ES2371426B1 - OLIGOMERIC PROTEINS AND ITS APPLICATIONS - Google Patents

Abstract

The oligomeric proteins comprise a plurality of fusion proteins and a label, each fusion protein comprising 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.

Description

Oligomeric proteins and their applications.

Field of the Invention

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.

Background of the invention

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, PET or PET (by Positron Emission Tomography) is a technique of a medical specialty called nuclear medicine and radiology, by 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 body, in a single study and regardless of the anatomical location where the neoplasm occurs (primary or metastatic), since 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 metabolism both in the heart and in the central nervous system.

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 marked, with a fluorescent molecule called a fluorophore. The sample is illuminated with light of a certain wavelength / s, which is absorbed by the fluorophores, causing a 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 fl uorophores are fl uorescein isothiocyanate (FITC), rhodamine derivatives (TRITC), comarin and cyanine, fl uorophores “Alexa Fluor” and “DyLight Fluor”, the cyanine family (eg, Cy3, Cy5 and Cy7).

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 (NIR) region, 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 (e.g., pentametine and heptametin). The probes used for in vivo fluorescence imaging can be directed or not directed. Targeting has the advantage that they can be directed specifically to 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 may 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 fl uorochrome that provides images of activated macrophages involved in inflammatory joint diseases. These molecules would be directed to the 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.

On the other hand, different systematic tests in vivo have provided a confirmation that size is an important parameter in pharmacokinetics 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; Cancer Biother 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 eliminated 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 scFv2-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 Fab2 fragments (110 kDa).

However, despite the good results obtained with these formats in several models [Williams et al., 2001; Cancer Biother 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 precisely oriented 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.

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.

Compendium of the invention

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 an antibody binding domain.

or of a functionally equivalent fragment thereof, and an oligomerization domain, subsequently labeled 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.

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 had an excellent antigen binding characteristic, which gives them a high functional affinity (greediness). Likewise, chemically conjugated anti-CEA trimerbodies with a fluorescent marker showed an efficient location of the tumor in an experimental model of human colorectal carcinoma in mice and the antilaminin trimerbodies showed excellent localization in several types of human cancer, including fi brosarcomas 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:

(to)

a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y

(b)

a polypeptide (B) comprising an oligomerization domain.

In another aspect, the invention relates to a process for obtaining said oligomeric protein.

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.

In another aspect, the invention relates to a composition comprising said oligomeric protein and an appropriate medium.

In another aspect, the invention relates to a pharmaceutical composition comprising said oligomeric protein and a pharmaceutically acceptable carrier.

In 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 fi gures

Figure 1 describes the molecular characterization of the purified trimerbodies. (A) Elution profile of the gel fi ltration experiment of the L36 [L36 trimerbody (darker lines) and L36 scFv (lighter line)] constructs. The exclusion (Vo) and total (VT) volumes are indicated. Elution volumes of selected molecular weight markers are indicated with arrows, as well as their corresponding molecular weights. To make it clear, the absorbance of both chromatograms has been scaled and moved in the fi gure. (B) The functionality of the purified trimerbodies was demonstrated by ELISA on plates covered with bovine serum albumin (BSA), murine laminin-1, BIP-conjugated NIP hapten (NIP10-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 B1.8 and trimerbody B1.8 to NIP10-BSA using BIAcore. The curves show the data obtained after the 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 B1.8 and trimerbody B1.8 trimerbodies (dil. 1200 and 800 nM) in HBS-EP buffer injected on 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-N1, pCR3.1-L36-NC1ES-, or pCEP4-B1.8-NC1ES− are compared with supernatants of 293T cells transfected with pCR3.1-L36-NC1ES− and pCEP4-B1. 8-NC1ES−, using direct ELISA (A), using plates upholstered with BSA, conjugates NIP10-BSA and laminin-1, and by sandwich ELISA (B) using plates upholstered with laminin.

Figure 3 describes the stability of the trimerbody molecules in serum. Purified L36 trimerbodies were incubated in human or mouse serum at 37 ° C, as detailed in Example 1 (materials and methods) and the functionality of the reaction mixtures was analyzed by ELISA.

Figure 4 describes the location of antibodies conjugated to a fluorophore (cyanine 5; Cy5) in an experimental gastric cancer (CEA-positive) model in nude mice. Near-infrared images in nude mice carrying a subcutaneous MKN45 gastric carcinoma (s.c.). Ventral (A, C) and dorsal (B, D) images taken 3, 24 and 48 h after intravenous (iv) injection with trimerbodies (B1.8, MFE-23 and L36) marked with Cy5 (A, B), or scFv L36 marked with Cy5 (C, D). b: bladder, t: tumor.

Figure 5 describes the location of trimerbodies conjugated to a fluorophore (cyanine 5; Cy5) in several experimental human (CEA-negative) cancer models in nude mice. Near-infrared images of nude mice carrying human HT1080 fi brosarcom s.c. (A) or human cervix carcinomas (HeLa) s.c. (B). The images were taken 3, 24 and 48 h 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.

Detailed description of the invention

De fi nitions

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 molecules of the major histocompatibility complex of 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 specific tumor mutation; alternatively, tumor antigens that are presented by both tumor cells and normal cells are referred to as "tumor associated antigens" (TAA) and, in general, come from a specific mutation of the tumor.

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 (VHH), 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 VHH fragment, which can be produced directly by recombinant host cells . In a particular and preferred embodiment, 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.

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).

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.

The term "VHH", 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 VHH. This domain (VHH) of approximately 15 kDa can be expressed in the periplasm of E. coli while maintaining its antigen binding capacity. VHH are more stable molecules than scFv, and unlike they rarely add. Like the mouse VL and VH domains, the VHH 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 markings can be radioactive or non-radioactive. Radioactive or isotopic labeling has been used more frequently, although it is progressively displaced by non-radioactive methods. Although there is no consensus about the ideal marking, in most cases it is done with 32P. Since the short half-life of this isotope (14 days) creates difficulties in the preparation, commercialization and routine use of the tests, other alternatives are also used (35S, 3H, 125I, 14C, etc.). Regarding 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: measure of the light emitted by reaction products that possess a fluorescent molecule, 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.

Specifically, fluorometry can be used for in vivo labeling. In this way, a component of interest can be specifically marked in a sample, with a fluorescent molecule, called a fluorophore. One of the most commonly used fluorophores is fl uorescein isothiocyanate (FITC), a reagent derived from fl uorescein, which is chemically coupled 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 fl uorophores 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 fl uorophores for near infrared (NIR), the most common are polymetins of general formula (I)

where

R1 and R2, independently of each other, represent C1-C10 alkyl, C1-C10 sulfoalkyl, C3-C10 cycloalkyl, C1-C10 alkoxy, or aryl;

R3 and R4, independently of each other, represent C1-C10 sulfoalkyl, C1-C10 haloalkyl, or C1-C10 hydroxycarbonylalkyl;

YesC, O, S; and

n is an integer between 1 and 10.

Among the most useful polymethines are the cyanines pentametine (n = 1) and heptametin (n = 2) comprising a benzoxazole, benzothiazole, indolyl, 2-quinolin or 4-quinolin group.

Oligomeric protein of the invention

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:

(to)

a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y

(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 area near the infrared eg Cy5, Cy7 etc .; (ii) a radionuclide for radioimmunoscintigraphy [compounds that emit gamma particles: 99mTc, 111In, 131I, rhenium 186 (186Re)] or for positron emission tomography (PET) (PET marker); etc. In a particular embodiment, said marker (M) is a fluorine, such as Cy5, Cy7, etc., or a PET marker, such as 64Cu, 68Ga, 18F, 86Y, 76Br, 89Zr, 124I, etc.

In general, the use in clinical practice of recombinant antibodies for diagnostic purposes requires the use of PET-like markers (“immuno-PET”), which, due to their characteristics, will allow the detection of small lesions, including micrometastases, located in deep areas; alternatively, to detect more superficial lesions, fl uorophores, e.g., NIR fl uorescent 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.

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.

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.

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 (A1) 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 (e.g., 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 that comprise a polypeptide

(TO)

comprising an antibody or a functionally equivalent fragment of said antibody, and a polypeptide

(B)

comprising an oligomerization domain, in which said oligomerization 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, triespecifics, if it recognizes three different targets or antigens, etc.

The polypeptide (A) 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 the EDB domain of fi bronectin (Ebbinghaus C et al. Curr Pharm Des. (2004); 10 (13): 153 7-49).

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 or in its complete recombinant format (Fab + Fc), or a VHH monodomain antibody preferably, an scFv (Example 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, 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).

In a particular embodiment, said polypeptide (A) comprises a recombinant scFv derived from the AcM L36 antilaminin (Example 1) [scFv L36] containing the heavy chain variable region (VH) of the fused monoclonal antibody L36, through a spacer , such as a peptide comprising the sequence (Gly-Ser) 4 (or a sequence of 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 al. 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 coding sequence of said linker and the 3 ′ end of the coding nucleotide sequence of said linker 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, Vol. 22 (7): 1508-1517].

In another particular embodiment, said polypeptide (A) comprises a recombinant scFv derived from the N1 hapten specific AcM B1.8 (Example 1) containing the heavy chain (VH) variable region of the fused AcM B1.8, through a spacer, to the variable region of the light chain (VL) of the AcM B1.8; 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.

In another particular embodiment, said polypeptide (A) comprises a recombinant scFv derived from the human Carcinoembryonic antigen (CEA) specific MMA23 (Example 1) containing the heavy chain variable region (VH) of the fused AcM MFE23, through 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.

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.

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 NC1 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 NC1 domain of collagen XV or the NC1 domain of collagen XVIII which contains the trimerization domain but to which the ES domain (NC1ES−) has been removed as well as all or part of the hinge peptides between these domains. The sequences of said NC1 domains of collagen XV and collagen XVIII are known; by way of illustration, the sequence of the NC1 domain of collagen XVIII has been previously described by 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].

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)

a polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof; Y

(ii)

a polypeptide (B) comprising the NC1ES- domain of mammalian collagen XVIII.

However, any other domain similar to the NC1 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.

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 of "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 to each other 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.

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 Likewise, in order to facilitate the isolation and purification of the fusion protein of the invention, said fusion protein may, if desired, contain a peptide (D) capable of being used for the purpose of isolation or purification of the protein from fusion, 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 fusion protein :, resulting by chromatography immunoaffinity, such as tag peptides, for example, epitopes derived from the hemagglutinin (HA) of the influenza virus, C-myc, FLAG, V5, etc.

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)

a polypeptide (A) comprising an antibody or a functionally equivalent fragment thereof; Y

(ii)

a polypeptide (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 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.

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.

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 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.

For use in such applications, the oligomeric protein of the invention will be in an appropriate and suitable medium for administration.

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 solution, 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.

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.

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 has excellent stability and binding capacity to the target (antigen), recognizing with high efficiency both purified antigens immobilized on a plate 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 (e.g., the anti-NIP oligomeric protein -Example 1) has a functional affinity for the much higher 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 oligomeric proteins of the bispecific 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.

This gain in affinity through avidity makes the oligomeric proteins of the invention very attractive reagents for in vivo imaging techniques as alternative, and preferred, agents to dimeric antibodies (diabodies and minibodies). As is known, for complete avidity in multivalent antibodies directed to surface bound molecules, 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. The 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 in fl uence about 11 times greater, approximately, than other bivalent formats, such such as diabodies and minibodies, increasing the likelihood of a second effective interaction. The fl exibility 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

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 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.

The kit of the invention is a product that contains the different products (e.g., 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 the formulations of the invention to be kept 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.

Fusion protein [polypeptide (A) -polypeptide (B)]

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:

(to)

a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y

(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 fl exible binding peptide between said polypeptides (A) and (B) and / or a peptide (D) to facilitate isolation or purification of the fusion protein.

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.

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 the gene constructs, expression cassettes and corresponding vectors will be generated. 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 (e.g., 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).

Therefore, in another aspect, the invention relates to a gene construct, hereinafter referred to as a gene construct of the invention, comprising at least:

a) a first nucleic acid sequence (A ’), comprising the nucleotide sequence encoding a polypeptide (A), wherein said polypeptide (A) comprises an antibody or a functionally equivalent fragment of said antibody; Y

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 ').

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 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.

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, e.g., an AcM, an AcP, a scFv, a bispecific or diabody antibody, a VHH, etc. In a particular embodiment, said nucleic acid sequence (A ') encodes a polypeptide (A) comprising a recombinant scFv derived from the anti-laminin Ac36 L36 [scFv L36], or a recombinant scFv derived from the hapten specific M1 B1.8 NIP, or a recombinant scFv derived from the specific MFE23 AcM of the human carcinoembryonic antigen (CEA) (Example 1).

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.

The nucleic acid sequence (B ') comprises the nucleotide sequence that encodes a polypeptide (B) comprising an oligomerization domain. As previously mentioned, an oligomerization domain allows the formation of oligomers (e.g., 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 NC1 domain of collagen XVIII or collagen XV; in a specific embodiment, said oligomerization domain is the trimerization domain of the NC1 domain of collagen XVIII or of collagen XV to which all or part of the ES domain has been removed. Thus, in a particular and preferred embodiment, the nucleic acid sequence (B ') comprises the nucleotide sequence encoding the NC1 domain of mammalian collagen XVIII or the NC1 domain of mammalian collagen XV, wherein, optionally, all or part of the ES domain has been removed. The sequences of said NC1 domains of collagen XV and XVIII are known; by way of illustration, the sequence of the NC1 domain of collagen XVIII has been previously described by 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].

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 ’).

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'). Therefore, 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 to 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 Gly 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 peptide Leu-Glu-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser. 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.

Likewise, 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 only, 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.

The gene construct of the invention can be obtained by employing techniques widely known in the state of the art [Sambrook et al., "Molecular cloning, a Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989 Vol 1-3]. Said gene construct of the invention may, operably linked, incorporate a sequence regulating the expression of nucleotide sequences encoding 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 frame of correct reading under the control of the control or regulatory expression sequences.

Therefore, in another aspect, the invention provides an expression cassette comprising the gene construct of the invention operatively 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, the 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.

Advantageously, said expression cassette further comprises a marker or gene that encodes a motif.

or for a phenotype that allows the selection of the transformed host cell 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.

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 that, when introduced into a host cell, is integrated or not into the genome of said 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.

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.

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 the 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 can be expressed, if desired, in an appropriate host cell by co-transformation, co-transfection or co-infection using vectors containing the appropriate coding sequences.

The following Example serves to illustrate the invention and should not be considered as limiting its scope.

Example 1

In vivo localization and visualization of tumors by oligomeric proteins comprising antibody fragments and collagen derived sequences

I. Materials and methods

Antibodies and reagents

Monoclonal antibodies (AcMs) used included the AcM 9E10 (Abcam, Cambridge, R. Kingdom) specific for human c-myc, and the AcM NCRC23 (AbD Serotec, Kidlington, R. Kingdom) specific for 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). Purified laminin-1 from the murine EHS 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 (NIP10: BSA) a previously described protocol was followed [Reth, M. et al. (1979). Eur. J. Immunol. 9: 1004-1013].

Cells and culture conditions

HEK-293 cells (human embryonic kidney epithelial cells; CRL-1573), and their 293T derived cells (CRL-11268), as well as HT-1080 cells (human fi brosarcoma; CCL-121), MKN45 (human gastric adenocarcinoma ; 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). HeLaCEA cells (Compte, M. et al. 2007; Cancer Gene Ther. 14: 380-388) were cultured in DCM supplemented with 750 μg / ml of G418 (Invitrogen).

Construction of expression vectors

The expression vectors pCR3.1-L36 and pCR3.1-L36-NC1ES− 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].

Plasmid pVOM1.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 NotI 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 that encodes Ac B1.8 (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-NC1ES− and pCEP4-B1.8-NC1ES− the 252 base pair fragment (pb) derived from plasmid pCR3.1-L36-NC1ES−, obtained by digestion with NotI, cloned in plasmids pCEP4MFE-23 or pCEP4-B1.8 respectively.

Cellular transfections and puri fi cation of recombinant antibodies

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-NC1ES - respectively, were selected in DCM supplemented with 0.5 mg / ml neomycin (G418) (Promise). HEK-293 cells transfected with the vectors pCEP4-MFE-23-NC1ES− and pCEP4-B1.8-NC1ES−, 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. The stable transfectants of the HEK-293 cells were used to obtain serum-free conditioned medium (MCLS) (approximately 1 liter) which was concentrated (x10) with a fi lter of 10,000 MWCO Viva fl ow 50 (Vivascience AG, Hannover, Germany), dialyzed versus phosphate buffered saline (PBS) (pH 7.4) and loaded onto a 1 ml HisTrap HP column using the ÄKTA 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 -20 ° C until use.

Gel filtration analytical chromatography

The analyzes were performed using Superdex 200 10 / 300GL (GE Healthcare) columns equilibrated with PBS (pH7.4) in a ÄKTA FPLC (GE Healthcare) equipment. 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.

ELISA

The ability to recognize and bind recombinant proteins (antibody trimers (scFv) - sometimes identified in this description as "trimerbodies") purified [L36, MFE23 or B1.8] to laminin-1 minin, human CEA or NIP10 conjugates -BSA, was studied by ELISA [Sanz, L. et al. 2003; EMBO J., 22: 150817]. The multivalence of the trimerbodies was studied by ELISA using the supernatant of 293T cells transfected with either a single vector (pCR3.1-L36-NC1ES− or pCEP4-B1.8-NC1ES−) or with 2 vectors (pCR3.1 -L36-NC1ES− and pCEP4-B1.8-NC1ES−). 96-well plates Maxisorp (NUNC Brand Products, Roskilde, Denmark) were coated with laminin (0.5 μg / well) by a 16-hour incubation at 4 ° 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-NC1ES− or pCEP4-B1.8-NC1ES−) or with 2 vectors (pCR3.1-L36-NC1ES− and pCEP4-B1.8-NC1ES−) for 1 hour at room temperature. After 3 washes, 100 μl of NIP10-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.

Flow cytometry, detection of antigens on the cell surface

CEA expression in the HeLa and HeLaCEA tumor lines and the binding of the recombinant antibodies (trimerbodies) was analyzed following a protocol already described [Blanco B et al., (2003). J. Immunol. 171: 1070-1077] by indirect immuno fl uorescence. Briefly, the 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). The studies were performed on an EPICS XL cytometer (Coulter Electronics).

Affinity studies by surface plasmon resonance

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; P20 0.005% Surfactant) previously fi ltered through were used of a 0.22 μm fi lter and degassed before use. Recombinant proteins (trimerbodies) were dissolved in 10 mM sodium acetate (pH 4.5). The NIP10-BSA conjugate was immobilized directly on the surface of the sensor chip following the manufacturer's instructions in independent fl ow cells at 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 fl ow 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.

For the kinetic analyzes, a flow cell with small amounts of NIP10-BSA (approximately 100 UR) was used in order to minimize the effects of mass transport and re-union. The individual samples, consisting of scFv or purified trimerbodies, were passed over the chip surface 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.

Comparative protein modeling

The 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, H.

M. et al. 2000; Nucleic Acids Res. 28: 235-42]. The structure of the NC1 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

To determine whether the trimerbody maintained its functional capacity in serum, 500 ng of purified L36 trimerbody was incubated at 37 ° C for 72 hours, with 12.5% murine serum from BALB / c mice (Harían Ibérica, Barcelona, Spain). Samples were removed for analysis at 3 h, 24h and 72h 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.

Conjugation of recombinant antibodies with cyanine 5

scFv L36 and purified trimerbodies were labeled with the fluorochrome Cyanine 5 (Cy5) NHS esters following the manufacturer's instructions (GE Healthcare). The labeling 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-10 columns, GE Healthcare) and concentrated on a 10,000 MWCO Vivaspin 500 filter (Vivascience) 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 the specific antigens.

Tumor localization assays in mice carrying recombinant antibodies by molecular imaging - Infrared immunophotodetection in tumor bearing mice

MKN45, HT1080 or HeLa cells (1-2x106) were implanted subcutaneously (s.c.) in the dorsal region of nude nude female nu / nu mice of 6 weeks Hsd (Nude-Fox1nu -Harlan Iberian nude mice). The dimensions of the nodules were used to calculate the tumor volume using the formula: (width) 2 x (length) x 0.52. When the tumors reached an appropriate size (0.2-0.4 cm3), 100 µl of a solution of purified recombinant antibody in trimerbody format labeled with Cy5 was administered intravenously (iv) in the tail vein. PBS (5 mg / kg). Fluorescence images in vivo were taken at various times (3, 24 and 48 hours) after i.v. of the labeled antibody, with a Hamamatsu system equipped with a high resolution digital camera with coupled load devices (CCD) ORCA-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. Results

Design and expression of trimeric constructions

Structural analyzes of the NC1 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 fl exible region sensitive to proteases; 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 L36 antilaminin scFv fused to the NC1 amino terminal domain of murine collagen XVIII was produced and functionally secreted by HEK-293 cells [Sánchez-Arévalo, LV et al. (2006). Int. J. Cancer 119: 455-462]. In addition, to provide sufficient spatial fl exibility to the scFv located in the amino terminal region, an arti fi cial 21 amino acid connector was inserted [Sánchez-Arévalo, LV et al. (2006), cited supra].

The trimeric nature of the scFv-NC1 fusion protein was demonstrated by ultracentrifugation [Sánchez-Arévalo, LV et al. (2006), cited above] and analytical gel filtration chromatography (Figure 1A). 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). On the contrary, the 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."

In the present invention, the concept has been extended by the design of trimerbodies with different specificities for the NIP hapten or human CEA. The coding genes of the scFvs derived from the anti-NIP (B1.8) 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 trimerbodies B1.8 and MFE-23 eluted from the column with single peaks (data not shown) comparable to those shown in Figure 1A.

Antigen binding studies

The functionality of the purified trimerbodies was demonstrated by ELISA against the conjugated immobilized antigens NIP-BSA (NIP10-BSA), murine EHS laminin and human CEA (Figure 1B). Its ability to detect its antigen in a cellular context was investigated by immuno fl uorescence labeling of human tumor cells that express the CEA antigen on their cell surface. Fluorescence was observed after incubation with the MFE-23 trimerbody, followed by reaction with the anti-myc AcM and detection with the goat anti-mouse IgG Ac conjugated to FITC. On the contrary, the incubation of cells expressing CEA (CEA +) with trimerbody B1.8

or incubation with HeLa cells that do not express CEA (CEA−) with the MFE-23 trimerbody showed no fluorescence (Figure 1C). These results indicate that trimerbodies not only recognize the immobilized antigen, but also recognize the native antigen expressed on the surface of tumor cells.

The SPR tests were used to determine the influence of the scFv or trimerbody format on the functionality of Ac B1.8. The binding kinetics of each Ac format were compared using three different densities of the NIP10-BSA immobilized on the chip surface. BSA coupled to dextran was used as a reference for nonspecificity. To compare the binding responses during the association-dissociation processes, several concentrations of Ac B1.8 (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 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 1D).

For kinetic studies, a chip coated with approximately 100 UR of NIP10-BSA was used. In these trials, scFv B1.8 behaved very differently than trimerbody B1.8, with slower association and dissociation ratios faster. The kinetic constants of association (ka) and dissociation (kd) were determined by simultaneous adjustment using the Langmuir 1: 1 interaction model. For scFv B1.8, the apparent association (ka) and dissociation (kd) constants were ka: 1.0 x 104 ± 1.7x102 M − 1s − 1 ykd: 2x10−3 ± 4x10−5 s− 1 (Chi2 = 0.189). Using KD = kd / ka, the equilibrium dissociation constant for scFv B1.8, 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 NIP10 - Immobilized 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 were adjusted for trivalent B1.8 binding. The data of the curves with resonance values that approximate Rmax would be close to those obtained for the monovalent junction. In this case, as the concentration of trimerbody B1.8 increased, the ka decreased from 4 x 105 M − 1s − 1 (12.5 nM trimerbody) to 2 x 104 M − 1s − 1 (800 nM trimerbody). These changes are compatible with changes in the proportion of antibody that binds to the ligand mono- or multivalently, since an approximation to surface saturation leads to a greater proportion of monovalent antibody binding. The apparent ka has a narrower range, from 6x10-4 (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 KD (kd / ka) of 1.5 x10-9 M or higher .

To ensure that there are at least two active binding sites in the same trimerbody molecule, 293T cells were transfected with the L36, B1.8 trimerbodies coding plasmids or with both plasmids in a co-transfection. Western blotting trials showed that both trimerbodies were expressed in the supernatant of the cells 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 NIP10-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 NIP10-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 NIP10-BSA.

In vitro serum stability tests

A very important aspect to determine the potential in vivo application of the designed Ac fragments is to check the stability against serum. The researchers have determined the functionality and structural stability of the trimerbody L36 after incubation with human and murine serum at 37 ° C for prolonged periods of time (equal to or greater than 72 hours), by Western and ELISA transfer assays. As shown in Figure 3, trimerbody L36 maintains 80-90% of its binding capacity to its antigen after 72 hours of incubation.

In vivo specific tumor localization assays

To evaluate the biodistribution of, and the specific location of tumors by, the anti-NIP, anti-CEA and anti-laminin trimerbodies, an optical molecular imaging system was used, 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 the near-infrared emission fluorochrome Cy5 and injected intravenously (iv) into the tail vein of nude female nude mice nu / nu carrying human tumors MKN45 (gastric adenocarcinoma), HT-1080 (fi brosarcoma ), 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 4A and 4C).

Trimerbody B1.8 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 injection i.v. 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 trilabody antilaminin showed specific tumor location in all the 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 antilaminin scFv also showed specific tumor location but at a much lower level than that of the trilabody antilaminin (Figures 4B and 4D).

III. Discussion

Previous studies by 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 NC1 N-terminal sub-domain of collagen XVIII, responsible for the nocovalent 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 shown that using a scFv (L36) that recognizes a laminin epitope associated with angiogenesis and that inhibits tumor angiogenesis and growth, the trimeric L36 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").

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 media 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 plaque, 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 gain in affinity 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 sterically possible, then the apparent gain in functional affinity is likely to be lower 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). The 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 ˚

If the conformation is fully extended, it 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 about 11 times greater, approximately, 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" thereby 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.

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 intermediate sized multivalent molecules that have high stability in 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 antilaminin 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 fl exible area, which corresponds 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. Cell. 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 trimerbodies specific for tumor-associated antigens (for example, the human epidemic growth factor receptor 2, prostate-specific antigen), or directed to the tumor stroma (for example, phospholblast 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 (19)

1. An oligomeric protein comprising a plurality of fusion proteins, the same or different, and a marker (M), wherein each fusion protein comprises:

(to)

a polypeptide (A) comprising an antibody or a functionally equivalent fragment of said antibody; Y

(b)

a polypeptide (B) comprising an oligomerization domain.

2.

Oligomeric protein according to claim 1, wherein said marker (M) is a fluorophore or a radionuclide.

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 antigenic epitope or determinant of an antigen.

4. Oligomeric protein according to claim 3, wherein said antigen is an antigen that is de novo expressed or is overexpressed in a pathological disorder.

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.

Oligomeric protein according to claim 1, wherein said oligomerization domain present in said polypeptide (B) comprises NC1 domain of collagen XVIII or mammalian collagen XV, optionally totally or partially devoid of the endostatin (ES) domain.

7. Oligomeric protein according to claim 1, consisting of 2 or more fusion proteins, the same or different. 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; Y ii) a polypeptide (B) comprising an oligomerization domain, with a marker (M) under conditions that allow the binding of said marker (M) to the oligomeric protein.

9.

Use of an oligomeric protein according to any one of claims 1 to 7, in a method for the detection, visualization or localization of a target.

10.

Use according to claim 9, wherein said target is an antigen that expresses de novo or is overexpressed in a pathological alteration.

eleven.

A method for the detection, visualization or localization of a target comprising the use of an oligomeric protein according to any of claims 1-7.

12. Method according to claim 11, wherein said method is based on an imaging technique.

13.

Method according to claim 11, wherein said target is an antigen that is de novo expressed or overexpressed in a pathological alteration.

14.

A composition comprising an oligomeric protein according to any one of claims 1 to 7, together with at least one appropriate medium.

fifteen.

A pharmaceutical composition comprising an oligomeric protein according to any one of claims 1-7, together with at least one pharmaceutically acceptable carrier.

16.

A kit comprising an oligomeric protein according to any one of claims 1-7.

17.

Use of a kit according to claim 16, for the detection, visualization or location of a target.

18.

Use according to claim 17, wherein said target is an antigen that expresses de novo or is overexpressed in a pathological alteration.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901027 SPAIN Date of submission of the application: 04.20.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

X X

EP 1798240 A1 (IND TECH RES INST.) 06.20.2007, Claims 1, 4-8. Figures 4A, 4B. Example 2. Sánchez-Arévalo Lobo Víctor J. et al. "Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events". International journal of cancer. 15.07. 2006. Vol. 119. No. 2. Pages 455-462. ISSN 0020-7136 (Print). 1-5,7,9-18 1-18

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 01.12.2011

Examiner J. Manso Tomico Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200901027 CLASSIFICATION OBJECT OF THE APPLICATION  C07K16 / 28 (2006.01) C07K16 / 30 (2006.01) C07K19 / 00 (2006.01) C07K14 / 435 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C07K Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, EMBASE, BIOSIS. State of the Art Report Page 2/4  WRITTEN OPINION Application number: 200901027 Date of Completion of Written Opinion: 01.12.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-18 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-18 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 200901027 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

EP 1798240 A1 (IND TECH RES INST.) 06/20/2007

D02

Sánchez-Arévalo Lobo Víctor J. et al. "Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events". International journal of cancer. 15.07. 2006. Vol. 119. No. 2. Pages 455-462. ISSN 0020-7136 (Print).

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application discloses an oligomeric protein formed by the combination of several fusion proteins and their use in the detection or visualization of target molecules. Claims 1-7 characterize the product called oligomeric protein, and claim 8 characterizes the process for obtaining it. Claims 9-13 characterize the use of the oligomeric protein for the detection of a target molecule. Claims 14-15 refer to a pharmaceutical composition containing the oligomeric protein. Similarly, claims 16-18 refer to a kit containing the oligomeric protein and its uses. D01 discloses (claims 1, 4-8) a trimeric antigen binding protein complex comprising three polypeptides, wherein each polypeptide comprises: a triple helix loop scaffold domain, a fused heterologous domain, which may be an antibody, and optionally A second domino. In Example 2, the generation of three fusion polypeptides is described: OKT3_scFv-Col, erb_scFv-Col, and erb_NSPD-scFv. In the case of scFv-Col, the scFv-Col region includes the NC1 domain of collagen XXI. In studies of affinity to antigen, where the binding antigen is the epidermal growth factor EGFR, carried out by ELISA, chemiluminescent detection was used after the binding of the appropriate marker. D02 describes, in Figure 1, the structure of the L36 scFv-collagen XVIII NC1 fusion protein. In its schematic description it is shown that said fusion protein is formed by a trimeric antibody next to the domain called NC1 of collagen XVIII. Thus, the prior art documents disclose fusion proteins that contain all the technical characteristics of the claimed oligomeric protein. In addition, these fusion proteins are used for the detection of antigens and more particularly in cancer therapy. Therefore, claims 1-18 would lack novelty and inventive activity, as mentioned in arts. 6 and 8 of Law 11/1986. State of the Art Report Page 4/4