WO2010076334A2

WO2010076334A2 - Intraoperative diagnosis of primary tumors and secondary tumors or metastases

Info

Publication number: WO2010076334A2
Application number: PCT/EP2009/068060
Authority: WO
Inventors: Aurélie DUTOUR; Raphaël ROUSSEAU
Original assignee: Centre Leon Berard
Priority date: 2008-12-31
Filing date: 2009-12-31
Publication date: 2010-07-08
Also published as: WO2010076334A3

Abstract

The invention relates to the improvement of the detection of the surgical margins of primary tumors as well as to the improvement of the resection of secondary tumors (metastases), most notably pulmonary metastases. The invention notably relates to the use of a RAFT scaffold on which have been grafted both a marker detectable in the near infrared and a ligand of the α_vβ₃ integrins.

Description

Intraoperative diagnosis of primary tumors and secondary tumors or metastases

The invention relates to improving the detection of the surgical margins of primary tumors as well as improving the resection of secondary tumors (metastases) , notably pulmonary metastases .

The goal of tumor resection is the complete excision of the tumor with adequate safety margins of healthy tissue. Among patients bearing a primary tumor, the quality of tumor control is correlated with survival. To date, only preoperative evaluation by means of classic imaging techniques (MRI, scanner ) enables the surgeon to determine the extent of the safety margins to anticipate. Thus, it is sometimes difficult for the surgeon to distinguish peπtumoral edema or inflammation from the tumor itself. This uncertainty generally leads the surgeon to wide resection margins of healthy tissue, making functional recovery problematic (notably in the case of limb resection) . To date, no intraoperative method is available or reliable enough to help the surgeon to delimit m situ (i.e., during the procedure) with sufficient precision the extension of the tumor in healthy tissue.

Among patients in metastatic relapse, the surgical control of metastatic localizations, notably pulmonary, is also correlated with survival. Currently, the surgical indication is guided by the results of conventional imaging (MRI, CT scanner) whose sensitivity is limited by the sometimes subcentimeter size of the metastatic lesions. With regard to pulmonary metastases, no reliable technique for intraoperative detection exists beyond manual palpation of the pulmonary tissue. In the field of mammary adenocarcinomas, the sentinel node technique makes it possible to detect the propagation of the breast cancer toward the axillary lymph nodes. However, the purpose of the sentinel node technique is not tumor resection. The purpose of this technique is to determine if the breast cancer has propagated toward the axillary lymph nodes. In this technique, 450 microcuries of technetium are injected around the tumor followed by 5 ml of isosulfan blue (1%, Lymphazuπn dye) 5-10 minutes before axillary dissection. As the sentinel lymph node becomes mildly radioactive, the surgeon uses a Geiger counter to precisely locate the sentinel lymph node. This enables the surgeon to make a very small incision above the sentinel node. Thus, this is not an intraoperative diagnostic method. These methods have the disadvantage of using radioactive markers and do not have as a goal a true visualization of the tumor but rather aim to provide an indication of the localization of the tumor. Moreover, tumor binding of markers or tracers is generally not uniform and it is not suitable for precisely delimiting surgical margins.

Conventional methods for determining tumor margins by preoperative imaging and anatomopathological analyses of the excised tumor are thus quite insufficient. To date, genuine intraoperative diagnostic methods for guiding the surgeon during tumor excision have not been described.

In contrast to the intraoperative field, a number of techniques have been described for the preoperative diagnosis of primary or secondary tumors as well as for the specific targeting of tumors with imaging markers. Others specific ligands of tumor cells have been described for carrying and targeting therapeutic molecules toward tumors.

Thus, RAFT (regioselectively addressable functionalized template) is a cyclodecapeptide possessing functionalizable amino acid side chains. RAFT is a multifunctional molecule that combines the ability to target specifically and to carry a diagnostic or therapeutic molecule. The RAFT molecule has two functional domains. On the lower face, two motifs are for grafting diagnostic or therapeutic molecules. The upper face or "targeting domain" of the RAFT enables the grafting of four target-specific ligands. RAFT maintains spatial separation between the "targeting" and "therapeutic" functional domains, thus avoiding the situation m which one of the functions disturbs the other (WO 2004/026894) .

In particular, a RAFT scaffold has been described bearing on one of these faces four cyclic RGD motifs for targeting α_vβ₃ integrins and on the other face the cyanine 5 (Cγ5) fluorophore: RAFT-c (RGD) ₄-Cy5 (Garanger et al .) . Due to its conformation, RAFT enables the combination targetmg/drug/imaging. A single molecule specifically carries the drug and is used to image the tumor or, subsequently, to quantify the real activity of the carried drug by using "intelligent probes." The multivalent presentation of cRGD ligands by the RAFT cyclodecapeptide (RAFT-cRGD) enables specific targeting of tumor neovascularization and tumor cells overexpressmg α_vβ₃ lntegπns. Binding of the cRGD motif to α_vβ₃ integrin is followed by internalization of the RAFT by endocytosis, thus enabling delivery of the therapeutic or diagnostic agent within the target cell. In several animal models, it has thus been shown that it is possible not only to target tumors and their vascularization but also to deliver in a specific and above all targeted manner a diagnostic marker or a molecule of therapeutic interest (Jin et al.) . Thus, RAFT has been envisaged essentially as a system for carrying and targeting molecules of therapeutic interest toward target tumor cells. The use of RAFT or RAFT-cRGD for intraoperative diagnosis has neither been described nor envisaged.

Labeled ligands bound to α_vβ₃ integrins can be used for detection of tumors and for molecular imaging to detect specific targeting of therapeutic molecules to tumors in clinical trials.

A great number of markers, labels and ligands may be used for the detection and molecular imaging of tumors. However, for these various markers it remains to be determined to what extent such molecules enable satisfactory detection of tumor margins in per operative conditions. As discussed above, intra-operative or per- operative diagnostic methods for guiding the surgeon during tumor excision have not been described.

The essential difficulty is to delimit in situ with sufficient precision the extension of the tumor in healthy tissue.

Another problem arising is that expression of some receptors/molecules on tumor cells may vary especially after chemotherapy. This has been reported for αvβ3 integrins for which chemotherapeutic treatments induce a significant decrease m integrin expression. Labeled ligands of the integrins were therefore not envisioned as particularly useful in per operative detection of tumor margins as patients are often treated by chemotherapy before performing surgery. Surprisingly, it has now been shown that a RAFT on which is grafted both a ligand of the α_vβ₃ integrins (the RGD motif) and a marker can be used for the mtra-operative or per-operative diagnosis of primary and secondary tumors. Even more surprisingly, treatment by chemotherapy does not alter the detection of tumor margins in per-operative conditions.

The technigues of the present invention make it possible to delimit with much better precision the margins of primary and secondary tumors, to facilitate the surgical procedure and, finally, to limit extension of the resection in healthy tissue all while maintaining tumor control.

Moreover, the inventive techniques make it possible to detect intraoperatively the existence of pulmonary micrometastases invisible to the naked eye or not palpable by the surgeon.

SUMMARY OF THE INVENTION

The present invention is related to a composition comprising a tracer for use in surgery wherein the tracer comprises a molecular scaffold having two faces, a marker detectable m the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face.

In a first embodiment, the invention is related to a composition comprising a tracer for use in surgery for the intraoperative detection of the surgical margins of a tumor m an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said intraoperative detection of the surgical margins of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer in the individual and c) detection of the tumor margins in near infrared light.

In a second embodiment, the invention is related to a composition comprising a tracer for use in surgery for the resection of a tumor in an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said resection of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer in the individual, c) detection of the tumor margins in the near infrared and d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control .

In a third embodiment, the invention relates to a composition comprising a tracer for use m surgery for the resection of a tumor in an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable m the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said resection of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer m the individual, c) detection of the tumor margins in near infrared light, d) resection of the tumor while limiting the extension of the resection m healthy tissue all while maintaining tumor control, and e) verification of the precision of the resection of the tumor in near infrared light.

Advantageously, said resection of a tumor in an individual comprises a repetition of steps c) to e) for the resection of the same tumor and / or for the resection of several distinct tumors.

In some embodiments, the tumor is an osteosarcoma, an uncategorized sarcoma, a myxoid liposarcoma, a nondifferentiated liposarcoma, a chondrosarcoma, a Ewing' s sarcoma, a rhabdomyosarcoma, a hepatic carcinoma, a colorectal carcinoma, a gastrointestinal stromal tumor (GIST) , a mammary adenocarcinoma, an ovarian carcinoma or a neuroblastoma. In other embodiments, the tumor is a secondary tumor or a metastasis .

In another embodiment, the tumor is a pulmonary metastasis. Preferably, the molecular scaffold is a cyclopeptide with two faces, the marker detectable m the near infrared being grafted on one of the faces and the ligand of the α_vβ₃ integrins being grafted on the other face. Preferably, the molecular scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Lys-Lys-Pro-Gly-] or the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Ala-Lys-Pro-Gly-] .

Advantageously, the marker detectable m the near infrared is a fluorophore.

More preferred, the marker detectable m the near infrared is Indocyanme Green (ICG) .

Advantageously, the ligand of the α_vβ₃ integrins is selected among the peptide Arg-Gly-Asp (RGD) , the motif cyclo [RGDfK] and the motif cyclo [RGDyK] .

Preferably, the molecular scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Lys-Lys-Pro-Gly-] having two faces, four cyclo [RGDfK] motifs being grafted on one face and a

Indocyanme Green flurophore being grafted on the other face. Even more preferred, the molecular scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Lys-Lys-Pro-Gly-] having two faces, four cyclo [RGDfK] motifs being grafted on one face on the lysine in positions 1, 3, 6, 8 and a Indocyanme Green flurophore being grafted on the other face on the lysine (s) in position 2 and/or 7.

The invention relates to a method for the intraoperative detection of the surgical margins of a tumor in an individual comprising the following steps: a) administration in the individual of a sufficient quantity of a tracer comprising a molecular scaffold on which is grafted a marker detectable m the near infrared and a ligand of the α_vβ₃ integrins, b) circulation of the tracer in the individual, c) detection of the tumor margins m near infrared light.

Another object of the invention is a method for the resection of a tumor in an individual comprising the following steps: a) administration in the individual of a sufficient quantity of a tracer comprising a molecular scaffold on which is grafted a marker detectable m the near infrared and a ligand of the α_vβ₃ integrins, b) circulation of the tracer in the individual, c) detection of the tumor margins m the near infrared, and d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control.

Another object of the invention is a method for the resection of a tumor in an individual comprising the following steps: a) administration in the individual of a sufficient quantity of a tracer comprising a scaffold on which is grafted a marker detectable in the near infrared and a ligand of the α_vβ₃ integrins, b) circulation of the tracer in the individual, c) detection of the tumor margins in near infrared light, d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control, and e) verification of the precision of the resection of the tumor in the near infrared.

In a particular embodiment of the invention, the inventive method comprises a repetition of steps c) to e) for the resection of the same tumor and / or for the resection of several distinct tumors.

In an advantageous embodiment, the tumor is an osteosarcoma, an uncategorized sarcoma, a myxoid liposarcoma or a nondifferentiated liposarcoma. In other embodiments, the tumor is a chondrosarcoma, a Ewmg's sarcoma, a rhabdomyosarcoma, a hepatic carcinoma, a colorectal carcinoma, a gastrointestinal stromal tumor (GIST) , a mammary adenocarcinoma, an ovarian carcinoma or a neuroblastoma .

In another embodiment, the tumor is a secondary tumor or a metastasis . Advantageously, the tumor is a pulmonary metastasis.

Advantageously, the molecular scaffold is a cyclopeptide with two faces, the marker detectable in the near infrared being grafted on one of the faces and the ligand of the α_vβ₃ integrins being grafted on the other face. Preferentially, the scaffold is the cyclic decapeptide c[- Lys (Boc) -Lys (Alloc) -Lys-Lys (Boc) -Pro-Gly-Lys (Boc) -Lys (Alloc) - Lys (Boc) -Pro-Gly-] . Advantageously, the marker detectable in the near infrared is a fluorophore.

In a preferred embodiment, the ligand of the α_vβ₃ mtegrms is selected among the peptide Arg-Gly-Asp (RGD) , the motif cyclo [RGDfK] and the motif cyclo [RGDyK] .

Still more advantageously, the scaffold carries four cyclo [RGDfK] motifs on one face and an ICG' marker on the other face .

DESCRIPTION OF THE INVENTION

The invention relates both to compositions comprising a tracer for use m surgery and to intraoperative methods or surgical methods combining a molecular scaffold, a ligand of the α_vβ₃ mtegrms, a marker detectable in the near infrared and a near infrared camera. These compositions and methods make it possible to precisely define tumor margins diagnostically and lntra- operatively or per operatively (i.e. during surgery) .

The present invention is related to compositions comprising a tracer for use m surgery wherein the tracer comprises a molecular scaffold having two faces, a marker detectable m the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ mtegrms being grafted on the other face. The compositions comprising a tracer according to the invention are for use m per- operative or intraoperative diagnostic methods for detection of the surgical margins of a tumor during surgery.

The compositions and methods of the present invention provide for improvement of tumor resection thanks to a precise detection of the margins of a tumor during surgery. In a first embodiment, the invention is related to a composition comprising a tracer for use m surgery for the intraoperative detection of the surgical margins of a tumor m an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable m the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ mtegrms being grafted on the other face, and wherein said intraoperative detection of the surgical margins of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer in the individual and c) detection of the tumor margins in near infrared light.

In a second embodiment, the invention is related to a composition comprising a tracer for use in surgery for the resection of a tumor in an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said resection of a tumor m an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer in the individual, c) detection of the tumor margins in the near infrared and d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control . In a third embodiment, the invention relates to a composition comprising a tracer for use in surgery for the resection of a tumor in an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable m the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said resection of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer m the individual, c) detection of the tumor margins in near infrared light, d) resection of the tumor while limiting the extension of the resection m healthy tissue all while maintaining tumor control, and e) verification of the precision of the resection of the tumor in near infrared light.

The invention relates also to methods for the intraoperative detection of the surgical margins of a tumor in an individual comprising the following steps: a) administration m the individual of a sufficient quantity of a tracer comprising a molecular scaffold on which is grafted a marker detectable m the near infrared and a ligand of the α_vβ₃ integrins, b) circulation of the tracer in the individual, c) detection of the tumor margins m near infrared light. The term "tracer" means the combination marker-scaffold- ligand of the ot_vfi₃ lntegπns. Surprisingly, it has been shown in the present invention that this tracer circulates and is distributed m the organism and tumor tissues m such a way that tumor margins can be detected with precision without observing background noise.

The tracer can be administered m the individual by all commonly used routes of administration. Preferably, the administration is by intravenous route.

A sufficient or efficient amount/quantity of tracer is administered to the individual. This amount is determined according to standard techniques.

Preferably, the quantity of tracer administered to the individual is between 50-1000nmol/kg, preferably between 75- 500nmol/kg and even more preferably between 75-150 nmol/kg.

In order to enable distribution or circulation of the tracer in the individual, the tracer is administered for a sufficient length of time before the intraoperative diagnosis of the tumor. This distribution time is typically between 6 and 24 hours, more preferentially between 12 and 24 hours and still more preferentially the distribution time is 18 hours. In preferred embodiments, administration of the tracer is performed at least 6,

9, 12, 18 or 24 hours before surgery to allow for adequate distribution of the tracer in the individual.

The marker is a marker detectable m the near infrared and consequently the detection of tumor margins takes place in near infrared light with a near infra red camera. Advantageously, this detection is carried out using a near infrared camera connected to a computer and a display for visualizing the surgical margins. Such a system can be implemented by placing the camera on an articulated arm that the surgeon can reposition during the operation .

The invention also relates to a method for the resection of a tumor in an individual comprising the following steps: a) administration m the individual of a sufficient quantity of a tracer comprising a molecular scaffold on which is grafted a marker detectable in the near infrared and a ligand of the α_vβ3 integnns, b) circulation of the tracer in the individual, c) detection of the tumor margins in the near infrared, and d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control.

The precise definition of the tumor margins obtained using the tracer and detection in the near infrared provides a significant improvement in surgical methods for the resection of primary and secondary tumors such as pulmonary metastases.

The invention also relates to a method for the resection of a tumor in an individual comprising the following steps: a) administration in the individual of a sufficient guantity of a tracer comprising an RGD scaffold on which is grafted a marker detectable m the near infrared and a ligand of the α_vβ₃ integnns, b) circulation of the tracer in the individual, c) detection of the tumor margins in near infrared light, d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control, and e) verification of the precision of the resection of the tumor in near infrared light. The intraoperative detection of tumor margins enables the surgeon to move back and forth between the resection and the detection of the tumor in order to be sure of the complete resection of the tumor while limiting the resection of healthy tissue . Thus, steps c) to d) can be repeated for the resection of the same tumor and / or for the resection of several distinct tumors.

In the above described methods, detection of the tumor margins and precise resection of the tumor may further be controlled by histopathology analysis. The inventive compositions for use in surgery and the intraoperative diagnostic methods prove particularly effective when the tumor is an osteosarcoma, an uncategorized sarcoma, a myxoid liposarcoma, a nondifferentiated liposarcoma, a chondrosarcoma, a Ewing' s sarcoma, a rhabdomyosarcoma, a hepatic carcinoma, a colorectal carcinoma, a gastrointestinal stromal tumor (GIST) , a mammary adenocarcinoma, an ovarian carcinoma or a neuroblastoma . The inventive compositions for use in surgery and the intraoperative diagnostic methods have also been shown effective for secondary tumors or metastases, notably pulmonary metastases.

The tracer used in the inventive methods consists of a molecular scaffold comprising two faces. Persons skilled in the art are familiar with these molecular scaffolds and will understand that the scaffold can take various forms. The function of the scaffold is to avoid interference between the marker detectable in the near infrared and the ligand or ligands of the α_vβ₃ integrins. In addition, the scaffold improves the presentation of the ligands of the α_vβ₃ integrins and their binding with the tumor cells or tumors. Typically, the scaffold has two faces, a marker detectable in the near infrared being grafted on one face and a ligand of the α_vβ3 integrins being grafted on the other face.

In a preferred embodiment, the molecular scaffold is a cyclopeptide with two faces, the marker detectable in the near infrared being grafted on one of the faces and the ligand (s) of the α_vβ₃ integrins being grafted on the other face.

Preferably, the scaffold is a RAFT scaffold (RAFT cyclic decapaptide) as described in WO 2004/026894 or by Garanger et al. RAFT (regioselectively addressable functionalized template) is a cyclodecapeptide possessing functionalizable amino acid side chains. RAFT is a multifunctional molecule that combines the ability to target specifically and to carry a diagnostic or therapeutic molecule. The RAFT molecule has two functional domains. On the lower face, one or two motifs are for grafting diagnostic or therapeutic molecules. The upper face or "targeting domain" of the RAFT enables the grafting of four target-specific ligands. RAFT maintains spatial separation between the "targeting" and "therapeutic" functional domains, thus avoiding the situation in which one of the functions disturbs the other.

In preferred embodiments the scaffold is a RAFT cyclic decapeptide. Advantageously, the scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Lys-Lys-Pro-Gly-] .

Particularly advantageously, the scaffold is the cyclic decapeptide c [-Lys (Boc) -Lys (Alloc) -Lys (Boc) -Pro-Gly-Lys (Boc) - Lys (Alloc) -Lys (Boc) -Pro-Gly-] wherein protection of the lysine in positions 1, 3, 6 or 8 and with a protective group such as Boc and protection of the lysine in positions 2 and 7 with a protective group such as Alloc results m RAFT molecules having two orthogonally addressable domains pointing on either side of the cyclopeptide backbone. Typically, four ligands of the αvβ3 integrins (such as an RGD motif or a cyclic RGD motif ) are grafted on the lysines in positions 1, 3, 6 and 8 for recognition and binding to the integrin whereas a marker detectable in near infrared is grafted on the lysine in position (s) 2 and/or 7.

In other embodiments, the scaffold is c [-Lys-Lys-Lys-Pro-Gly- Lys-Ala-Lys-Pro-Gly- ] . Typically, four ligands of the αvβ3 integrins (such as an RGD motif or a cyclic RGD motif) are grafted on the lysine m positions 1, 3, 6 and 8 for recognition and binding to the integrin where as a marker detectable in near infrared is grafted on the lysine m position 2. Other scaffolds or vectors can be envisaged for the tracer used in the inventive compositions and methods. In particular, liposome vectors comprising in their membrane specific antibodies to tumor epitopes can be envisaged.

The tracer also comprises a marker detectable in the near infrared (NIR) . This is typically a near infrared emitting fluorophore such as Cy5 or ICG (Indocyanine Green) . Indocyanine

Green has excitation/emission wavelengths of 780-830 nm.

Advantageously, this fluorophore has received regulatory approval.

The α_vβ₃ integrins have been the subject of a number of studies due to their role in tumor angiogenesis and in the formation of metastases. Ligands of the α_vβ3 integrins have thus been studied and implemented for the preoperative detection of tumors, for therapy or for carrying and targeting therapeutic molecules toward tumors. In the present invention the extraordinary effectiveness of ligands of the α_vβ₃ integrins for marking and defining tumor margins is shown in an astonishing manner. Numerous ligands of the α_vβ₃ integrins have been described in the literature and are well known to persons skilled m the art. In a preferred embodiment of the invention, the ligand of the α_vβ₃ integrins is selected among the peptide Arg-Gly-Asp (RGD) or among cyclic RGD motifs such as cyclo [RGDfK] and cyclo [RGDyK] . The motif cyclo [RGDfK] or cyclo (-Arg-Gly-Asp-D-Phe-Lys) has been described by Van Hagen et al . Int. J. Cancer 90, 186 (2000) and Hu et al . Biochem.39, 2284 (2000) . The motif cyclo [RGDyK] or cyclo (-Arg-Gly- Asp-D-Tyr-Lys) has been described by Chen et al. Bioconjug. Chem 15, 41 (2004) . Particularly advantageously the ligand is the RAFT cyclopeptide cyclo [RGDfK] .

Preferably, the tracer implemented in the inventive compositions and methods has several copies of the cyclic RGD (cRGD) motifs, which have a greater affinity for the integrins than the non-cyclic RGD motif.

Advantageously, the tracer is ICG-RAFT-C(RGD)₄ or ICG-RAFT- c (-RGDfK-) ₄; RAFT-c (-RGDfK-) ₄ notably has been described by Garanger et al. and in WO 2004/026894. Four cyclo [RGDfK] motifs are exposed on one of the faces of a RAFT scaffold to which they are bound by an oxime bond (-0-N=C-) . The other face of the RAFT carries a marker detectable in the near infrared such as Indocyanine Green (ICG or ICG') .

In preferred embodiments of the invention, the tracer comprises a scaffold which is a RAFT cyclic decapeptide having the sequence c [-Lys-Lys-Lys-Pro-Gly-Lys-Lys-Lys-Pro-Gly-] or c[-Lys- Lys-Lys-Pro-Gly-Lys-Ala-Lys-Pro-Gly- ] . The tracer further comprises four cyclic RGD (cRGD) motifs grafted on the lysine m positions 1, 3, 6 and 8. Preferably, the four cyclic RGD motifs are c (RGDfK) motifs. A marker detectable m near infrared (NIR) is grafted on the other surface of the cyclic decapaptide on the lysine (s) in positions 2 and/or 7.

The multivalent presentation of cRGD ligands by the RAFT cyclodecapeptide (RAFT-cRGD) makes it possible to specifically target tumor neovascularization and tumor cells overexpressmg the α_vβ₃ integrins. Binding of the cRGD motif to the α_vβ3 integπn is followed by internalization of the RAFT by endocytosis, thus enabling the delivery of the diagnostic agent within the target cell. The invention is also related to a tracer or to a composition comprising a tracer as described herein above.

FIGURES

Figure 1 : Specific tumor localization of RAFT-cRGD-ICG ' administered by IV route. Exposure of the tumor zone under the NIR camera makes it possible to distinguish transcutaneously the tumor/muscle limit (B) invisible under white light (A) . Tumor localization specificity is confirmed by detection of the light signal in the tumor only whereas healthy muscle in contact (M) has no interfering signal in situ (C) . Image analysis (D) shows the accumulation of RAFT-cRGD-ICG' in the tumor and its absence in healthy tissues in direct contact.

Figure 2: The combination of RAFT-cRGD-ICG' with the NIR probe makes it possible to significantly decrease (p<0.01) the size of the tumor fragments resected compared to the size of the fragments determined by preoperative imaging.

Figure 3: Detection of pulmonary metastases using the near infrared probe. After thoracotomy, exposure of the lungs under near infrared light makes it possible to distinguish millimeter- size metastases invisible under white light (arrow) . Comparison of number of metastases (M PuIm) detected according to the conventional technique or with the near infrared probe (RAFTs + NIR) for 9 rats is also shown in table I.

Figure 4: Tumor resection under near infrared lighting after intravenous injection of RAFT-cRGD-ICG'. (A) The 4-5 mm primary tumor, invisible under white light, is visible directly beneath the skin when the zone of interest is exposed to near infrared light. (B) The tumor margins appear clearly under near infrared lighting; no signal is detected in the muscles surrounding the tumor. This distinction makes it possible to perform an optimal resection of the primary tumor (C) . The specificity and sensitivity of RAFT-cRGD-ICG' are demonstrated by the detection of millimeter-size pulmonary metastases under near infrared light whereas no signal is detectable in the surrounding tissue (D) .

Figure 5: Quantification of the fluorescence signal in tumor and healthy tissues. No significant difference in the quantity of intra-tumor signal is observed between the control group (mean tumor signal: 302+/- 25 RLU/pix/ms) and the group having received the chemotherapy treatment (mean tumor signal: 273 RLU/pix/ms) .

Figure 6: Relative expression of genes of αv mtegπn/reference gene (HPRT) and genes of β3 lntegπn/reference gene (HPRT) m control groups (A) and those treated with ifosfamide (B) .

EXAMPLES

Example 1

All elements of the procedure will be carried out by experimenters experienced in the handling of animals, according to good laboratory practices, under suitable anesthesia and analgesics, and after agreement of the ethics committee of the Lyon veterinary school .

Objective 1. Improve the detection of the surgical margins of the primary tumor by means of diagnostic nanoparticles .

We will use an animal model that accurately reproduces the clinical operational context. Animal model. Our model of metastatic osteosarcoma m the rat has been described previously. In brief, the primary tumor is obtained by the transplantation of a tumor graft (0.5 mm³) in the paratibial position on 3-week-old immunocompetent rats. This model has the advantage of perfectly mimicking the human osteosarcoma due to its rapid growth, its hypervasculaπzed and chemoresistant character and its high metastatic potential. Four weeks after implantation of the tumor graft pulmonary metastases are observed in 90% of the animals. We have recently shown that non-mvasive medical imaging techniques make it possible to follow the response of the tumor to a therapy. Determination of doses and administration times of RAFT-cRGD-ICG '

RAFT-cRGD-ICG' molecules will be injected by intravenous route according to a dose escalation protocol (three levels: 0.25 nraol/g, 0.5 nmol/g and 0.75 nmol/g, 3 rats per level) . We will measure by in vivo spectrometry the lntra-turaor accumulation kinetics of RAFT-cRGD-ICG ' in order to determine the optimal intra-tumor signal/noise ratio. Detection of tumor margins

We will compare the quality and effectiveness of the surgical resection using in vivo intraoperative imaging with conventional techniques (i.e., quality of resection margins by fluorescent marking on anatomopathological slices after excision visually) . The primary tumor will be resected 10 days after implantation of the tumor graft. The animals of the treated group will receive four hours before the procedure an IV injection of RAFT-cRGD-ICG' at the established concentration. The surgical procedure will be carried out using the near infrared probe. The comparison with the control group (i.e., RAFT-cRGD without ICG') will involve the anatomopathological analysis of the tumor margins (comparison of the fluorescence due to the presence of ICG' nanoparticles and classic H&E counterstainmg) and the comparison of resected tumor volumes . Expected results

At the end of these experiments, we expect to have determined: i) the improvement of the tumor margins and of the resection of the primary tumor (e.g., less resection of healthy tissue, better tumor control) in the rats when the tumor margins are visualized using RAFT-cRGD-ICG' molecules detected by the near infrared probe compared to conventional methods for determining tumor margins (i.e., preoperative imaging and anatomopathological analyses of the excised tumor) . Objective 2. Improve the resection of pulmonary metastases by means of diagnostic nanoparticles .

On the basis of the RAFT-cRGD-ICG' doses and injection times established above, we will compare the sensitivity of the near infrared probe with the standard surgical technique (i.e., detection visually and by palpation) for detecting pulmonary metastases in our model of metastatic osteosarcoma in the rat. The animals of the control group will receive RAFT-cRGD without ICG'. The animals of the experimental group will receive RAFT-cRGD-ICG' . Four hours after administration of RAFT, the animals will be euthanized, the lungs will be removed and the number of pulmonary metastases will be determined according to the conventional method (counting of detectable metastases visually and by palpation) or by means of the near infrared probe. Next, the resection of pulmonary metastases will be performed on the anesthetized animals. The surgical procedure will be carried out using the near infrared probe for the animals of the treated group (having received an intravenous injection of RAFT-cRGD-ICG ' ) or according to the conventional technique for the control group. The quality of the intraoperative resection by means of the near infrared probe will be evaluated by comparing the number of persistent metastases/not detected m situ. This comparison will involve anatomopathological and fluorescence analyses. To us it does not appear possible to evaluate the benefit to the survival of animals in which pulmonary metastases have been resected by one or the other of the approaches as the rat model does not lend itself to keeping alive animals having undergone a thoracic surgical approach . Expected results At the end of these experiments, we expect to have determined the improvement in the detection of pulmonary metastases by means of RAFT-cRGD-ICG' compared to simple visualization and to intraoperative palpation. Due to the limitations of the animal model, a benefit in terms of survival could be determined only during a future phase I test in humans. RESULTS OBTAINED

We confirmed the specific tumor localization of RAFT-cRGD- ICG' molecules injected by intravenous route (Figure 1) . Exposure of the tumor zone under NIR light makes it possible to visualize transcutaneously the tumor/muscle limit (Figure IB) , invisible under white light (Figure IA) . Tumor localization specificity of RAFT-cRGD-ICG' is confirmed by the detection of the fluorescent signal only in the tumor (Figure 1C—D) , whereas no signal is present in the healthy muscles in contact with the tumor (Figure IC-D) .

Detection of tumor margins . We resected the primary tumor of 9 rats bearing osteosarcoma using intraoperative imaging. Anatomopathological analyses (localization of fluorescence due to the presence of ICG' nanoparticles and classic H&E counterstainmg) confirmed the quality of the resection. The comparison of resected tumor volumes using intraoperative imaging compared to those determined by conventional preoperative imaging (MRI) shows a significant benefit (i.e., a significant decrease in the size of the resected fragment, p<0.01) m favor of the resection carried out under intraoperative guidance (Figure 2) .

Objective 2. Improve the resection of pulmonary metastases by means of diagnostic nanoparticles.

We were able to show that the near infrared probe makes it possible to visualize in situ millimeter-size metastases invisible under white light (Figure 3) . We established that the number of metastases visualized by the near infrared probe is significantly (p<0.01) greater than that detected by the standard technique {i.e., detection visually and by palpation) . This is shown by the table below.

Table I

Example 2: Results obtained with RAFT-CRGD₄

In several animal models, we show that it is possible not only to target tumors (lung, osteosarcoma, prostate) and their vascularization but also to deliver m a specific and above all targeted way a diagnostic marker. The figure shows the specificity of RAFT-cRGD for the α_vβ₃ integrins and the sensitivity of RAFT- cRGD-Cγ5 in the in situ detection of a peritoneal carcinosis of an ovarian tumor in a mouse model.

Preliminary experiments using ICG' (lndocyanine green), a fluorophore similar to Cy5 and approved for clinical use, made it possible to show a specific tumor targeting of RAFT-cRGD-ICG ' m the rat model of grafted osteosarcoma.

Tumors 4-5 mm in diameter, undetectable in visible light, are detected under exposure to near infrared light after administration of RAFT-cRGD-ICG' (fig. 4A) . Dissection of the region of interest under near infrared light makes it possible to clearly discriminate the limit between muscles and tumor (fig.

4B) . The total resection of the tumor with minimal healthy margins is obtained under near infrared light (fig. 4C) . Moreover, use of the near infrared camera enables the detection of millimeter-size pulmonary metastases, invisible under white light, after intravenous administration of RAFT-cRGD-ICG' .

Example 3 : Procedure

As currently applied for the resection of the primary tumor in the rat:

Eighteen hours before the procedure, the animals receive an injection of RAFT-cRGD by intravenous route.

During the operation the animals are maintained under anesthesia by isoflurane inhalation. The tumor region is placed under the field of the near infrared camera. The tumor is examined/visualized by NIR on the display in order to begin to visualize the extension of the tumor in surrounding tissues (tumor visible transcutaneously) . The resection of the tumor is carried out by NIR guidance. The best approach to the tumor is given by virtue of the near infrared image displayed on the screen. After incision of the skin (generally m the posterior muscle cavity) , the tumor is freed from the muscles, the successive layers of muscles located around the tumor (which appear non-fluorescent by near infrared imaging) are removed according to the limits of the fluorescent signal: the last muscle freed (not resected, left in situ) corresponds to the last non-fluorescent muscle nearest the tumor (muscle in contact) . Next, the resection margins are determined on the bones: the limit of resection is set by the detection of fluorescence: the bones are cut at the limits of the fluorescence visualized on the display. The tumor, freed of all muscles, is removed and the non- resected region around the tumor (i.e., the healthy region) is observed under near infrared light to verify the quality of the resection (verification of the absence of the fluorescence indicative of total tumor resection) .

Example 4: Clinical protocol This phase I/II clinical protocol (toxicity/feasibility study) is comprised of three parts:

1) Study of the toxicity of the diagnostic nanoparticles . In a dose escalation protocol (3 doses; 4 patients per dose level) , we will evaluate the toxicity (determine the dose limit) of clinical grade RAFT-cRGD₄-ICG ' administered by

IV route m patients with a refractory or relapsing osteosarcoma .

2) In this same cohort of patients we will compare the sensitivity of detection of tumor margins of RAFT-CRGD₄- ICG' to the sensitivity of detection of the conventional technique (anatomopathological analyses) . Clinical grade RAFT-CRGD₄-ICG' will be administered by IV route to patients 18 hours before the surgical procedure. The resection of the tumor will be carried out according to the conventional technique (preoperative imaging for the determination of margins) . Tumor margins will be determined by near infrared imaging after the resection (exposure of the resected tissue under the near infrared camera) and according to the conventional method (anatomopathology) . We think that the use of RAFT-CRGD4-

ICG' will make it possible to determine tumor margins with a sensitivity equal to (or better than) the conventional technique.

3) Determination of the sensitivity of intraoperative determination of tumor margins of RAFT-cRGD4-ICG ' and of the near infrared camera. In a second cohort of patients, we will verify that the clinical grade RAFT-cRGD4-ICG' administered by IV route before the surgery makes it possible to correctly determine the margins of the tumor (or to determine exactly the extension of the tumor in healthy tissues) when the tumor region is exposed under near infrared light. Resection of the tumor will be guided by intraoperative imaging. Conventional anatomopathological and fluorescent analyses of the resected tissues will make it possible to determine the quality of the resection of the tumors and to verify the margin detection sensitivity of RAFT-cRGD₄-ICG' . We think that the use of RAFT-cRGD₄-ICG' and the intraoperative detection probe will improve the resection of the tumor (will make it possible to ensure local control of the tumor while avoiding the sacrifice of too much healthy surrounding tissue) . 4) Typically, the computer and display are placed on a cart and the camera on an articulated arm that the surgeon is able to reposition.

Example 5: Extrapolation to other tumors overexpressmg αvβ3 integrins

The results obtained m the rat osteosarcoma model have made it possible to: i) establish the sensitivity and specificity of tumor targeting by RAFT-c (RGD) 4-Fluo and ii) determine the feasibility of tumor surgery assisted m situ by use of the RAFT- c (RGD) 4-Fluo/detection probe combination peroperatively .

In a model of orthotopic and metastatic rat osteosarcoma, we have shown that it is possible to significantly improve tumor resection quality by virtue of assistance by near infrared imaging after intravenous injection of RAFT-c (RGD) 4-Fluo . Comparison of resected tumor volumes shows a significant increase (i.e., a significant decrease in resected fragment size; p<0.01) in favor of resection performed under peroperative guidance. We have also shown that the RAFT-c (RGD) 4-Fluo/near infrared probe combination makes it possible in situ to visualize metastases that are invisible under white light.

In view of the promising results obtained in the rat graftable osteosarcoma model, we defined the field of application of the peroperative RAFT-c (RGD) -Fluo/detection probe. More particularly concerning sarcomas, we defined for which types of sarcomas surgery under peroperative guidance will be applicable .

We believe that the peroperative RAFT-c (RGD) -Fluo/detection probe can be used to aid the resection of all tumors that overexpress αvβ3 integrins.

To determine precisely which tumors overexpress these integrins, we evaluate the expression of αvβ3 integrins, targets of RAFT-c (RGD) 4-Fluo, by immunohistochemistry on samples of human sarcomas having or not having received neoadjuvant chemotherapy and sampled at the time of surgery.

Results obtained

Up to now, it has been possible for us to evaluate by immunohistochemistry the expression of αvβ3 integrins on 12 human sarcoma samples including liposarcomas and uncategorized sarcomas

(Table II) . These initial analyses reveal that all sarcomas do not express αvβ3 integrins in an identical manner and that within a given histological subtype (liposarcoma) differences can be observed. Dedifferentiated or even differentiated liposarcomas do not express this molecule whereas nondifferentiated or mixed liposarcomas express it weakly and its expression is clearly detected in uncategorized sarcomas.

Sample ID Histological Integrin

Type labeling

Sard Uncategorized ++ sarcoma

Differentiated 0 liposarcoma

Sarc3 Myxoid ++ liposarcoma

Sarc4 Uiff^rentist^<i D

1ipoaarcoma

Sarc5 +

Uncategorized sarcoma

Sarc7 Myxoid ++ liposarcoma

Sarcδ Liposarcoma ++ 5

0 differentiated liposarcoms

SarclO Liposarcoma +

10

Sarcll Uncategorized ++ sarcoma

Sarcl2 Liposarcoma +

Table II: Semi-quantitative analysis of the expression of αvβ3 integrins by immunohistochemistry. It arises from this analysis that all sarcomas do not express integrins with the same intensity. 0: no expression detected, +: weak expression of integrins, ++ : average expression; +++: strong expression.

In view of these results it appears to us that the peroperative RAFT-c (RGD4) -Fluo/detection probe can be used at least for uncategorized sarcomas, myxoid liposarcomas and nondifferentiated liposarcomas. This approach can then be extended to any tumor overexpressing αvβ3 integrins. For example, it could be applied to certain neuroendocrine tumors and to ovarian carcinomas. Indeed, it has been shown by qRT-PCR in neuroendocrine tumors that the level of expression of integrins - especially β3 integrin - is extremely variable and weaker in these tumors than in hepatic metastases of colorectal cancer. (Oxboel J. et al . Oncol rep. 2009; 21 (769-775) . In the case of ovarian carcinomas, a team has demonstrated that in all cases the αv and β5 integrin subunits (αvβ5 being the other dimmer target of the cRGD motif with αvβ3) are expressed in these carcinomas. Expression of the β3 subunit is more variable (Maubant et al . J MoI Histol. 200 (36) : 119-129) . These results led us to think that RAFT-c (RGD4 )- Fluorophore would target these tumors. The literature and our results confirm the possibility of expanding use of the peroperative RAFT-c (RGD) -Fluorophore/detection probe combination to other sarcomas (rhabdomyosarcoma, Ewing sarcoma, leiomyosarcomas, etc.) and tumors expressing the target pre- and post-chemotherapy treatment.

Example 6: Neoadjuvant chemotherapies do not affect the binding of RAFT-CRGD4 -ICG' to tumors

In the rat osteosarcoma model, we tested the sensitivity of RAFT-c(RGD)-Fluo injected by IV route to enable the detection in situ of primary tumors in animals having received chemotherapy treatments (ifosfamide) .

The treatment was as follows: 21 days after tumor implantation, the rats with a progressive tumor receive an IP injection of ifosfamide (increasing doses from 5 mg/kg to 10 mg/kg) . One week after treatment, the primary tumor is resected using the peroperative RAFTc (RGD) 4-Fluo/detection probe combination .

We have demonstrated the specific tumor localization of RAFT- c (RGD) 4-Fluo injected by intravenous route on animals having received chemotherapy. Exposure of the tumor region under NIR light makes it possible to visualize transcutaneously the tumor/muscle limit which is invisible under white light. The specificity of tumor localization is confirmed by detection of the light signal only m the tumor whereas healthy muscle m contact has no parasitic signal in situ.

The specific tumor localization of RAFT- (cRGD) 4-Fluo is confirmed by detection of the fluorescent signal only in the tumor (Figure 5) , whereas no signal is present in the healthy muscles m contact with the tumor (Figure 5) . The intensity of the fluorescence signal detected in tumors m control animals (i.e., having received no treatment) and that detected in tumors of animals having received ifosfamide displays no significant difference (Figure 5) . These results indicate that chemotherapy does not modify the specific tumor targeting of RAFT (cRGD) 4-Fluo .

In parallel to these tests, and to verify the expression of post-chemotherapy lntegnns, we analyze by qRT-PCR the expression of αvβ3 integrins in the tumors of animals from each group

(control and treated with ifosfamide) .

The results obtained to date and presented in Figure 6 indicate that chemotherapy does not diminish the expression of these molecules. An increase in their expression is observed after treatment with ifosfamide (Figure 6 B) .

This result is surprising. Indeed, it has been reported m ovarian carcinomas that, although the expression of β3 integrins does not vary as a function of the response to chemotherapy, that of αv integπn does vary according to response to chemotherapy.

(Guo HY et al. Zhonghua Fu Chan Ke Za Zhi . 2004 ; 39 : 750-753 ) .

Moreover, in the case of osteosarcoma, a team analyzed by immunohistochemistry the expression of integrins and described that polychemotherapeutic treatments (adriamycine /cisplatine/ ifosfamide) induce a significant decrease in αv mtegrin expression.

REFERENCES

Chen et al . Bioconjug. Chem 15, 41 (2004) .

Garanger et al . , Molecular Therapy, vol. 16, no. 6, 1168-

1174, 2005

Guo HY et al. Zhonghua Fu Chan Ke Za Zhi . 2004 ; 39 : 750-753

Hu et al. Biochem. 39, 2284 (2000)

Jm et al., Molecular Imaging, vol. 5, no. 3, 188-197, 2006

Jm et al., Molecular Cancer, 6: 41, 2007

Maubant et al . J MoI Histol. 200 (36) : 119-129

Oxboel J. et al . Oncol rep. 2009; 21 (769-775)

Van Hagen et al . Int. J. Cancer 90, 186 (2000)

PATENT REFERENCES

WO 2004/026894

Claims

1. A composition comprising a tracer for use in surgery wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face .

2. A composition comprising a tracer for use in surgery according to claim 1 for the intraoperative detection of the surgical margins of a tumor in an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said intraoperative detection of the surgical margins of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer in the individual and c) detection of the tumor margins in near infrared light.

3. A composition comprising a tracer for use in surgery according to claim 1 for the resection of a tumor m an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ₃ integrins being grafted on the other face, and wherein said resection of a tumor in an individual comprises a) administration to the individual of said tracer, b) circulation of the tracer in the individual, c) detection of the tumor margins in the near infrared and d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control.

4. A composition comprising a tracer for use in surgery according to claim 1 for the resection of a tumor in an individual, wherein the tracer comprises a molecular scaffold having two faces, a marker detectable in the near infrared being grafted on one of the faces and a ligand of the α_vβ3 integrins being grafted on the other face, and wherein said resection of a tumor in an individual comprises a) administration to the individual of a sufficient quantity of said tracer, b) circulation of the tracer in the individual, c) detection of the tumor margins in near infrared light, d) resection of the tumor while limiting the extension of the resection in healthy tissue all while maintaining tumor control, and e) verification of the precision of the resection of the tumor in near infrared light.

5. A composition comprising a tracer for use in surgery according to claim 4 wherein said resection of a tumor in an individual comprises a repetition of steps c) to e) for the resection of the same tumor and / or for the resection of several distinct tumors.

6. A composition comprising a tracer for use in surgery according to anyone of claims 1-5 wherein the tumor is an osteosarcoma, an uncategorized sarcoma, a myxoid liposarcoma, a nondifferentiated liposarcoma, a chondrosarcoma, a Ewing' s sarcoma, a rhabdomyosarcoma, a hepatic carcinoma, a colorectal carcinoma, a gastrointestinal stromal tumor (GIST), a mammary adenocarcinoma, an ovarian carcinoma or a neuroblastoma.

7. A composition comprising a tracer for use in surgery according to anyone of claims 1-5 wherein the tumor is a secondary tumor or a metastasis.

8. A composition comprising a tracer for use in surgery according to claim 7, wherein the tumor is a pulmonary metastasis .

9. A composition comprising a tracer for use in surgery according to anyone of the preceding claims wherein the molecular scaffold is a cyclopeptide with two faces, the marker detectable m the near infrared being grafted on one of the faces and the ligand of the α_vβ₃ integrins being grafted on the other face.

10. A composition comprising a tracer for use m surgery according to claim 9 wherein the molecular scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro- Gly-Lys-Lys-Lys-Pro-Gly-] or the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Ala-Lys-Pro-Gly- ] .

11. A composition comprising a tracer for use m surgery according to anyone of the preceding claims wherein the marker detectable m the near infrared is a fluorophore.

12. A composition comprising a tracer for use m surgery according to claim 11 wherein the marker detectable m the near infrared is Indocyanine Green (ICG) .

13. A composition comprising a tracer for use m surgery according to anyone of the preceding claims wherein the ligand of the α_vβ3 integrins is selected among the peptide Arg-Gly-Asp (RGD) , the motif cyclo [RGDfK] and the motif cyclo [RGDyK] .

14. A composition comprising a tracer for use in surgery according to anyone of the preceding claims wherein the molecular scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro-Gly-Lys-Lys-Lys-Pro- GIy-] having two faces, four cyclo [RGDfK] motifs being grafted on one face and a Indocyanine Green flurophore being grafted on the other face.

15. A composition comprising a tracer for use in surgery according to claim 14 wherein the molecular scaffold is the cyclic decapeptide c [-Lys-Lys-Lys-Pro- Gly-Lys-Lys-Lys-Pro-Gly-] having two faces, four cyclo [RGDfK] motifs being grafted on one face on the lysine in positions 1, 3, 6, 8 and a Indocyanine Green flurophore being grafted on the other face on the lysine (s) in position 2 and/or 7.