que expresan CEA (CEA+) con el trimerbody B 1.8 o la incubación con células HeLa que no expresan CEA (CEA ) con el trimerbody MFE-23 no mostró fluorescencia alguna (Figura IC). Estos resultados indican que los trimerbodies no sólo reconocen al antígeno inmovilizado, sino que también reconocen el antígeno nativo expresado en la superficie de células tumorales.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. In contrast, incubation of cells expressing CEA (CEA + ) with trimerbody B 1.8 or incubation with HeLa cells that do not express CEA (CEA) with trimerbody MFE-23 showed no fluorescence (Figure IC). These results indicate that trimerbodies not only recognize the immobilized antigen, but also recognize the native antigen expressed on the surface of tumor cells.
Los ensayos de SPR sirvieron para determinar la influencia del formato de scFv o de trimerbody sobre la funcionalidad del Ac Bl.8. Se compararon las cinéticas de unión de cada formato de Ac utilizando tres densidades diferentes del NIPio-BSA inmovilizado sobre la superficie del chip. Se utilizó BSA acoplado a dextrano como referencia de inespecificidad. Para comparar las respuestas de unión durante los procesos de asociación-disociación, se inyectaron varias concentraciones de Ac B 1.8 (desde 9 a 1.200 nM para el scFv y de 6 a 800 nM para el trimerbody). Bajo estas condiciones, solo el trimerbody alcanzó la saturación de la superficie del antígeno, mientras que el scFv se unió lentamente y con una disociación aparente más rápida. Los sensogramas indican que el trimerbody tiene una mayor capacidad de unión que su versión monovalente (Figura ID).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).
Para los estudios cinéticos, se utilizó un chip recubierto con aproximadamente 100 UR de NIPio-BSA. En estos ensayos, el scFv Bl.8 se comportó de forma muy diferente al trimerbody Bl.8, con cocientes de asociación más lentos y de disociación más rápidos. Las constantes cinéticas de asociación (ka) y de disociación (ka) se determinaron mediante ajuste simultáneo utilizando el modelo de interacción Langmuir 1 :1. Para el scFv B 1.8, las constantes cinéticas de asociación (ka) y de disociación (ka) aparentes fueron ka: 1,0 x 104 ± 1,7 x 102 M'V1 y kd: 2 x 10~3 ± 4 x 10"5 s"1 (Chi2 = 0,189). Utilizando KD = kd/ka, la constante de disociación en equilibrio para scFv Bl.8, se estimó en 2 x 10"7 M. Dependiendo de la concentración de Ac utilizado en cada ensayo, el trimerbody inyectado en condiciones de baja densidad de NIPio-BSA inmovilizado podría probablemente unirse de forma mono- y multivalente, e incluso las constantes cinéticas de unión aparentes podrían incluir efectos de avidez por unión multivalente. Usando un modelo de interacción 1 :1 en el que las fases de asociación y disociación se tratan de forma separada para cada concentración, se ajustaron los resultados experimentales para la unión de B 1.8 trivalente. Los datos de las curvas con valores de resonancia que se aproximan a Rmax serían próximos a los obtenidos para la unión monovalente. En este caso, a medida que la concentración del trimerbody B 1.8 aumentaba, la ka disminuyó de 4 x 105 M-1S"1 (12,5 nM trimerbody) a 2 x 104 M-1S"1 (800 nM trimerbody). Estos cambios son compatibles con los cambios en la proporción de anticuerpo que se une al ligando de forma mono- o multivalente, ya que una aproximación a la saturación de la superficie conduce a una proporción mayor de unión de anticuerpo monovalente. La ka aparente tiene un rango más estrecho, de 6 x 10"4 (12,5 nM) a 9 x 10"4 (800 nM). Considerando que a 12,5 nM, la mayor parte de los trimerbodies puede unirse de forma bi- o multivalente, la afinidad funcional del trimerbody calculada para esa concentración podría ser KD (kd/ka) de 1,5 x 10"9 M o superior.For kinetic studies, a chip coated with approximately 100 UR of NIPio-BSA was used. In these trials, scFv Bl.8 behaved very differently than trimerbody Bl.8, with slower association and dissociation ratios faster. The kinetic constants of association (k a ) and dissociation (ka) were determined by simultaneous adjustment using the Langmuir 1: 1 interaction model. For scFv B 1.8, the apparent association (k a ) and dissociation (ka) constants were k a : 1.0 x 10 4 ± 1.7 x 10 2 M ' V 1 and k d : 2 x 10 ~ 3 ± 4 x 10 "5 s " 1 (Chi 2 = 0.189). Using K D = kd / k a , the equilibrium dissociation constant for scFv Bl.8, was estimated at 2 x 10 "7 M. Depending on the concentration of Ac used in each test, the trimerbody injected under low density conditions of immobilized NIPio-BSA could probably be mono- and multivalently bound, and even apparent binding kinetic constants could include avidity effects by multivalent binding, using a 1: 1 interaction model in which the association and dissociation phases are treated separately for each concentration, the experimental results for the trivalent B 1.8 junction were adjusted.The data of the curves with resonance values that approximate R max would be close to those obtained for the monovalent junction. 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). Considering that at 12.5 nM, most of the trimerbodies can bind bi- or multivalently, 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.
Para asegurar que al menos hay dos sitios activos de unión en una misma molécula de trimerbody, células 293T fueron transfectadas con los plásmidos codificantes de trimerbodies L36, Bl .8 o con ambos plásmidos en una co-transfección. Ensayos de transferencia Western pusieron de manifiesto que ambos trimerbodies eran expresados en el sobrenadante de las células y que se unían a sus antígenos respectivos, y que la cantidad de trimerbody era más alta en células 293T co-transfectadas (con ambos plásmidos) que en células 293T monotransfectadas (datos no mostrados). El medio condicionado de las células 293T monotransfectadas unidas a NIPiO-BSA o laminina, así como el medio condicionado de las células 293T co-transfectadas reconocen ambos antígenos (Figura 2A). En un ensayo de ELISA, medio condicionado de las células 293T co-transfectadas (es decir, doblemente transfectadas), se añadió a placas recubiertas con laminina y, tras lavado, los trimerbodies unidos a laminina eran capaces de capturar NIPio-BSA soluble (Figura 2B), demostrando su multivalencia. Por el contrario, los trimerbodies en el sobrenadante de células 293T monotransfectadas con el plásmido que codifica el trimerbody L36 eran incapaces de unirse y capturar NIP10- BSA soluble.To ensure that there are at least two active binding sites in the same trimerbody molecule, 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). The 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). In an ELISA assay, conditioned medium of co-transfected 293T cells (i.e., double transfected), was added to laminin-coated plates and, after washing, laminin-linked trimerbodies were able to capture soluble NIPio-BSA (Figure 2B), demonstrating its multivalence. In contrast, trimerbodies in the supernatant of 293T cells monotransfected with the plasmid encoding trimerbody L36 were unable to bind and capture soluble NIP 10 -BSA.
Ensayos de estabilidad frente a suero in vitroIn vitro serum stability tests
Un aspecto muy importante para determinar la potencial aplicación in vivo de los fragmentos de Ac diseñados es comprobar la estabilidad frente a suero. Los investigadores han determinado la funcionalidad y la estabilidad estructural del trimerbody L36 tras incubación con suero humano y murino a 370C durante periodos prolongados de tiempo (igual o superiores a 72 horas), mediante ensayos de transferencia Western y ELISA. Tal como se muestra en la Figura 3, el trimerbody L36 mantiene un 80-90% de su capacidad de unión a su antígeno tras 72 horas de incubación.A very important aspect to determine the potential in vivo application of the designed Ac fragments is to check the stability against serum. Researchers have determined the functionality and the structural stability of L36 trimerbody after incubation with human and mouse serum at 37 0 C for extended periods of time (equal to or greater than 72 hours), using assays and Western blot and ELISA. As shown in Figure 3, trimerbody L36 maintains 80-90% of its binding capacity to its antigen after 72 hours of incubation.
Ensayos de localización específica de tumores in vivoIn vivo specific tumor localization assays
Para evaluar la biodistribución de, y la localización específica de tumores por, los trimerbodies anti-NIP, anti-CEA y anti- laminina, se utilizó un sistema óptico de imagen molecular, que permite la evaluación de la cinética de localización del tumor y del aclaramiento por el Ac en un mismo animal y en diferentes tiempos del ensayo. Los trimerbodies fueron conjugados con el fluorocromo de emisión cercana al infrarrojo Cy5 e inyectados por vía intravenosa (i.v.) en la vena de la cola de ratones hembra atímicos desnudos nu/nu portadores de tumores humanos MKN45 (adenocarcinoma gástrico), HT-1080 (fibrosarcoma), o HeLa (adenocarinoma de cérvix) (n =4/grupo). Todos los trimerbodies mostraron un rápido aclaramiento renal tras la inyección i.v., con un máximo de intensidad de señal a las 3 horas, siendo indetectable a las 48 horas post-inyección (Figuras 4 A y 4C). El trimerbody B 1.8 no mostró localización detectable de tumor en ninguno de los tres tipos de tumores estudiados (Figuras 4B y 5). Se observó una selectiva y fuerte acumulación en los tumores que expresan CEA (CEA+) del trimerbody MFE-23. Tras la inyección i.v. del trimerbody MFE-23-Cy5, el máximo de señal registrada se produjo a las 3 horas, decayendo la señal a las 24 horas y manteniéndose detectable a las 48 horas (Figura 4B). El trimerbody anti-laminina mostró localización específica de tumor en todos los modelos estudiados (sean o no tumores que expresasen CEA) (Figuras 4B y 5). Sin embargo, la cinética de intensidad de señal resultó diferente al trimerbody MFE- 23, siendo el máximo de señal a las 24 horas, algo menor que el máximo obtenido por el trimerbody MFE-23. El scFv anti-laminina también mostró localización específica de tumor pero a un nivel mucho más bajo que el del trimerbody anti-laminina (Figuras 4B y 4D).To evaluate the biodistribution of, and the specific location of tumors by, the anti-NIP, anti-CEA and anti-laminin trimerbodies, an optical system of molecular image, which allows the evaluation of the kinetics of tumor location and clearance by Ac in the same animal and at different times of the test. The trimerbodies were conjugated with Cy5 infrared near-emission fluorochrome and injected intravenously (iv) into the tail vein of nude female nude mice nu / nu carrying human tumors MKN45 (gastric adenocarcinoma), HT-1080 (fibrosarcoma ), or HeLa (cervical adenocarinoma) (n = 4 / group). All 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. After iv injection of the MFE-23-Cy5 trimerbody, 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). However, 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).
III. DISCUSIÓNIII. DISCUSSION
Estudios previos de los investigadores mostraban la capacidad de las secuencias derivadas de colágeno de promover la trimerización de anticuerpos [Sánchez-Arevalo VJ et al. (2006) Int J Cáncer 119:455-462]. En dichos estudios, se mostraba que la fusión del subdominio NCl N-terminal de colágeno XVIII, responsable de la trimerización no- covalente de las cadenas alfa del colágeno XVIII al extremo C-terminal de un anticuerpo scFv, confiere un estado trimérico al anticuerpo generado ("trimerbody"). Así, se demostró que utilizando un scFv (L36) que reconoce un epítopo de laminina asociado a angiogénesis y que inhibe la angiogénesis y el crecimiento del tumor, el L36 trimérico era más efectivo que el correspondiente monomérico en la inhibición de la morfogénesis capilar in vitro, e inhibiendo el crecimiento del tumor in vivo [Sánchez- Arevalo VJ et al. (2006), citado supra]. Otro grupo investigador [Fan CY et al. (2008). FASEB J 22:3795- 3804] ha utilizado una aproximación similar para multimerizar anticuerpos y ha demostrado que un esqueleto de péptidos cortos similares a colágeno era capaz de promover la trimerización de fragmentos scFv fusionados ("collabody").Previous studies of the researchers showed the ability of collagen-derived sequences to promote antibody trimerization [Sánchez-Arevalo VJ et al. (2006) Int J Cancer 119: 455-462]. In these studies, it was shown that the fusion of the N-terminal NCl sub-domain of collagen XVIII, responsible for the non-covalent trimerization of the alpha chains of collagen XVIII to the C-terminal end of an scFv antibody, confers a trimeric state to the generated antibody ("trimerbody"). Thus, it was demonstrated that using a scFv (L36) that recognizes a laminin epitope associated with angiogenesis and that inhibits angiogenesis and tumor growth, the trimeric L36 it was more effective than the corresponding monomer in inhibiting capillary morphogenesis in vitro, and inhibiting tumor growth in vivo [Sánchez-Arevalo VJ et al. (2006), cited supra]. Another research group [Fan CY et al. (2008). FASEB J 22: 3795- 3804] has used a similar approach to multimerize antibodies and has shown that a skeleton of short collagen-like peptides was capable of promoting trimerization of fused scFv fragments ("collabody").
En la presente invención, se presenta una caracterización detallada, tanto in vivo como in vitro de la fusión del anticuerpo-dominio de oligomerización, y se extiende el concepto produciendo moléculas análogas con especificidad frente al hapteno NIP y frente a un antígeno asociado a tumores (CEA). En particular, se han desarrollado unas proteínas triméricas (trímeros) que comprenden 3 proteínas de fusión, comprendiendo cada proteína de fusión un fragmento de un anticuerpo y una secuencia derivada de colágeno que comprende el dominio de trimerización; dichas proteínas oligoméricas (trímeros) caen dentro del concepto "trimerbody" acuñado en esta descripción. Todos los trimerbodies se aislaron en una forma activa a partir de medio condicionado obtenido de células HEK293 transfectadas y fueron fácilmente purificados utilizando cromatografía de afinidad por metal inmovilizado. Los trimerbodies son triméricos en solución, y poseen una excelente estabilidad y capacidad de unión al antígeno. Los trimerbodies son muy eficientes reconociendo antígenos purificados inmovilizados sobre una placa, o expresados en la superficie de una célula tumoral. Análisis mediante SPR demostró que el trimerbody tenía una señal de unión mayor que el anticuerpo monomérico y aparentemente una disociación menor, consistente con la unión multivalente al antígeno. Los investigadores calcularon que el trimerbody anti-NIP tiene una afinidad funcional por el antígeno (conjugados NIP-BSA) unas 100 veces mayor, comparado con la versión monovalente. Este resultado sugiere que esta ganancia de afinidad podría ser debida al efecto de avidez de un segundo sitio de combinación en la molécula de trimerbody. La presencia de al menos dos sitios de unión funcionales en una sola molécula de trimerbody fue posteriormente demostrada mediante la generación de trimerbodies biespecíficos. Trimerbodies bifuncionales anti-laminina y anti-NIP estables fueron fácilmente producidos por co-expresión de dos construcciones diferentes de trimerbodies en células humanas. La ganancia en afinidad a través de la avidez hace a los trimerbodies atractivos para técnicas de imagen in vivo como agentes alternativos a los anticuerpos diméricos. Se podría, por tanto, especular que los trimerbodies se preferirán sobre los anticuerpos diméricos (diabodies y minibodies), aunque esta propiedad puede depender de la estructura y densidad del antígeno reconocido por los scFv. Para una completa avidez en anticuerpos multivalentes dirigidos a moléculas unidas a la superficie, los sitios de unión del antígeno deben apuntar hacia la misma dirección. Si la unión múltiple simultánea no es posible estéticamente, entonces la ganancia aparente en afinidad funcional es probable que sea menor y debida únicamente al efecto de unión aumentado, que es dependiente en tasas de difusión y la concentración del antígeno de superficie [Lawrence LJ et al. (1998) FEBS letters 425:479-484]. El análisis del modelo de trimerbody sugiere una estructura con forma de trípode con los dominios scFv orientados hacia fuera (Figura 6). La flexibilidad entre los sitios de unión al antígeno es otro aspecto importante en el diseño de anticuerpos multivalentes, requeridos para el entre cruz amiento de receptores de superficie, bien en la misma célula o en adyacentes. El espaciador de 21 residuos, con una longitud máxima de 79,8 Á si la conformación es completamente extendida, es muy flexible, permitiendo numerosas geometrías de unión. Cuando una interacción antígeno- anticuerpo ocurre, la posibilidad de establecer una segunda interacción depende de la valencia, orientación y flexibilidad del sitio de unión del antígeno. Según los cálculos de los inventores, en la molécula de trimerbody, los scFv que permanecen sin interaccionar tienen un área de influencia unas 11 veces mayor, aproximadamente, que otros formatos bivalentes, tales como los diabodies y los minibodies, aumentando la probabilidad de una segunda interacción efectiva. Uniones múltiples pueden, efectivamente, reducir las "off rates " aumentando así el tiempo de retención del anticuerpo unido al antígeno diana. A este respecto, una ventaja mayor del trimerbody sobre otros formatos triméricos (como el collabody) es la flexibilidad. La estructura más compacta/rígida del collabody reduce la accesibilidad de los scFv, que es un parámetro crítico para la localización de tumores in vivo.In the present invention, a detailed characterization is presented, both in vivo and in vitro of the fusion of the oligomerization antibody-domain, and the concept is extended by producing analogous molecules with specificity against the NIP hapten and against a tumor associated antigen ( CEA). In particular, trimeric (trimer) proteins comprising 3 fusion proteins have been developed, each fusion protein comprising a fragment of an antibody and a sequence derived from collagen comprising the trimerization domain; said oligomeric proteins (trimers) fall within the "trimerbody" concept coined in this description. All 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. The presence of at least two functional binding sites in a single trimerbody molecule was subsequently demonstrated by the generation of bispecific 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. For complete avidity in multivalent antibodies directed to surface bound molecules, the antigen binding sites must point in the same direction. If simultaneous multiple binding is not aesthetically possible, then the apparent gain in functional affinity is likely to be less and due solely to the increased binding effect, which is dependent on diffusion rates and the concentration of the surface antigen [Lawrence LJ et al . (1998) FEBS letters 425: 479-484]. The analysis of the trimerbody model suggests a tripod-shaped structure with the scFv domains oriented outwards (Figure 6). Flexibility between antigen binding sites is another important aspect in the design of multivalent antibodies, required for cross-linking of surface receptors, either in the same cell or in adjacent ones. The spacer of 21 residues, with a maximum length of 79.8 Á if the conformation is fully extended, is very flexible, allowing numerous joining geometries. When an antigen-antibody interaction occurs, the possibility of establishing a second interaction depends on the valence, orientation and flexibility of the antigen binding site. According to the calculations of the inventors, in the trimerbody molecule, scFv that remain without interaction have an area of influence approximately 11 times greater, than other bivalent formats, such as diabodies and minibodies, increasing the probability of a second effective interaction Multiple binding can effectively reduce the "off rates" thus increasing the retention time of the antibody bound to the target antigen. In this respect, a major advantage of the trimerbody over other trimeric formats (such as collabody) is flexibility. The more compact / rigid collabody structure reduces the accessibility of scFv, which is a critical parameter for tumor localization in vivo.
Así, la multimerización de las construcciones scFv presenta ventajas para las aplicaciones in vivo. Los anticuerpos recombinantes, tal como los diabodies y minibodies han demostrado su potencial como agentes de localización in vivo [Holliger P et al. (2005) Nat Biotechnol 23:1126-1136]. Los trimerbodies son moléculas multivalentes de tamaño intermedio que presentan una alta estabilidad en condiciones fisiológicas. El potencial de los trimerbodies para la localización in vivo se estudió en modelos experimentales de cáncer humano en ratones desnudos. Los trimerbodies anti-CEA localizan rápida y específicamente en los tumores CEA-positivos. La máxima señal en el tumor se alcanzaba a las 3 horas tras la inyección y era lentamente eliminada a lo largo del tiempo. La fluorescencia era aún detectable en el tumor tras 48 horas tras la inyección con el trimerbody. Es de destacar que el anticuerpo trimerbody anti-laminina L36 se localiza en todos los tumores estudiados independientemente de su estirpe histológica. El máximo de absorción de los trimerbodies anti-laminina fue a las 24 horas tras la administración del mismo. A pesar de que los scFv L36 anti-laminina presentaban una acumulación específica en el tumor, la acumulación en el tumor era menor, probablemente debido a su eliminación más rápida de la sangre (con una vida media menor de 15 minutos) y por su naturaleza monovalente (que implica bajos tiempos de retención) [Adams GP et al. (1995). Cáncer Immunol. Immunother. 40:299-306]. Según resultados previos, el epítopo reconocido por el anticuerpo L36 está localizado en la parte media del brazo largo de la laminina, en un área altamente flexible, que corresponde a un sitio susceptible de proteasas. Los inventores han postulado que este epítopo es únicamente accesible durante el ensamblaje de la membrana basal (MB) [Sanz L et al. (2003) EMBO J. 22:1508-1517], donde la laminina intacta durante el proceso de polimerización actúa como soporte para el reclutamiento de otros componentes de la MB [Sasaki T et al. (2004) J. CeIl. Biol. 164:959-963]. La expresión restringida de este epítopo a situaciones asociadas con la remodelación de la MB, explicaría la captación más lenta de los trimerbodies L36 en comparación con los trimerbodies MFE23 que reconocen un antígeno expresado por la célula tumoral. Más allá de sus aplicaciones diagnósticas, los trimerbodies ofrecen oportunidades terapéuticas prometedoras basadas en la liberación selectiva de moléculas bioactivas en los tejidos diana. Alguna de las aplicaciones inmediatas de los trimerbodies específicos de antígenos asociados a tumores (por ejemplo, el receptor 2 del factor de crecimiento epidemial humano, antígeno específico de próstata), o dirigidos al estroma tumoral (por ejemplo, proteína de activación de fibroblasto) incluyen el desarrollo de proteínas de fusión con inhibidores angiogénicos [Sánchez-Arevalo VJ et al. (2006) Int J Cáncer 119:455-462], citoquinas, enzimas, o receptores truncados, y conjugación con radionucleidos [Sanz L et al. (2004) Trends Immunol 25:85-91]. Thus, the multimerization of scFv constructions has advantages for in vivo applications. Recombinant antibodies, such as diabodies and minibodies, have demonstrated their potential as in vivo localization agents [Holliger P et al. (2005) Nat Biotechnol 23: 1126-1136]. 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. It is noteworthy that the 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]. According to previous results, 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]. The restricted expression of this epitope to situations associated with the remodeling of MB, would explain the slower uptake of L36 trimerbodies compared to MFE23 trimerbodies that recognize an antigen expressed by the tumor cell. Beyond their diagnostic applications, trimerbodies offer promising therapeutic opportunities based on the selective release of bioactive molecules in target tissues. Some of the immediate applications of tumor-specific antigen-specific trimerbodies (e.g., the human epidemic growth factor receptor 2, prostate-specific antigen), or targeting the tumor stroma (e.g., fibroblast activation protein) 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].

Claims

REIVINDICACIONES
1. Una proteína oligomérica que comprende una pluralidad de proteínas de fusión, iguales o diferentes, y un marcador (M), en la que cada proteína de fusión comprende:1. An oligomeric protein comprising a plurality of fusion proteins, the same or different, and a marker (M), wherein each fusion protein comprises:
(b) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente de dicho anticuerpo; y(b) a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y
(b) un polipéptido (B) que comprende un dominio de oligomerización.(b) a polypeptide (B) comprising an oligomerization domain.
2. Proteína oligomérica según la reivindicación 1, en la que dicho marcador (M) es un fluoróforo o un radionucleido.2. Oligomeric protein according to claim 1, wherein said marker (M) is a fluorophore or a radionuclide.
3. Proteína oligomérica según la reivindicación 1, en la que dicho polipéptido (A) comprende un anticuerpo que reconoce una diana, o un fragmento funcionalmente equivalente de dicho anticuerpo que reconoce una diana, en donde dicha diana es un epítopo o determinante antigénico de un antígeno.3. Oligomeric protein according to claim 1, wherein said polypeptide (A) comprises an antibody that recognizes a target, or a functionally equivalent fragment of said antibody that recognizes a target, wherein said target is an epitope or antigenic determinant of a antigen.
4. Pro teína oligomérica según la reivindicación 3, en la que dicho antígeno es un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica.4. Oligomeric protein according to claim 3, wherein said antigen is an antigen that is de novo expressed or overexpressed in a pathological alteration.
5. Proteína oligomérica según la reivindicación 1, en la que dicho polipéptido (A) comprende un anticuerpo monoclonal, un anticuerpo policlonal, un fragmento Fab, F(ab')2 o Fab', un fragmento Fv de cadena única (scFv), un diabody o un anticuerpo monodominio (VHH).5. Oligomeric protein according to claim 1, wherein said polypeptide (A) comprises a monoclonal antibody, a polyclonal antibody, a Fab, F (ab ') 2 or Fab' fragment, a single chain Fv fragment (scFv), a diabody or a monodomain antibody (VHH).
6. Proteína oligomérica según la reivindicación 1 , en la que dicho dominio de oligomerización presente en dicho polipéptido (B) comprende el dominio NCl de colágeno XVIII o del colágeno XV de mamífero, opcionalmente desprovisto total o parcialmente del dominio de endostatina (ES). 6. Oligomeric protein according to claim 1, wherein said oligomerization domain present in said polypeptide (B) comprises the NCl domain of collagen XVIII or mammalian collagen XV, optionally totally or partially devoid of the endostatin (ES) domain.
7. Pro teína oligomérica según la reivindicación 1, constituida por 2 ó más proteínas de fusión, iguales o diferentes.7. Oligomeric protein according to claim 1, consisting of 2 or more fusion proteins, the same or different.
8. Un procedimiento para la obtención de una proteína oligomérica según cualquiera de las reivindicaciones 1 a 7, que comprende poner en contacto una proteína oligomérica que comprende una pluralidad de proteínas de fusión, iguales o diferentes, en la que cada proteína de fusión comprende: i) un polipéptido (A) que comprende un anticuerpo o un fragmento funcionalmente equivalente del mismo; y ii) un polipéptido (B) que comprende un dominio de oligomerización, con un marcador (M) bajo condiciones que permiten la unión de dicho marcador (M) a la proteína oligomérica.A method for obtaining an oligomeric protein according to any one of claims 1 to 7, which comprises contacting an oligomeric protein comprising a plurality of fusion proteins, the same or different, in which each fusion protein comprises: i) a polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof; and ii) a polypeptide (B) comprising an oligomerization domain, with a label (M) under conditions that allow said label (M) to bind to the oligomeric protein.
9. Empleo de una proteína oligomérica según cualquiera de las reivindicaciones 1 a 7, en un método para la detección, visualización o localización de una diana.9. Use of an oligomeric protein according to any of claims 1 to 7, in a method for the detection, visualization or localization of a target.
10. Empleo según la reivindicación 9, en el que dicha diana es un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica.10. Use according to claim 9, wherein said target is an antigen that expresses de novo or is overexpressed in a pathological alteration.
11. Un método para la detección, visualización o localización de una diana que comprende el empleo de una proteína oligomérica según cualquiera de las reivindicaciones 1 a 7.11. A method for the detection, visualization or localization of a target comprising the use of an oligomeric protein according to any one of claims 1 to 7.
12. Método según la reivindicación 11, en el que dicho método se basa en una técnica de imagen.12. Method according to claim 11, wherein said method is based on an imaging technique.
13. Método según la reivindicación 11, en el que dicha diana es un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica.13. A method according to claim 11, wherein said target is an antigen that is de novo expressed or overexpressed in a pathological alteration.
14. Una composición que comprende una proteína oligomérica según cualquiera de las reivindicaciones 1 a 7, junto con, al menos, un medio apropiado. 14. A composition comprising an oligomeric protein according to any one of claims 1 to 7, together with at least one appropriate medium.
15. Una composición farmacéutica que comprende una proteína oligomérica según cualquiera de las reivindicaciones 1 a 7, junto con, al menos, un vehículo farmacéuticamente aceptable.15. A pharmaceutical composition comprising an oligomeric protein according to any one of claims 1 to 7, together with at least one pharmaceutically acceptable carrier.
16. Un kit que comprende una proteína oligomérica según cualquiera de las reivindicaciones 1 a 7.16. A kit comprising an oligomeric protein according to any one of claims 1 to 7.
17. Empleo de un kit según la reivindicación 16, para la detección, visualización o localización de una diana.17. Use of a kit according to claim 16, for the detection, display or location of a target.
18. Empleo según la reivindicación 17, en el que dicha diana es un antígeno que se expresa de novo o se sobreexpresa en una alteración patológica. 18. Use according to claim 17, wherein said target is an antigen that expresses de novo or is overexpressed in a pathological alteration.
PCT/ES2009/070107 2009-04-20 2009-04-20 Oligomeric proteins and the uses thereof WO2010122181A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/ES2009/070107 WO2010122181A1 (en) 2009-04-20 2009-04-20 Oligomeric proteins and the uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/ES2009/070107 WO2010122181A1 (en) 2009-04-20 2009-04-20 Oligomeric proteins and the uses thereof

Publications (1)

Publication Number Publication Date
WO2010122181A1 true WO2010122181A1 (en) 2010-10-28

Family

ID=43010703

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2009/070107 WO2010122181A1 (en) 2009-04-20 2009-04-20 Oligomeric proteins and the uses thereof

Country Status (1)

Country Link
WO (1) WO2010122181A1 (en)

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

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031540A1 (en) * 1994-05-16 1995-11-23 Medical Research Council Trimerising polypeptides, their manufacture and use
WO2006048252A1 (en) * 2004-11-02 2006-05-11 Universidad Autónoma de Madrid Multifunctional and multivalent angiogenesis inhibitors
EP1798240A1 (en) * 2005-12-15 2007-06-20 Industrial Technology Research Institute Recombinant triplex scaffold-based polypeptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031540A1 (en) * 1994-05-16 1995-11-23 Medical Research Council Trimerising polypeptides, their manufacture and use
WO2006048252A1 (en) * 2004-11-02 2006-05-11 Universidad Autónoma de Madrid Multifunctional and multivalent angiogenesis inhibitors
EP1798240A1 (en) * 2005-12-15 2007-06-20 Industrial Technology Research Institute Recombinant triplex scaffold-based polypeptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FAN C. ET AL.: "Production of multivalent protein binders using a self-trimerizing collagen-like peptide scaffold.", THE FASEB JOURNAL., vol. 22, November 2008 (2008-11-01), pages 3795 - 3804 *
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 *
KORTT A.A. ET AL.: "Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting.", BIOMOLECULAR ENGINEERING., vol. 18, 2001, pages 95 - 108, XP004305907, DOI: doi:10.1016/S1389-0344(01)00090-9 *
SÁNCHEZ-ARÉVALO V.J. ET AL.: "Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events.", INT. J. CANCER., vol. 119, 2006, pages 455 - 462, XP009107338, DOI: doi:10.1002/ijc.21851 *

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

Similar Documents

Publication Publication Date Title
JP7127008B6 (en) Novel PD-L1 binding polypeptides for imaging
US20220184239A1 (en) Methods and compositions for 18f-radiolabeling of biologics
JP6959616B2 (en) Site-specific RI-labeled antibody with IgG-binding peptide
Ayo et al. Peptide-based strategies for targeted tumor treatment and imaging
Zhu et al. Novel glypican-3-binding peptide for in vivo hepatocellular carcinoma fluorescent imaging
Chen et al. Melanocortin-1 receptor-targeting ultrasmall silica nanoparticles for dual-modality human melanoma imaging
US20240335571A1 (en) Pre-targeting strategies for molecular imaging and/or radioimmunotherapy
WO2012162418A1 (en) Methods and compositions for heterodimeric targeting ligands
TWI795415B (en) A novel anti-human CEACAM5 antibody Fab fragment
Sirk et al. Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2
Du et al. Developing PEGylated reversed D-peptide as a novel HER2-targeted SPECT imaging probe for breast cancer detection
Li et al. Application of phage-displayed peptides in tumor imaging diagnosis and targeting therapy
Pung et al. Generation of peptides using phage display technology for cancer diagnosis and molecular imaging
KR20210009421A (en) Multispecific antibody construct
Hemmerle et al. Tumor targeting properties of antibody fusion proteins based on different members of the murine tumor necrosis superfamily
Li et al. Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer
US10981980B2 (en) Polypeptide targeting aptamers for characterization, capture, and clinical management of circulating tumor cells
Al-Baradie Nanobodies as versatile tools: A focus on targeted tumor therapy, tumor imaging and diagnostics
Cao et al. Development of a novel HER2-targeted peptide probe for dual-modal imaging of tumors
KR20220083785A (en) Humanized Antibodies and Methods of Using the Same
WO2010122181A1 (en) Oligomeric proteins and the uses thereof
ES2371426B1 (en) OLIGOMERIC PROTEINS AND ITS APPLICATIONS
CN111247249A (en) DNA, polypeptide, anti-mesothelin antibody, tumor imaging agent and complex
Kumar et al. Functionalized nanobody-based delivery systems for cancer diagnosis and therapeutic applications
ES2831651T3 (en) Use of antibodies and antagonists directed against Lrg1 in a treatment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09843588

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09843588

Country of ref document: EP

Kind code of ref document: A1