Netrin-1 detection, concomitant testing and radiation-based therapy

Abstract

FIELD: biotechnology.SUBSTANCE: netrin-1 binding compound is provided, comprising an antibody against netrin-1 or an antigen-binding fragment thereof and a chelating group linked to a radioisotope. The invention also relates to the use of said compound in methods of imaging, diagnosing or treating cancer expressing netrin-1 by means of internal radiation therapy. The present invention is based on the discovery that netrin-1 is retained in a more sticky manner in the cellular matrix at the cellular periphery of cancer cells, and that netrin-1 is also expressed in adults, particularly in certain tumors. Netrin-1 has also been shown to be expressed at a very early stage during tumor formation.EFFECT: early detection of tumors expressing netrin-1.17 cl, 23, dwg, 2 tbl

Description

[0001] Field of technology to which the invention relates

[0002] The present invention relates to a method and reagents for detecting and localizing or visualizing netrin-1 in tumors, as well as a method and means for treating cancer based on the presence of netrin-1. The present invention, in particular, relates to a new diagnostic test that can be a companion test, and a new cancer therapy that can be combined with the companion test.

[0003] Technology Level

[0004] Currently, a wide range of methods are used to treat each type of cancer, including surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. Successful cancer therapy targets the primary tumor and its metastases, whether clinically apparent or microscopic.

[0005] It's important for patients to detect cancer as early as possible, localize it, and determine the type of cancer to treat. Cancer diagnosed at an early stage has a better chance of successful treatment. If the cancer spreads, effective treatment becomes more difficult, and survival chances are often greatly reduced. Therefore, it's important to know when to immediately initiate a more rigorous and aggressive treatment protocol to prevent the spread of aggressive cancer.

[0006] Furthermore, even if treatment is highly successful, tumor cells or tumor stem cells may remain in some areas. Identifying and localizing these cells is also crucial.

[0007] Patients may also be interested in receiving targeted anticancer therapy. However, in the case of targeted therapy, there is a real need for reagents that can detect and localize cancer in vivo, as well as determine certain molecular characteristics of the cancer, so that appropriate targeted therapy can be provided at the earliest possible stage or as an adjunctive therapy only for those patients whose cancer is amenable to such therapy.

[0008] Netrin-1 plays a crucial role in development, particularly in the formation of the central nervous system. It also acts as a conduit for commissural neurons. Netrin-1 has been described for many years in neuronal development as a secreted diffusible molecule. Signaling pathways are mediated through receptors called "deleted in colorectal carcinoma" (DCC), receptors from the uncoordinated-5 (UNC-5) family, and neogenins. All of netrin-1's molecular pathways suggest that it acts pleiotropically in a wide range of diseases or signaling mechanisms.

[0009] Netrin-1 levels have also been shown to be elevated in many types of cancer, such as breast cancer, NSCLC, and medulloblastoma. It has been suggested that such overexpression in tumor cells acts as a molecular mechanism blocking cell death induced by the activity of dependent receptors DCC and the Unc-5 family. These receptors act as tumor suppressor genes and trigger apoptosis in the absence of their ligand. To counteract this protective mechanism, tumor cells activate netrin-1 expression, leading to overexpression of this protein to suppress cell death, for example, after chemotherapy. Thus, reactivation of this molecular mechanism is becoming a therapeutic target in oncology. Therefore, therapeutic strategies have been developed to block netrin-1, or more precisely, to inhibit the interaction between netrin-1 and its receptors on the surface of cancer cells.A Phase I-II clinical trial has begun to evaluate NP137, a humanized monoclonal antibody capable of blocking the Unc5-B / netrin-1 interaction. Interim results show encouraging signs of clinical activity as monotherapy. Thus, netrin-1 blockade appears to be effective in a subset of patients, but there is no predictive value for patients using an approved simple companion test, as all biopsy-based tests are prone to errors and limitations due to the invasive nature of tissue collection.

[0010] J. Wischhusen et al. (Theranostics 2018, 8(18): 5126–5142) showed that netrin-1 co-localizes with endothelial CD31 in netrin-1-positive breast tumors. Netrin-1 localizes to the vascular endothelium of these tumors. Ultrasound molecular imaging (USMI) has been proposed as a non-invasive companion diagnostic method for netrin-1 interference therapy in breast cancer. Netrin-1 is detected on the surface of endothelial cells and is visualized in a very short time (on the order of ten minutes), allowing for the visualization of netrin-1 sequestered on the endothelial cells that make up the vessels. The results for tumor incorporation were very low, with a very low background-to-incorporation ratio: Fig. 5A it ranges from 32% to 45%, i.e. a real increase of 1.4 times.

[0011] Radiation tomography and internal radiotherapy are used to target membrane receptors or surface molecules. These methods typically do not target secreted factors or ligands, which are generally considered to be more or less diffusible.

[0012] J. Wischhusen et al. (infra) did not show that netrin-1 is sequestered in the cellular matrix at the periphery of cancer cells, and the simple demonstration that netrin-1 is sequestered on endothelial cells that make up vessels does not qualify netrin-1 detection as a reliable tool for the detection and localization of netrin-1-expressing tumors.

[0013] Netrin-1, which is primarily defined as a secreted protein or protein sequestered on the surface of endothelial cells in vessels, is not a primary candidate for imaging and / or targeted therapy.

[0014] Essence of the invention

[0015] This paper presents unexpected and extensive data showing that netrin-1 is retained more adherently in the cellular matrix at the cell periphery of cancer cells, as evidenced by netrin-1 accumulation in tumors. Interestingly, netrin-1, which is expressed at the embryonic stage, is also expressed in adults, particularly in certain tumors. Combined with the fact that netrin-1 is retained at the tumor site in the extracellular matrix (ECM), netrin-1 unexpectedly emerges as a highly specific target for imaging and / or targeted therapy. Sequestration of netrin-1 in the cellular matrix of tumor cells opens the way to imaging methods with long acquisition times, such as 24 to 96 hours, which allows for the visualization of netrin-1 sequestered in the extracellular matrix of the tumor itself, thereby providing reliable and powerful imaging of the entire tumor, in contrast to USMI described by J. Wischhusen et al.In particular, the background-to-inclusion ratio with a method such as SPECT can be high, for example, approximately 5.8-fold, as demonstrated for 4T1 cells. Unexpectedly, it was also shown here that netrin-1 is expressed very early during tumor formation, allowing for the detection, localization, and / or therapeutic intervention of netrin-1-expressing tumor cells at a very early stage, even before the appearance of a small lesion or before palpation, such as that seen in breast cancer.

[0016] Some aspects of the invention relate to compounds per se, useful in imaging or diagnostics, particularly in concomitant diagnostics, or in targeted therapy. These compounds are based on compounds comprising an antibody against netrin-1 or an antigen-binding fragment thereof and a chelating group linked to this antibody or fragment, wherein this chelating group is not necessarily linked to a radioisotope. The radioisotope itself may dictate the use of the compound in either imaging or targeted therapy.

[0017] Disclosure of the essence of the invention

[0018] In a first aspect of the present invention, there are provided compounds comprising:

[0019] an antibody against netrin-1 or its antigen-binding fragment and

[0020] a chelating group associated with a given antibody or fragment,

[0021] wherein the chelating group is not necessarily linked to a radioisotope.

[0022] Typically, the antibody or fragment thereof is covalently linked to the chelating group. According to this embodiment, these compounds are conjugates. In one embodiment, the chelating group is linked to the side chain of one amino acid of the antibody or fragment thereof, particularly to the side chain of a lysine residue.

[0023] Typically, the radioisotope is covalently bonded to the chelating group.

[0024] The compounds of the invention are particularly useful because they are capable of specifically binding to netrin-1 in vivo, allowing for tumor imaging or targeting by binding the antibody to netrin-1 in the cellular matrix at the periphery of cancer cells. This is particularly advantageous for determining cancer localization and / or monitoring cancer growth or regression. Radiolabeled compounds are primarily used for continuous imaging using various technologies such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET). Radiolabeled compounds can also be used for radiation therapy or for both imaging and therapy.

[0025] Antibodies

[0026] The compounds preferably comprise a monoclonal antibody (mAb) or an antigen-binding fragment thereof, wherein the mAb or fragment specifically binds to netrin-1. The mAb may be a murine, chimeric, humanized, or fully human monoclonal antibody. The fragment may be any type of mAb fragment that substantially retains the ability of the intact antibody to bind to netrin-1, such as an Fab or F(ab′)2.

[0027] Examples of useful murine, chimeric and humanized monoclonal antibodies are given in U.S. Pat. No. 10,494,427, which is incorporated herein by reference. Specific embodiments disclosed in this previous document and which can be used according to the present invention are the following antibodies listed in Table 1. The first of the antibodies listed in Table 1 corresponds to murine mAb 4C11, the second of the listed antibodies - HUM00 - corresponds to an antibody grafted with the murine CDRs of antibody 4C11 on human IgG1. Ten mAbs from HUM01 to HUM10 correspond to humanized mAbs derived from HUM00 with specific modifications in the FR regions of human IgG. HUM03 is also called NP137. Sequences C H Human IgG1 was obtained from Genbank AEL33691.1 with the R97K modification. Sequences C L(kappa) human IgG1 were obtained from Genbank CAC20459.1. Other allotypes can also be used. Specific binding of all these mAbs, Fab fragments, and F(ab′)2 fragments to netrin-1 was demonstrated in US 2018 / 0072800.

[0028] Table 1

[0029] 4C11 (mouse) SEQ ID NO: 6 SEQ ID NO: 7 Humanized VH SEQ ID NO: Heavy chain constant region (CH) VL SEQ ID NO: Light chain constant region (CH) HUM00 21 Human IgG1 13 Human IgG1 HUM01 14 Human IgG1 8 Human IgG1 HUM02 15 Human IgG1 9 Human IgG1 HUM03 16 Human IgG1 10 Human IgG1 HUM04 17 Human IgG1 11 Human IgG1 HUM05 18 Human IgG1 11 Human IgG1 HUM06 19 Human IgG1 10 Human IgG1 HUM07 20 Human IgG1 11 Human IgG1 HUM08 16 Human IgG1 11 Human IgG1 HUM09 19 Human IgG1 12 Human IgG1 HUM10 15 Human IgG1 10 Human IgG1

[0030] In one embodiment, the antibody is a monoclonal antibody or antigen-binding fragment thereof, comprising:

[0031] variable domain V H , including:

[0032] - H-CDR1 with the sequence shown in SEQ ID NO: 1;

[0033] - H-CDR2 with the sequence shown in SEQ ID NO: 2;

[0034] - H-CDR3 with the sequence shown in SEQ ID NO: 3;

[0035] variable domain V L , including:

[0036] - L-CDR1 with the sequence shown in SEQ ID NO: 4;

[0037] - L-CDR2 with YAS sequence;

[0038] - L-CDR3 with the sequence shown in SEQ ID NO: 5;

[0039] or

[0040] variable domain V H , including:

[0041] - H-CDR1 with the sequence shown in SEQ ID NO: 22;

[0042] - H-CDR2 with the sequence shown in SEQ ID NO: 23;

[0043] - H-CDR3 with the sequence shown in SEQ ID NO: 24;

[0044] variable domain V L , including:

[0045] - L-CDR1 with the sequence shown in SEQ ID NO: 25;

[0046] - L-CDR2 with the sequence shown in SEQ ID NO: 26;

[0047] - L-CDR3 with the sequence shown in SEQ ID NO: 5.

[0048] Preferably, the antibody is a monoclonal antibody or antigen-binding fragment thereof comprising the V pair sequences H and VL , selected from the following pairs: SEQ ID NO: 21 and 13, SEQ ID NO: 14 and 8, SEQ ID NO: 15 and 9, SEQ ID NO: 16 and 10, SEQ ID NO: 17 and 11, SEQ ID NO: 18 and 11, SEQ ID NO: 19 and 10, SEQ ID NO: 20 and 11, SEQ ID NO: 16 and 11, SEQ ID NO: 19 and 12, SEQ ID NO: 15 and 10. More preferably, the antibody is a monoclonal antibody or antigen-binding fragment thereof comprising the sequences of the V pair H and V L by SEQ ID NO: 16 and 10.

[0049] The anti-netrin-1 antibody or antigen-binding fragment thereof may also contain a heavy chain constant region (C H ) human IgG1 and / or light chain constant region (C L ) human IgG1. In one embodiment, sequence C H Human IgG1 was obtained from Genbank AEL33691.1 with the R97K modification. Sequences C L(kappa) human IgG1 were obtained from Genbank CAC20459.1. In one embodiment, the mAb is NP137 and comprises SEQ ID NOs: 16 and 10 as V sequences H and V L , respectively, and specific to them C H and C L IgG1.

[0050] Table 2. Description of sequences

[0051]

[0052]

[0053]

[0054] In Table 1, the CDR sections for IMGT are highlighted in bold where appropriate.

[0055] As antibodies against netrin-1 that can be used according to the invention, other antibodies, in particular monoclonal antibodies or antigen-binding fragments thereof, developed against human netrin-1 or against animal netrin-1, can also be mentioned, since netrin-1 is highly homologous between species. The following antibodies from Abcam can be mentioned: ab126729, ab122903, ab201324, ab39370; AF1109, AF6419, AF128.

[0056] Chelating groups

[0057] A "chelating group" or "chelating agent" or "chelator" in the present invention refers to a compound capable of chelating any radioisotopes. The chelating group captures the corresponding free radioisotopes, typically from aqueous solutions, allowing these isotopes to be used for specific biological applications. Such chelating molecules are bifunctional chelators. A "bifunctional chelator" or "bifunctional chelating agent" in the present invention refers to a compound that has the function of a metal-binding molecule and a chemically reactive functional group that enables binding to an antibody.

[0058] Numerous bifunctional chelators are known in this field. Many of them are commercially available and regularly used as PET imaging agents. Examples of bifunctional chelating agents: NODAGA (1,4,7-triazacyclononane-1-glutaro-4,7-diacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, diethylenetriaminepentaacetic acid (DTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), NOTA (4,7-triazacyclononane-1,4,7-triacetic acid.

[0059] Bifunctional chelators are preferably esters of these chelating agents. Preferably, the chelator is NODAGA-NHS (NODAGA N-hydroxysuccinimide ester) or DOTA-NHS (DOTA N-hydroxysuccinimide ester).

[0060] Radioisotopes

[0061] "Radioisotope" as used herein means a type of chemical element that has an unstable nucleus and emits radiation when it decays to a more stable form. Radioisotopes in the compounds of the present invention may be those used in imaging or radionuclide therapy.

[0062] Radioisotopes useful in the invention include, but are not limited to, 68 Ga, 64 Cu, 89 Zr, 186 Re, 188 Re, 153 Sm, 111 In, 99m Tc, 123 I, 177 Lu, 90 Y, 131 I, 213 Bi, 212 Bi, 211 At, 225 Ac.

[0063] For visualization, you can specify, in particular, 68 Ga, 64 Cu, 89 Zr, 186 Re, 188 Re, 153 Sm, 111 In, 99m Tc, 123 I.

[0064] The radionuclides for therapy can be selected, in particular, from among the radionuclides used in internal radiotherapy, which are metals that induce cytotoxicity. β-emitting radionuclides, such as lutetium-177 ( 177 Lu), yttrium-90 ( 90 Y) and iodine-131 ( 131 I). It is also possible to use α-emitting radionuclides, such as bismuth-213 ( 213 Bi), bismuth-212 ( 212 Bi), astatine-211 ( 211 At) and actinium-225 ( 225 Ac).

[0065] These radioisotopes are preferably selected for their half-life, which is preferably long, making them particularly suitable for in vivo applications such as PET / SPECT imaging or targeted radiotherapy.

[0066] Compounds and compositions

[0067] One or more, for example, from 2 to 10 chelators or chelating groups, may bind to a single antibody. Thus, the compounds of the invention may include:

[0068] an antibody against netrin-1 or its antigen-binding fragment and

[0069] one or more, in particular from 2 to 10 chelating groups linked to the antibody or fragment,

[0070] and these chelating groups are not necessarily associated with a radioisotope.

[0071] In one embodiment, one or more chelating groups are linked to the radioisotope. The anti-netrin-1 antibody or antigen-binding fragment thereof may be any of the monoclonal antibodies or antigen-binding fragments thereof described above. In one specific embodiment, the antibody is NP137.

[0072] Another aspect of the invention is compositions containing such compounds, which include:

[0073] an antibody against netrin-1 or its antigen-binding fragment and

[0074] one or more, in particular from 2 to 10 chelating groups linked to the antibody or fragment,

[0075] and these chelating groups are associated with a radioisotope,

[0076] and a pharmaceutically acceptable carrier.

[0077] In one embodiment, one or more chelating groups are associated with the radioisotope.

[0078] In one embodiment, the compositions may include an anti-netrin-1 antibody or antigen-binding fragment thereof to which no chelating group is attached.

[0079] These compounds and compositions can be prepared by well-known methods such as those set forth herein.

[0080] These compositions may further contain a pharmaceutically acceptable carrier or diluent.

[0081] Getting a connection

[0082] In a further aspect, the present invention provides a method for producing a compound of the invention. This method comprises the steps of:

[0083] a) conjugating a chelating group to an antibody or a fragment thereof;

[0084] and

[0085] b) isolation of the conjugate of the antibody or its fragment with the chelator.

[0086] Conjugation is accomplished by incubating an amine-reactive chelating group with the antibody or its fragment. Incubation is carried out for a time sufficient to achieve chelation, typically ranging from 5 minutes to 2 hours. Incubation is carried out at a temperature that does not denature the antibody or its fragment. The temperature is typically between 35 and 42°C, preferably between 37 and 40°C.

[0087] Amino-reactive chelator structures for the radioisotopes described here are commercially available, such as DOTA-NHS and NODAGA-NHS esters. It is expected that NHS esters (N-hydroxysuccinimide esters) will react with primary amines at the N-terminus and in the side chains of lysine amino acid residues (Lys, K) in antibodies, as occurs with peptides. Therefore, there is no need to describe their binding in detail here.

[0088] One or more, for example, 2 to 10 chelating groups, can bind to one antibody containing a series of lysine amino acids.

[0089] Preferably, the method for producing the compound of the invention further comprises the step of:

[0090] c) incubating a conjugate of an antibody or its fragment and a chelator with a complementary radioisotope;

[0091] This produces a compound of the invention. The compound can then be isolated and incorporated into a pharmaceutical carrier or liquid carrier.

[0092] Incubation c) is carried out for a time sufficient to ensure binding of the radioisotope. Typically, the duration ranges from 5 minutes to 2 hours. Incubation is carried out at a temperature that does not denature the antibody or its fragment. The temperature is typically between 35 and 42°C, preferably between 37 and 40°C.

[0093] Visualization

[0094] In a further aspect of the present invention, there is provided a method for visualizing the presence or localization of netrin-1 in a subject, or visualizing the presence or accumulation of netrin-1 in organs or tissues, or visualizing a netrin-1-expressing cancer, by administering to an organism (an animal, in particular a mammal, in particular a human) an effective amount of a compound, wherein the compound comprises a metal isotope suitable for visualization.

[0095] In a further aspect, the present invention provides compounds comprising an anti-netrin-1 antibody or antigen-binding fragment thereof, a chelating group linked to the antibody or fragment, and a radioisotope linked to the chelating group, for use in in vivo cancer imaging. Netrin-1 is predominantly found in the cellular matrix at the periphery of cancer cells, where netrin-1 accumulates.

[0096] In one embodiment, imaging provides information about the relative level of presence or expression of netrin-1 in the examined area (e.g., in an organ or tissue) using a compound of the invention.

[0097] "Netrin-1 accumulation" specifically refers to the accumulation of netrin-1 in the cellular matrix at the periphery of cancer cells. Thus, netrin-1 can be located and accumulated in tissues or organs near or surrounding cancer cells or tumors.

[0098] Imaging can be performed using any suitable method known to those skilled in the art that allows for detection and / or imaging, in particular PET or SPECT, especially in combination with CT scanning (computed tomography). A radionuclide, such as that obtained from a cyclotron or generator, is attached to a biologically active molecule, forming a radioactive tracer, for example, a tracer for SPECT or PET. In the case of the present invention, the molecule is a compound consisting of an antibody or a fragment thereof and a chelator, and upon binding of the radionuclide to it, a radioactive tracer is formed. The radioactive tracer is then administered to the patient, preferably by injection, for example, intravenous (IV) injection.

[0099] In one aspect of the invention, there is provided a method for visualizing the presence or localization of netrin-1 (e.g., by imaging) in a subject, comprising:

[0100] a) administering, preferably by injection, a compound described herein to the subject;

[0101] b) detection or localization of the compound by in vivo imaging, preferably PET or SPECT imaging.

[0102] In one aspect of the invention, there is provided a method for detecting and localizing cancer in a subject, comprising:

[0103] a) administering to the subject a compound comprising:

[0104] an antibody against netrin-1 or its antigen-binding fragment,

[0105] a chelating group linked to the antibody or fragment, and

[0106] a radioisotope bound to a chelating group,

[0107] b) detection and localization of this cancer by in vivo imaging in the cellular matrix at the periphery of cancer cells.

[0108] In one aspect, a time interval between steps a) and b) is maintained before detection or localization. This time interval or data collection time is between 4 and 172 hours, in particular between 12 and 172 hours, preferably between 24 and 96 hours or between 24 and 48 hours, allowing the compound to bind to netrin-1 sequestered in the extracellular matrix. More specifically, this time interval is sufficient for the administered compound to exit the bloodstream, penetrate the tumor or tumors, and reach netrin-1 sequestered in the tumor cell matrix. This enables the subsequent step of detection or localization of the bound compound during in vivo imaging.

[0109] In step b) or "use for" in vivo imaging, the presence or accumulation of the compound is detected or demonstrated in at least one part of the body, such as an organ or tissue. This presence or accumulation is specific in that the compound binds to netrin-1 accumulated in that part of the body. It is specific because a certain time interval elapses between the administration of the compound and imaging.

[0110] The latency or time interval between administration and detection is selected so that detection or visualization occurs at the moment the antibody or its fragment specifically binds to netrin-1. Indeed, after administration, the compound begins to spread throughout the body and its organs, and only after some time will the presence of the compound in an organ or body part be specific for the presence of netrin-1 and its binding. The time interval can range from 4 hours to 168 hours; typically, it ranges from 4 hours to 96 hours. In practice, the observed time interval should be compatible with the half-life of the radioisotope, and vice versa.

[0111] A further aspect of the invention provides the compounds described herein for use in imaging as a compound with a radioactive isotope. This use is particularly intended for imaging the presence or localization of netrin-1 in a subject, as described above. The compounds are particularly intended for use in in vivo imaging, preferably by PET or SPECT.

[0112] In one embodiment, the method or use comprises imaging a part of the body, in particular imaging an organ or tissue or parts thereof (e.g., lungs, pancreas, bladder, spleen, kidneys, stomach, colon, small intestine, intestines, esophagus, muscles, skin, brain) and optionally surrounding tissues or organs.

[0113] In one embodiment, the method or use involves imaging an anatomical part of the body, in particular, imaging a leg, arm, chest, abdomen, head and parts thereof.

[0114] In one embodiment, the method or use involves imaging the entire body.

[0115] In PET, the system detects pairs of gamma rays emitted indirectly by a radionuclide (tracer) injected into the body along with the radioactive tracer. Computer analysis is then used to create three-dimensional images of the tracer concentration in the body. In modern PET-CT scanners, three-dimensional imaging is often achieved using an X-ray CT scan of the patient, performed during the same session on the same machine.

[0116] PET can calculate a standard uptake value, which allows for a quantitative assessment of the indicator in the examined region (e.g., tissue or organ). This allows for a specific quantitative assessment of the presence or expression of netrin-1 in the examined region. Alternatively, a radiologist may professionally detect indicator uptake in a specific region simply by observing it, and this uptake is distinct from background noise. This is referred to here as a "positive uptake detection."

[0117] Single-photon emission computed tomography (SPECT) is a nuclear medicine imaging technique similar to PET. It also uses a radioactive tracer and is based on gamma ray detection. Unlike PET, the radioactive tracer used in SPECT emits γ-radiation, which is measured directly. When combined with a CT scanner, SPECT-CT also provides three-dimensional imaging.

[0118] SPECT imaging can be used to compare the area being examined (e.g., tissue or organ) with the liver. This results in a result defined as above, equal to, or below the liver level. A radiologist can expertly detect tracer uptake in a specific area during simple observation, and this uptake is distinct from background noise. This is referred to here as a "positive uptake detection."

[0119] Due to the short half-life of most positron-emitting radioisotopes, radioactive tracers (tracers) are traditionally produced in a cyclotron in close proximity to the PET or SPECT imaging facility. The half-life of fluorine-18 is long enough that fluorine-18-labeled tracers can be produced commercially away from the recipient and shipped to imaging centers. Alternatively, it is possible to obtain 68Ga in the generator, thereby eliminating the need for a cyclotron. Furthermore, the half-life of gallium-68 is close to the half-life of 18 F, which makes this radionuclide particularly useful for PET imaging.

[0120] In one embodiment, the radionuclide used is 111 In. It emits low-energy γ-photons during radioactive decay, and its half-life is 2.8 days. It is typically produced using a cyclotron. Its half-life is long enough that tracers labeled with it can be produced commercially far from the recipient and shipped to imaging centers.

[0121] The imaging method is suitable for the detection and localization of netrin-1 in cancer tissue, a cancerous organ, or in the body of a cancer patient. In the present invention, the term "cancer" refers to a physiological condition in mammals that is typically characterized by unregulated cell proliferation. The terms "cancer" and "cancerous" in the present invention shall encompass all stages of the disease. In the present invention, "cancer" refers to any malignant neoplasm that results from the unwanted growth, invasion, and, under certain conditions, metastasis of damaged cells in the body. Cells that give rise to cancer have genetic abnormalities and typically lose the ability to control cell division, cell migration, differentiation state, and / or cell death mechanisms. Cancer typically begins at a primary site, giving rise to primary cancer. Cancer that spreads locally or to distant parts of the body is called metastasis.The imaging method described here enables detection and localization of netrin-1-expressing solid tumors at any stage.

[0122] The compounds of the invention are also useful for diagnosing cancer in patients. In this aspect, the invention provides a method for diagnosing cancer in patients, which comprises the steps of:

[0123] a) administering a compound described herein or a pharmaceutically acceptable salt thereof to the subject;

[0124] b) detecting or localizing the compound by in vivo imaging, preferably PET or SPECT imaging; and

[0125] c) diagnosis of cancer based on stage b).

[0126] In step b), in vivo imaging detects or demonstrates the presence or accumulation of the compound in at least one part of the body, such as an organ or tissue. This presence or accumulation is specific in that the compound binds to netrin-1 accumulated in that part of the body. It is specific because a certain time interval elapses between administration of the compound and imaging, as described above.

[0127] A further aspect of the invention provides the compound described herein for use as a radioisotope compound for diagnostic imaging. This use can serve to visualize the presence or localization of netrin-1 in vivo in a subject, as described above. This can facilitate the diagnosis of cancer. The compound is particularly intended for use in in vivo imaging, preferably in PET or SPECT imaging.

[0128] The antibodies or fragments thereof of the invention bind only netrin-1. Therefore, any signals detected during PET or SPECT imaging indicate the presence of netrin-1. Due to the accumulation of netrin-1 in the cellular matrix at the periphery of cancer cells and the sensitivity of the labeled compounds used, it is possible to identify cancer cells in a patient's body and thereby diagnose cancer, confirm cancer, localize cancer, and / or determine the cancer type. The cancer type includes the name of the organ or tissue affected by the cancer.

[0129] In a further aspect of the present invention, there is provided a method for predicting cancer in a patient, which comprises the steps of:

[0130] a) administering a compound described herein or a pharmaceutically acceptable salt thereof to the subject;

[0131] b) detecting the compound by in vivo imaging, preferably PET or SPECT imaging; and

[0132] c) cancer prognosis based on stage b) detection.

[0133] In step b), in vivo imaging detects or demonstrates the presence or accumulation of the compound in at least one part of the body, such as an organ or tissue. This presence or accumulation is specific in that the compound binds to netrin-1 accumulated in that part of the body. It is specific because a certain time interval elapses between administration of the compound and imaging, as described above.

[0134] The said method includes an additional step of making a medical prognosis, for example, in a patient who is undergoing or has undergone treatment in accordance with anti-cancer therapy.

[0135] A further aspect of the invention provides the compound described herein for use as a radioisotope compound for prognostic imaging. This use is particularly intended for imaging the presence or localization of netrin-1 in vivo in a subject, as described above, and for cancer prognosis. The compound is particularly intended for use in in vivo imaging, preferably in PET or SPECT imaging.

[0136] "Prognosis" in the present invention refers to the likelihood of recovery from a disease or a prediction of the likely development or outcome of a disease. For example, the larger the imaging area detected at stage b), the larger the cancer mass in the patient's body, the worse the prognosis.

[0137] In a further aspect of the present invention, there is provided a method for determining the location of cancer in a subject in need thereof, which comprises the steps of:

[0138] a) administering a compound described herein or a pharmaceutically acceptable salt thereof to the subject;

[0139] b) detecting the compound by in vivo imaging, preferably PET or SPECT imaging; and

[0140] c) visualization of the localization of the presence or accumulation of netrin-1.

[0141] In step b), the presence or accumulation of the compound is demonstrated in at least one body part, such as an organ or tissue, imaged in step c). In step c), the presence or accumulation of netrin-1 is demonstrated in the body part, such as an organ or tissue, imaged. If the presence or accumulation of netrin-1 is visualized in the body part, there is a strong presumption that cancer is present in that body part. This may be the detection of cancer in the patient or the identification of the cancerous body part, or both. This presence or accumulation is specific in the sense that the compound binds to netrin-1 accumulated in the body part. It is specific because a certain time interval elapses between the administration of the compound and the imaging, as described above.

[0142] A further aspect of the invention provides the compound described herein for use as a radioisotope compound in imaging. Such use is particularly intended for imaging the presence or localization of netrin-1 in vivo in a subject, as described above. The compound is particularly intended for use in in vivo imaging, preferably in PET or SPECT imaging. It is intended for the detection of netrin-1 in the cellular matrix at the periphery of cancer cells.

[0143] The method or use may further include the step of assessing the presence of cancer in the tissue or organ or in multiple tissues and / or organs, wherein the presence is indicated by the presence or accumulation of netrin-1.

[0144] It should be immediately apparent to those skilled in the art that the invention also enables the localization of cancer at its earliest stages. Notably, the present invention is particularly useful for identifying cancer sites that are too small to be detected by other means.

[0145] A pharmaceutical imaging composition or unit dosage form thereof comprises an effective amount of the compound described above. The composition of the invention or unit dosage form may contain from 5 to 3 GBq, in particular from 10 to 500 MBq, of the radionuclide-labeled imaging compound described above, in combination with a pharmaceutically acceptable carrier. The methods of use described above may include administering to a patient, in particular a human, a composition or unit dosage form containing from 0.1 mCi to 100 mCi of the radionuclide-labeled imaging compound described above.

[0146] Treatment

[0147] In a further aspect, a method for treating a netrin-1-expressing cancer in a subject in need thereof is provided, which comprises administering to the subject a therapeutically effective amount of one of the compounds described above. This method can be classified as internal radiation therapy.

[0148] In a further aspect, there is provided such a compound as described herein for use in treating a netrin-1 expressing cancer in a subject.

[0149] The compound comprises an antibody or fragment thereof conjugated to a chelating group to which a radionuclide is bound, wherein the antibody or fragment thereof specifically binds to netrin-1. The radionuclide may be selected from those commonly used in internal radiation therapy, which are metals that induce cytotoxicity. Common β-emitting radionuclides such as lutetium-177 ( 177 Lu), yttrium-90 ( 90 Y) and iodine-131 ( 131I). It is also possible to use α-emitting radionuclides such as bismuth-213 ( 213Bi ), bismuth-212 ( 212 Bi), astatine-211 ( 211 At) and actinium-225 ( 225 Ac).

[0150] In a further aspect, there is provided such a compound as described herein for treating a netrin-1 expressing cancer in a subject.

[0151] In one embodiment, the radionuclide used is 177 Lu is a γ- and β-emitter with a half-life of 6.7 days. It is typically produced in a cyclotron. Its half-life is long enough that radioactive preparations labeled with it can be produced commercially far from the recipient and shipped to treatment centers.

[0152] In one embodiment, the radionuclide used is 225 Ac. This radionuclide emits alpha particles, generating a total of 4 alpha particle isotopes in a short decay chain to stable 209Bi, so it can be called an alpha-particle nanogenerator. Its half-life is 10 days. For more information, specialists in this field can refer to M. Miederer et al., Adv Drug Deliv. Rev. 2008, 60(12): 1371-1382.

[0153] The compound is administered to the patient in a standard manner, preferably parenterally, such as by injection.

[0154] In one embodiment, the method or use is used to treat a patient who has been diagnosed with a cancer that expresses netrin-1. In particular, the patient is identified by the imaging method described herein.

[0155] The pharmaceutical composition for therapy or its unit dosage form comprises an effective amount of the compound described above. The composition according to the invention or the unit dosage form may contain from 5 to 1000 MBq, in particular from 10 to 500 MBq, of the radionuclide-labeled imaging compound described above, in combination with a pharmaceutically acceptable carrier.

[0156] Imaging (diagnosis) and treatment

[0157] Where appropriate, the tools and techniques previously presented in the Imaging and Treatment sections apply to the tools and techniques in this section.

[0158] In a further aspect, a method is provided for identifying cancer patients to be treated using a monoclonal antibody or fragment thereof, wherein the antibody or fragment thereof is capable of inhibiting the interaction of netrin-1 with its receptors on the surface of cancer cells, wherein the method comprises:

[0159] a) administering a compound described herein to the subject;

[0160] b) detecting the compound by in vivo imaging, preferably PET or SPECT imaging;

[0161] c) visualization of the localization of the presence or accumulation of netrin-1;

[0162] d) treatment of this patient for visualized cancer.

[0163] In a further aspect, there is provided a method for identifying cancer patients to be treated with targeted radiation therapy, which preferably comprises:

[0164] a) administering a compound described herein to the subject;

[0165] b) detecting the compound by in vivo imaging, preferably PET or SPECT imaging;

[0166] c) visualization of the localization of the presence or accumulation of netrin-1;

[0167] d) treatment of this patient for visualized cancer.

[0168] In a further aspect, there is provided a method for treating a netrin-1 expressing cancer, which comprises:

[0169] a) administering the imaging compound described herein to the subject;

[0170] b) detecting the compound by in vivo imaging, preferably PET or SPECT imaging;

[0171] c) visualization of the localization of the presence or accumulation of netrin-1;

[0172] d) treatment of this patient for visualized cancer.

[0173] These methods have an additional step between steps a) and b), which comprises waiting for the data collection time specified above, in particular from 4 to 172 hours, preferably from 24 to 96 hours, for the compound to bind to netrin-1 sequestered in the extracellular matrix of the tumor.

[0174] In these various aspects, the compound administered in step a) is one of the in vivo imaging compounds described herein.

[0175] In these various aspects, the treatment in step d) can be carried out using existing anti-cancer therapies. However, in a preferred embodiment, the treatment is carried out by a method that specifically targets a cancer expressing netrin-1. Therefore, such treatment can be carried out by administering an effective amount of an anti-netrin-1 antibody, as described in document U.S. Pat. No. 10,494,427. The antibody can be one of the monoclonal antibodies listed in Table 1 of the present description, in particular the antibody designated NP137. The method comprises administering a therapeutically effective amount of such a mAb or a fragment thereof. For information on the administration of such antibodies, one skilled in the art can refer to said U.S. Pat. No. 10,494,427.

[0176] In one embodiment, the therapy is internal radiation therapy as set forth above. Wherein the therapy comprises administering a therapeutically effective amount of a compound described herein to the subject. The compound comprises an antibody or fragment thereof conjugated to a chelating group to which a radionuclide is linked, wherein the antibody or fragment thereof specifically binds to netrin-1. Such an antibody may be one of the monoclonal antibodies listed in Table 1, in particular the antibody designated NP137. The antibody or fragment thereof is conjugated to a chelating group that is linked to a radionuclide, as set forth and described in detail herein. The compound is designed to bind to netrin-1 in a tumor, including netrin-1 sequestered in a cellular matrix, and the radiation therapy may have an effect on surrounding tumor cells or on the entire tumor.

[0177] In one embodiment, the radionuclide associated with the imaging compound is 111 In.

[0178] In one embodiment, the radionuclide associated with the compound for internal radiation therapy is 177 Lu or 225 Ac.

[0179] The dosages of the imaging compound and the therapeutic compound are described above.

[0180] Dosage forms

[0181] The compositions of the invention can be prepared as pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a material that is not biologically or otherwise undesirable, i.e., this material can be administered to a subject without causing any undesirable biological effects or a deleterious interaction with any other components of the pharmaceutical composition in which it is contained. The carrier, of course, should be selected so as to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as should be well known to those skilled in the art. Examples of pharmaceutically acceptable carriers and other components of pharmaceutical compositions are given, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.Some suitable pharmaceutical carriers will be known to the skilled person and include, for example, water (including sterile and / or deionized water), suitable buffers (such as PBS), saline, cell culture media (such as DMEM), artificial cerebrospinal fluid, etc.

[0182] The dosage of the compositions of the invention can be presented in unit dosage form. The term "unit dosage form" as used herein refers to physically discrete units suitable as a single dosage for animals (e.g., humans), wherein each unit contains a predetermined quantity of a compound of the invention calculated in an amount sufficient to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier, or liquid carrier. One skilled in the art will be able to readily determine the appropriate dose, regimen, and route of administration for a given dosage form of the composition being used to achieve the desired effective amount or effective concentration of the drug in a given patient.

[0183] When administered to an animal, particularly a human, the dosage of the composition described herein should be sufficient to produce at least a detectable level of diagnostic response in the individual within a reasonable period of time. The dosage will be determined by the presence of any adverse side effects that may accompany the use of the particular agent or its composition. Generally, it is desirable to minimize adverse side effects whenever possible.

[0184] When administered to an animal, particularly a human, for therapy, the dosage of the composition described herein should be sufficient to produce at least a detectable level of cytotoxicity to cancer cells, kill cancer cells, and reduce or regress cancer growth in the individual within a reasonable period of time. The dosage will be determined by the presence of any adverse side effects that may accompany the use of the particular agent or composition based on it. Generally, it is desirable to minimize adverse side effects whenever possible.

[0185] The pharmaceutical or radiopharmaceutical composition can be administered parenterally, i.e., by injection, most preferably in the form of an aqueous solution. "Pharmaceutically acceptable carrier" means a biocompatible solution that has the appropriate level of sterility, pH, isotonicity, stability, etc., and may include various solvents, diluents (including sterile saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection and other aqueous buffer solutions), dispersion media, coatings, antibacterial and antifungal agents, isotonizing agents, etc. The pharmaceutically acceptable carrier may also contain stabilizers, preservatives, antioxidants, or other additives that are well known to those skilled in the art, or other carriers known in the art.

[0186] Brief description of figures

[0187] Next, the present invention will be described in more detail by means of non-limiting examples with reference to the following drawings.

[0188] Fig. 1. Netrin-1 binds to and is retained in the extracellular matrix. Analysis of the binding of h-netrin-1 (recombinant human netrin-1) to extracellular matrix components: recombinant mouse laminin (m-laminin), recombinant human fibronectin (h-fibronectin), and recombinant human vitronectin (h-vitronectin) by biolayer interferometry.

[0189] Fig. 2. Characterization of conjugates with fragments. (a) Chemical structure of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) HS-ester molecules and NODAGA (1,4,7-triazacyclononane-1-glutaro-5,7-acetic acid) HS ester used for metal chelation. (b) Schematic representation of the whole anti-netrin-1 antibody (NP137), F(ab′)2, and Fab fragment conjugated to NODAGA or DOTA chelators. (c) NP137, F(ab′)2, and Fab conjugates were prepared by synthesis and enzymatic cleavage and subjected to electrophoresis under denaturing or non-denaturing conditions. (d) Biolayer interferometry analysis of NP137, F(ab′)2, and Fab after coupling of the NODAGA chelator. Numbers indicate the concentrations of NP137-NODAGA, F(ab′)2-NODAGA, and Fab-NODAGA.

[0190] Fig. 3. SPECT / CT analysis and detection of netrin-1 in tumors. (a) Quantitative analysis of netrin-1 expression by Q-RT-PCR on 4T1 and 67NR cell lines. (b) Maximum intensity projections of whole-body tomographic scintigraphy and X-ray CT of Balb / c mice bearing 4T1 tumors (netrin-1 positive) obtained, from left to right, at 4 h, 24 h, 48 h, and 72 h after intravenous administration of NP137-NODAGA- 111 In. (c) Maximum intensity projections of whole-body tomographic scintigraphy and X-ray CT of a Balb / c mouse bearing 67NR (netrin-1 negative) tumor, obtained, from left to right, 24 h, 48 h, and 72 h after intravenous administration of NP137-NODAGA- 111 In.

[0191] Fig. 4. Measurement of radioactivity accumulation. Biodistribution coefficient (a) 111 In-NODAGA-NP137-Fab, (b) 111 In-NODAGA-NP137-F(ab′)2i (c) 111In-NODAGA-NP137 in tumors in Balb / cJ mice bearing 4T1 (netrin-1 positive) xenografts compared to 67NR (netrin-1 negative) xenografts at 24 h, 48 h, 72 h, and 96 h. Radioactivity incorporation was determined as a percentage of the administered dose per 1 g of tumor. (d) Biodistribution parameters 111 In-NODAGA-NP137 in Balb / cJ mice bearing 4T1 xenografts was measured at 48 h, 72 h, and 96 h in all organs. Radioactivity incorporation was determined as a percentage of the administered dose per 1 g of organ (%ID / g).

[0192] Fig. 5. Measurement of radioactivity accumulation. (a) Maximum intensity projections of tomographic scintigraphy and whole-body X-ray CT of MMTV / neuT mouse genetically modified to produce mammary tumors, obtained, from left to right, 24 h, 48 h, and 72 h after administration of NP137-NODAGA- 111 In. (b) Schematic representation and location of 10 mammary glands in mice. (c) Biodistribution indices111 In-NODAGA-NP137 in MMTV / NeuT mice after 72 hours was measured in tumors and in all organs of the mice. Radioactivity incorporation was determined as a percentage of the administered dose per 1 g of organ (%ID / g).

[0193] Fig. 6. Novel anticancer therapy. (a) Balb / cJ mice were inoculated with EMT6 cells by subcutaneous injection of 1 million cells. Five days later, the animals were intravenously injected with PBS; DOTA-NP137 (anti-netrin-1 antibody); or DOTA-NP137- 177 Lu. n = 9 animals per group for PBS and DOTA-NP137; n = 12 animals per group for DOTA-NP137- 177 Lu; p<0.0001 between PBS and DOTA-NP137- 177 Lu and between DOTA-NP137 and DOTA-NP137- 177 Lu. (b) DOTA-NP137- 177 Lu increases survival of mice inoculated with EMT6 cells (see a). Kaplan-Meier survival curve analysis of mice treated or not with NP137. Mantel-Cox test; n = 9 animals per group for PBS and DOTA-NP137; n = 12 animals per group for DOTA-NP137. 177Lu; p<0.0001 between PBS and DOTA-NP137- 177 Lu and between DOTA-NP137 and DOTA-NP137- 177 Lu. (c) Balb / c mice were inoculated with 4T1 cells by subcutaneous injection of 1 million cells. After 8 days, the animals were intravenously injected with PBS; DOTA-NP137 (anti-netrin-1 antibody); or DOTA-NP137- 177 Lu. n=5 animals per group for PBS and DOTA-NP137; n=6 animals per group for DOTA-NP137- 177 Lu. (d) DOTA-NP137- 177 Lu increases survival of mice inoculated with 4T1 cells (see c). Kaplan-Meier survival curve analysis of mice treated or not with NP137. Mantel-Cox test; n = 9 animals per group for PBS and DOTA-NP137; n = 12 animals per group for DOTA-NP137. 177 Lu; p<0.0001 between PBS and DOTA-NP137- 177 Lu and between DOTA-NP137 and DOTA-NP137- 177Lu. (e) NMRI nude mice were inoculated with SYO1 cells by subcutaneous injection of 5 million cells. After 8 days, the animals were intravenously injected with PBS; DOTA-NP137 (anti-netrin-1 antibody); or DOTA-NP137- 177 Lu. n=9 animals per group for PBS and DOTA-NP137; n=12 animals per group for DOTA-NP137- 177 Lu; p<0.0001 between PBS and DOTA-NP137- 177 Lu and between DOTA-NP137 and DOTA-NP137- 177 Lu. (f) Effect of NP137- 177 Lu on survival of mice inoculated with H358 cells. Kaplan-Meier survival curves for mice treated or not with DOTA-NP137. Mantel-Cox test; n = 8 animals per group for PBS and DOTA-NP137; n = 9 animals per group for NP137- 177 Lu; p = 0.025 between PBS and NP137- 177 Lu and between DOTA-NP137 and NP137- 177 Lu.

[0194] Fig. 7. Quantitative analysis of netrin-1 in concentrated supernatants of cells treated or not with heparin.

[0195] Fig. 8. Maximum intensity projections of whole-body NMRI nude mice with H358 tumor (netrin-1 positive) obtained at 24 h, 48 h, 72 h and 96 h after intravenous administration of NP137-NODAGA- 111 In.

[0196] Examples

[0197] Materials and Methods

[0198] Tumor cell lines

[0199] 4T1 and 67NR mouse mammary carcinoma cells were obtained from ATCC and cultured in RPMI-1640 medium (ATCC) supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotics (streptomycin and penicillin). EMT-6 mouse mammary carcinoma cells were obtained from ATCC and cultured in Eagle's minimal essential medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotics (streptomycin and penicillin). H358 human lung adenocarcinoma cells were obtained from ATCC and cultured in RPMI-1640 medium (ATCC) supplemented with 10% BBS (Gibco) and antibiotics. Cells were maintained in culture at 37°C in a humidified atmosphere containing 20% ​​O2 and 5% CO2.

[0200] Western blotting

[0201] The cells in the monolayer state were washed with cold PBS and transferred to lysis buffer (10 mM Tris, pH 7.6; 5% SDS; 10% glycerol; 1% Triton X-100; 100 mM DTT). After sonication, protein was detected with Pierce 660nm Protein Assay Reagent (ThermoFisher Scientific), the protein was loaded onto 4-15% SDS-polyacrylamide gels (Bio-Rad), and then transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer (Bio-Rad). The membranes were blocked for 1 hour at room temperature with 5% dry skim milk for netrin-1 and 5% BSA. Staining was performed overnight with the primary antibody: anti-netrin-1 antibody (Ab126729, Abcam). After washing, the membranes were incubated with a second antibody: rabbit anti-goat antibody conjugated to HRP, for 1 hour at room temperature. A West Dura chemiluminescence system (Pierce) was used for signal amplification. Images were taken using a Chemidoch Touch (Bio-Rad).

[0202] For binding to netrin-1 in the cell matrix, 1×10 6 cells in a 100 mm flask 3 After 24 h, the cells were treated with 200 μg / ml of sodium heparin from porcine intestinal mucosa (H3147-100KU, Sigma) diluted in 4 ml of FBS-free medium. After overnight incubation, the supernatant was collected. Centricon centrifuge filters were used to concentrate the protein in the resulting supernatant. Protein concentration was then determined using Pierce 660nm Protein Assay Reagent (22660, ThermoFisher Scientific), and 30 μg of protein was applied to immunoblots.

[0203] Preclinical in vivo models

[0204] Human monoclonal antibody to NP137 (anti-netrin-1, HUM03) was kindly provided by Netris Pharma (Lyon, France). Eight-week-old female Balbc / J mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France). Total syngeneic breast cancer cells: 1×10 6 EMT-6; 5×10 5 4T1 and 1×106 67-NR was inoculated subcutaneously into the dorsal flank of 8-week-old female Balbc / J mice. Mice were maintained under pathogen-free conditions (Anican, Lyon, France, and Imthernat facility, HCL Lyon, France) and in sterile cages with filter lids. Their care and housing complied with European and French institutional guidelines as defined by the local CECCAP ethics committee. Human H358 cells (1×10 6 cells) or SKBR7 (2×10 6 cells) were inoculated into 8-week-old female NMRI immunocompromised mice and maintained under the same conditions.

[0205] Tumor volumes were determined by measuring two perpendicular tumor diameters with a caliper three times a week. Individual tumor volumes were calculated as follows: V = (a×b 2 ) / 2, where a is the largest diameter and b is the smallest. When tumors reached a volume of 200-400 mm 3 , mice were randomly divided into groups and subjected to treatment111 In-NODAGA-NP137, 111 In-NODAGA-NP137-Fab, 111 In-NODAGA-NP137-F(ab′)2or 177 Lu-DOTA-NP137, and then sent for imaging / therapy. For all experiments, mice were anesthetized using gas (isoflurane / oxygen = 2.5% / 2.5%).

[0206] Conjugation

[0207] 1 ml of the anti-netrin-1 monoclonal antibody NP137 (or its fragments, as required, under the same conditions for all of them) was applied to Amicon Ultra-15 50k (UFC905096). Diafiltration was performed against 0.1 M phosphate buffer (pH 8) containing 1.2 g / L Chelex 100. This operation was repeated 7 times using 10 ml of 0.1 M phosphate buffer solution (pH 8), with centrifugation for 25 minutes at 4900 rpm between each wash. The concentration of anti-netrin-1 antibody was then calculated using Nanodrop. The antibody concentration was then adjusted to 50 μM. Stock solutions of DOTA NHS ester and NODAGA (1,4,7-triazacyclononane-1-glutaro-5,7-acetic acid) NHS ester (CheMatech, C084) were diluted with ultrapure water to a concentration of 10 mg / mL (= 13.13 mM). Anti-netrin-1 antibody at 50 μM was mixed with the desired DOTA-NHS or NODAGA-NHS solution at a ratio of 1:25. Reactions were carried out at room temperature for 4 h and then transferred to 4°C for continuous stirring overnight.Diafiltration was then performed against PBS (Chelex). This process was repeated seven times, using 10 ml of 0.1 M phosphate buffer (pH 8), with centrifugation for 25 minutes at 4900 rpm between each wash. The concentration of the anti-netrin-1 antibody was then calculated using Nanodrop.

[0208] Radioactive labeling

[0209] NODAGA-NP137, NODAGA-NP137-Fab or NODAGA-NP137-F(ab′)2(40-70 μL, 5 mg / mL) were radioactively labeled by adding 400 μL of 100 mM acetate buffer pH 5 and 40-400 MBq of high-purity chloride 111 In (Covidien, Petten, The Netherlands). The mixture was incubated for 30 minutes at 37°C. The reaction was stopped with 100 μl of 1 mM DTPA solution. Free 111 In was removed on a PD-10 column. First, the column was washed with 15 ml of 0.1 M acetate buffer, then the labeled mixture was applied to it and eluted with acetate buffer. First, 111 In-NODAGA-NP137, 111 In-NODAGA-NP137-Fab or 111In-NODAGA-NP137-F(ab′)2. The radiochemical purity (RCP) of each 0.5 ml fraction was determined using ITLC-SG (Biodex, Tec-control black) and 50 mM citrate buffer (pH 5) as the mobile phase. Radioactively labeled NP-137 remained at its original position, while unbound 111 In migrated from R f 0.9-1. Fractions with the highest radiochemical purity were combined.

[0210] To test the stability of an aliquot of radioactively labeled 111 In-NODAGA-NP137, 111 In-NODAGA-NP137-Fab or 111 In-NODAGA-NP137-F(ab′)2 was incubated at 37°C in 2 ml phosphate-buffered saline (pH 7.4) and the radiochemical purity (RCP) of the labeled compounds was determined using ITLC-SG and 0.1 M citrate buffer pH5 as the mobile phase.

[0211] The same protocol can be applied to 111 In-DOTA-NP137, 111 In-DOTA-NP137-Fab or 111 In-DOTA-NP137-F(ab′)2.

[0212] The same protocol was used to obtain 177Lu-DOTA-NP137 via DOTA-NHS.

[0213] Biodistribution studies

[0214] Tumor-bearing mice (n=3 or 4 for each group) were intravenously injected with 1 to 10 MBq of radioactively labeled 111 In-NODAGA-NP137, 111 In-NODAGA-NP137-Fab or 111 In-NODAGA-NP137-F(ab′)2or 111In-DOTA-NP137 was administered in a maximum volume of 100 μl. Mice were sacrificed by cervical dislocation at the following times: 4 h, 24 h, 48 h, 72 h, and 96 h after injection. Tissues of interest (blood, heart, lungs, spleen, kidneys, muscle, brain, and skin) were removed, weighed, and radioactivity was measured for 5 minutes in a γ-scintillation counter (Wizard® gamma counter, Perkin Elmer, USA). Urine and feces were collected in individual metabolic containers for storage and measurement. Tissue distribution was expressed as a percentage of the administered dose per 1 gram (% ID / g). Renal and hepatobiliary elimination was expressed as cumulative radioactivity from the total administered activity.

[0215] Visualization

[0216] Images were acquired using a small animal Nano-SPECT / CT system (Bioscan, Washington, DC, USA). This system consists of four detectors (215×230 ​​mm 2NaI, 33 PMTs) equipped with replaceable multi-spot holes. SPECT / CT images were obtained after intravenous administration of 5-15 MBq (mega-Becquerel) of the radiolabeled molecule at different times (24 h, 48 h, 72 h, and 96 h). CT (tube voltage 55 kV, exposure 500 ms, and 180 projections) and SPECT / CT images were acquired in tumor-bearing mice in the supine position on a temperature-controlled couch (Minerve, Esternay, France) to maintain body temperature (set to 37°C). Data were recorded for 40 minutes with two 15% windows targeting the two peaks 111 In at 171 keV and 245 keV. All image data were reconstructed and analyzed using InVivo-Scope (Bioscan, Washington, DC, USA).

[0217] Obtaining Fab and F(ab′)2 fragments and synthesizing immunoconjugates with NODAGA and DOTA

[0218] NP137 proteolytic fragments were prepared using Pierce™ Fab and F(ab′)2 kits according to the manufacturer's instructions. For conjugation of DOTA or NODAGA to surface lysine residues, NP137 and its fragments were conjugated at a chelator:antibody molar ratio of 25:1 with DOTA NHS ester or NODAGA NHS ester (Chematech, Dijon, France) in metal-free buffers prepared using Chelex 100 resin. Briefly, buffer exchange was performed by diafiltration of 50 μM antibodies against 0.1 M phosphate buffer (pH 8) and then reaction with 1.25 mM DOTA NHS ester or NODAGA NHS ester for 4 h at 25°C on a rotator. The reaction mixture was then transferred to 4°C for continuous stirring overnight. Excess chelator was removed by diafiltration against PBS. Immunoconjugates were stored at 4°C.

[0219] Determination of antibody affinity

[0220] The affinity of antibody fragments to netrin-1 was determined by biolayer interferometry using an OctetRed96 system (ForteBio) at 30°C and constant shaking at 1000 rpm in PBS, 0.02% Tween-20, 0.1% BSA (BB). Briefly, recombinant human netrin-1-coated HIS1K biosensors (R&D) were incubated with a series of antibody or fragment concentrations, and association was recorded for 5 minutes. The biosensors were then incubated in BB for an additional 5 minutes, recording the dissociation of the complex. Binding kinetics were determined using the ForteBio Octet RED Evaluation 6.1 software on a 1:1 binding model, obtaining k values. on , k off and K D .

[0221] Results

[0222] Netrin-1 is a poorly diffusing matrix-binding protein

[0223] Immunohistochemistry (IHC) has long been the standard for characterizing target expression in cancer. However, this strategy has recently been challenged by recent data obtained with immune checkpoint inhibitors, as there is a significant discrepancy between target expression and patient response. For example, patients who respond to an anti-PDL-1 antibody may be negative for PDL-1 expression by IHC, and vice versa. This suggests that target expression is unstable over time, given that IHC is performed on paraffin blocks obtained at diagnosis of the primary tumor, while target expression differs in metastases. Therefore, new diagnostic strategies for real-time target expression analysis on a whole-body scale are needed to identify all the differences in protein expression between tumors and metastases.

[0224] The present inventors discovered that netrin-1 does not diffuse into tumor cells as predicted by the axonal outgrowth model. The inventors imaged netrin-1 using immunohistochemistry on paraffin-embedded tumor sections of human endometrial and ovarian tumors (data not shown). Netrin-1 in human tumors was found to be present in the basement membrane of cells after IHC staining, indicating its accumulation in the extracellular matrix of cells. To complete and confirm these new findings, the inventors characterized the molecular partners of netrin-1 in matrix components. The interaction of netrin-1 with matrix proteins was screened using biolayer interferometry (BLI). Netrin-1 was found to strongly bind to fibronectin, laminin, and vitronectin (Fig. 1).

[0225] Heparin blocks the interaction of netrin-1 with plastic materials. Although netrin-1 was not detected in the conditioned medium of netrin-1-expressing 4T1 / EMT6 cells under conditions without heparin treatment, netrin-1 was detected with the addition of heparin (Fig. 7).

[0226] All these data indicate that netrin-1 is sequestered in the extracellular matrix of cancer cells but does not diffuse.

[0227] Characteristics of a new companion test for real-time detection of netrin-1

[0228] Compounds were obtained in which NP137 fragments (Fab or F(ab′)2) were linked to indium-111 ( 111In) for detection by SPECT / CT molecular imaging (Fig. 2a). The three resulting molecules are believed to differ in their molecular activity in vivo: full antibodies have a longer half-life in the bloodstream, Fab are able to penetrate tumors faster, and F(ab′)2 is an intermediate form (Fig. 2b). More precisely, the antibody or its fragment was conjugated with a metal chelator (DOTA or NODAGA), which binds to lysine residues of the antibody or its fragment, and the chelator itself binds to an indium isotope, forming a radioactive tracer. After purification by steric exclusion chromatography, radiolabeled NP-137, Fab, and F(ab′)2 were obtained with a radiochemical purity (RCP) exceeding 98%. The radiochemical yield was 70% for 111 In-NODAGA-NP137, 60% for 111 In-NODAGA-NP137-Fab and 65% for 111In-NODAGA-NP137-F(ab′)2. After 5 days of incubation, the RCP still exceeded 95% in phosphate-buffered saline (pH 7.4), indicating adequate kinetic stability for in vitro and in vivo experiments. The authors demonstrated using biolayer interferometry that this chemical modification, necessary for isotope binding, does not impair the ability of these three forms to bind netrin-1 (Fig. 2d).

[0229] Calculations K D After analyzing the experiments shown in Fig. 2d using the biolayer interferometry method, the following values ​​of K were obtained D with high affinity:

[0230] NP137-NODAGA: 1.72×10 -10 ,

[0231] F(ab′)2-NODAGA: 1.51×10 -10 ,

[0232] Fab-NODAGA: 1.52×10 -10 .

[0233] To analyze the ability of these molecules to detect netrin-1 in vivo, two syngeneic tumor models were used: 4T1 cells, positive for netrin-1 expression, and 67NR cells, negative for netrin-1, as a negative control.

[0234] First, netrin-1 expression was quantitatively analyzed by Q-RT-PCR on 4T1 and 67NR cells. The results in Fig. 3a confirm that netrin-1 expression is observed only in 4T1 cells.

[0235] Second, the maximum intensity projections of the whole body tomographic scintigraphy and X-ray CT of the Balb / cJ mouse bearing 4T1 tumor (netrin-1 positive) were obtained at 24 h, 48 h, 72 h, and 96 h after intravenous administration 111 In-NODAGA-NP137-F(ab′)2, 111 In-NODAGA-NP137-Fab or 111In-NODAGA-NP137. Similarly, maximum intensity projections were obtained from tomographic scintigraphy and whole-body X-ray CT of Balb / c mice bearing 67NR tumor (netrin-1 negative) at 24 h, 48 h, and 72 h after intravenous administration. 111 In-NODAGA-NP137-F(ab′)2, 111 In-NODAGA-NP137-Fab or 111 In-NODAGA-NP137.

[0236] Strong tumor uptake is found in 4T1 tumors in the group 111 In-NODAGA-NP137 (Fig. 3b), whereas in the group 111 In-NODAGA-NP137-F(ab′)2i in the group 111 In-NODAGA-NP137-Fab exhibited slower uptake (data not shown). Interestingly, mice bearing 67NR tumors did not show uptake of all molecules, suggesting that tumor uptake is specific for tumor netrin-1 (compare Figs. 3B and 3C). Localized tumor uptake is also seen in the H358 tumor in Fig. 8.

[0237] The authors performed a quantitative analysis of the ratio of uptake in 67NR and 4T1 cells ex vivo, which indicates that the best specific incorporation is detected 48 hours after treatment (Fig. 4a-c). These data confirm that tumor incorporation will be better with the whole antibody than with other formulations.

[0238] Biodistribution parameters were measured 111 In-NODAGA-NP137 was administered to Balb / c mice bearing 4T1 xenografts at 48, 72, and 96 hours in all organs. Radioactivity incorporation was determined as a percentage of the administered dose per gram of organ. At the indicated time points, tumors showed strong uptake, close to 25% of the administered dose (ID). No nonspecific binding was observed when incorporating into other organs (Fig. 4d).

[0239] New diagnostic tool

[0240] Further study was performed on the MMTV-NeuT transgenic breast cancer model (20) expressing endogenous levels of netrin-1. Figure 5a shows the time evolution, from left to right, at 24 h, 48 h and 72 h after injection. 111 In-NODAGA-NP137.

[0241] Strong staining was detected in the fat pad tissue, namely in 10 mammary glands of the animals (see Fig. 5b).

[0242] It is very interesting to note that some tumors were visualized even before they were detected by breast palpation, confirming the relevance of this indicator as a tool for the early detection of netrin-1-expressing tumors. This result was unexpected. Strong tumor uptake, close to 8% of the administered dose (ID), was detected after measuring incorporation in all tumors, indicating that 111In-NODAGA-NP137 may be a good diagnostic tool to characterize netrin-1 expression when a small lesion appears within the tumor mass (Fig. 5c).

[0243] Fig. 8 shows strong accumulation of NP137-NODAGA- 111 In a netrin-1-positive mouse model of human H358 xenografts (a model of human non-small cell lung cancer), similar results (not shown) were obtained with EMT6 cells (a mouse mammary carcinoma cell line). Tumor uptake in both models was greater than 10% of the administered dose per gram (ID / g).

[0244] 177 Lu-DOTA-NP137 - a new theranostic compound for targeting resistant tumors

[0245] The authors developed an antibody linked to lutetium-177 (in Fig. 6 it is designated DOTA-NP137- 177Lu), which is designed to emit β-radiation, effectively damaging cancer cells. Lutetium emits high doses of radiation in the 1.8 mm range with high specificity when combined with targeted therapy.

[0246] The authors initially used this molecule to treat 4T1 and EMT6 cell lines. These cell lines are resistant to NP137 monotherapy and are known to be the most aggressive preclinical models. However, tumor growth was reduced when treated with a single dose of 10 MBq NP137-DOTA- 177 Lu compared to the control groups receiving either PBS or DOTA-NP137 (Fig. 6a, c and e), with increased survival of mice in the NP137-DOTA-treated group. 177 Lu (Fig. 6b and d). These results indicate that this new molecule may exert antitumor activity in vivo on netrin-1-expressing tumors. The therapeutic efficacy of NP137- 177Lu was also evaluated in the H358 human lung cancer xenograft model, where treatment again significantly reduced tumor growth rate, confirming its clear antitumor effect (p<0.001) (Fig. 6f).

[0247] Discussion

[0248] This study describes a novel companion test for the in vivo detection of netrin-1 using nuclear medicine SPECT / CT. Netrin-1 has been studied as a therapeutic target in several cancer types in ongoing clinical trials, but due to the lack of a traditional detection method (i.e., serum detection by ELISA, mass spectrometry, reliable detection of netrin-1 in FFPE pathology samples), the authors developed an innovative, simple, and reliable companion test to detect high netrin-1 expression in vivo in cancer cells. Based on these results, the authors can state that no non-specific binding is detected in their preclinical models. 111 In-NODAGA-NP137 or111 In-DOTA-NP137 or very low, as evidenced by the RCP value in the 67NR model with netrin-1-negative tumors. These results indicate that netrin-1 is not widely expressed in adults and exhibits high tumor tissue specificity. Therefore, targeting netrin-1 developmental genes, which are re-expressed during tumor formation, is a key solution for improving tumor imaging specificity.

[0249] To identify the best molecule for PET or SPECT imaging, we developed three different probes based on the clinically used human anti-netrin-1 monoclonal antibody NP137: full-length NP137-IgG1, NP137-F(ab′)2, and NP137-Fab. All of these molecules are capable of tightly binding to netrin-1. The best in vivo accumulation with tumor specificity was observed for a radioactive tracer containing full-length NP137-IgG1 or NP137-Fab; the tracer containing the full-length antibody was the best. The full-length NP137-IgG1 antibody showed the highest accumulation in tumors and the most promising results for clinical translation. Such translation can be expected for all metals and compounds used in molecular imaging.

[0250] In terms of more basic research, netrin-1 has been described for many years during neuronal development as a secreted molecule with a diffusion gradient. Netrin-1 is a ligand, so it is not the best choice as a target for imaging or internal radiation therapy.

[0251] Furthermore, based on the demonstrated tumor incorporation, the authors developed a new molecule in which NP137-DOTA combines with lutetium-177 to form NP137-DOTA- 177Lu. As a result, this molecule also specifically accumulated in tumors expressing netrin-1. Lutetium 177 is a β-emitter capable of delivering a strong radiation dose in the 1.8 mm range to tumor tissue. A significant reduction in tumor growth was observed, correlating with improved survival in tumor-bearing mice. Notably, these tumor types were completely resistant to NP137 monotherapy. Survival studies showed that treatment with this compound doubled the survival of mice compared to controls in mouse tumor models and in a human tumor model. Since NP137 proved safe when used as monotherapy, and the doses 177 Lu is well studied, so this molecule can easily be used to treat tumors.

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[0584] <INSDFeature_quals>

[0585] <insdqualifier id="q18">

[0586] <INSDQualifier_name>note< / INSDQualifier_name>

[0587] <INSDQualifier_value>VL aa sequence of humanized variant of

[0588] 4C11< / INSDQualifier_value>

[0589] < / insdqualifier>

[0590] < / INSDFeature_quals>

[0591] < / insdfeature>

[0592] < / INSDSeq_feature-table>

[0593] <INSDSeq_sequence>DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWFQQRPGQSPRRLIY

[0594] YASNRYTGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQDYSSPWTFGQG< / INSDSeq_sequence

[0595] >

[0596] < / insdseq>

[0597] < / sequencedata>

[0598] <sequencedata sequenceidnumber="10">

[0599] <insdseq>

[0600] <INSDSeq_length>101< / INSDSeq_length>

[0601] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0602] <INSDSeq_division>PAT< / INSDSeq_division>

[0603] <INSDSeq_feature-table>

[0604] <insdfeature>

[0605] <INSDFeature_key>source< / INSDFeature_key>

[0606] <INSDFeature_location>1..101< / INSDFeature_location>

[0607] <INSDFeature_quals>

[0608] <insdqualifier>

[0609] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0610] <INSDQualifier_value>protein< / INSDQualifier_value>

[0611] < / insdqualifier>

[0612] <insdqualifier id="q19">

[0613] <INSDQualifier_name>organism< / INSDQualifier_name>

[0614] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0615] < / insdqualifier>

[0616] < / INSDFeature_quals>

[0617] < / insdfeature>

[0618] <insdfeature>

[0619] <INSDFeature_key>REGION< / INSDFeature_key>

[0620] <INSDFeature_location>1..101< / INSDFeature_location>

[0621] <INSDFeature_quals>

[0622] <insdqualifier id="q20">

[0623] <INSDQualifier_name>note< / INSDQualifier_name>

[0624] <INSDQualifier_value>VL aa sequence of humanized variant of

[0625] 4C11< / INSDQualifier_value>

[0626] < / insdqualifier>

[0627] < / INSDFeature_quals>

[0628] < / insdfeature>

[0629] < / INSDSeq_feature-table>

[0630] <INSDSeq_sequence>DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGQAPRLLIY

[0631] YASNRYTGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQDYSSPWTFGQG< / INSDSeq_sequence

[0632] >

[0633] < / insdseq>

[0634] < / sequencedata>

[0635] <sequencedata sequenceidnumber="11">

[0636] <insdseq>

[0637] <INSDSeq_length>101< / INSDSeq_length>

[0638] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0639] <INSDSeq_division>PAT< / INSDSeq_division>

[0640] <INSDSeq_feature-table>

[0641] <insdfeature>

[0642] <INSDFeature_key>source< / INSDFeature_key>

[0643] <INSDFeature_location>1..101< / INSDFeature_location>

[0644] <INSDFeature_quals>

[0645] <insdqualifier>

[0646] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0647] <INSDQualifier_value>protein< / INSDQualifier_value>

[0648] < / insdqualifier>

[0649] <insdqualifier id="q21">

[0650] <INSDQualifier_name>organism< / INSDQualifier_name>

[0651] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0652] < / insdqualifier>

[0653] < / INSDFeature_quals>

[0654] < / insdfeature>

[0655] <insdfeature>

[0656] <INSDFeature_key>REGION< / INSDFeature_key>

[0657] <INSDFeature_location>1..101< / INSDFeature_location>

[0658] <INSDFeature_quals>

[0659] <insdqualifier id="q22">

[0660] <INSDQualifier_name>note< / INSDQualifier_name>

[0661] <INSDQualifier_value>VL aa sequence of humanized variant of

[0662] 4C11< / INSDQualifier_value>

[0663] < / insdqualifier>

[0664] < / INSDFeature_quals>

[0665] < / insdfeature>

[0666] < / INSDSeq_feature-table>

[0667] <INSDSeq_sequence>DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYLQKPGQSPQLLIY

[0668] YASNRYTGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQDYSSPWTFGQG< / INSDSeq_sequence

[0669] >

[0670] < / insdseq>

[0671] < / sequencedata>

[0672] <sequencedata sequenceidnumber="12">

[0673] <insdseq>

[0674] <INSDSeq_length> 101< / INSDSeq_length>

[0675] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0676] <INSDSeq_division> PAT< / INSDSeq_division>

[0677] <INSDSeq_feature-table>

[0678] <insdfeature>

[0679] <INSDFeature_key>source< / INSDFeature_key>

[0680] <INSDFeature_location>1..101< / INSDFeature_location>

[0681] <INSDFeature_quals>

[0682] <insdqualifier>

[0683] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0684] <INSDQualifier_value>protein< / INSDQualifier_value>

[0685] < / insdqualifier>

[0686] <insdqualifier id="q23">

[0687] <INSDQualifier_name>organism< / INSDQualifier_name>

[0688] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0689] < / insdqualifier>

[0690] < / INSDFeature_quals>

[0691] < / insdfeature>

[0692] <insdfeature>

[0693] <INSDFeature_key>REGION< / INSDFeature_key>

[0694] <INSDFeature_location>1..101< / INSDFeature_location>

[0695] <INSDFeature_quals>

[0696] <insdqualifier id="q24">

[0697] <INSDQualifier_name>note< / INSDQualifier_name>

[0698] <INSDQualifier_value>VL aa sequence of humanized variant of

[0699] 4C11< / INSDQualifier_value>

[0700] < / insdqualifier>

[0701] < / INSDFeature_quals>

[0702] < / insdfeature>

[0703] < / INSDSeq_feature-table>

[0704] <INSDSeq_sequence> DIVMTQTPLSLPVTPGEPASISCKASQSVSNDVAWYQQKPGQAPRLLIY

[0705] YASNRYTGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQDYSSPWTFGQG < / INSDSeq_sequence

[0706] >

[0707] < / insdseq>

[0708] < / sequencedata>

[0709] <sequencedata sequenceidnumber="13">

[0710] <insdseq>

[0711] <INSDSeq_length> 101< / INSDSeq_length>

[0712] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0713] <INSDSeq_division> PAT< / INSDSeq_division>

[0714] <INSDSeq_feature-table>

[0715] <insdfeature>

[0716] <INSDFeature_key>source< / INSDFeature_key>

[0717] <INSDFeature_location>1..101< / INSDFeature_location>

[0718] <INSDFeature_quals>

[0719] <insdqualifier>

[0720] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0721] <INSDQualifier_value>protein< / INSDQualifier_value>

[0722] < / insdqualifier>

[0723] <insdqualifier id="q25">

[0724] <INSDQualifier_name>organism< / INSDQualifier_name>

[0725] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0726] < / insdqualifier>

[0727] < / INSDFeature_quals>

[0728] < / insdfeature>

[0729] <insdfeature>

[0730] <INSDFeature_key>REGION< / INSDFeature_key>

[0731] <INSDFeature_location>1..101< / INSDFeature_location>

[0732] <INSDFeature_quals>

[0733] <insdqualifier id="q26">

[0734] <INSDQualifier_name>note< / INSDQualifier_name>

[0735] <INSDQualifier_value>VL aa sequence of humanized variant of

[0736] 4C11< / INSDQualifier_value>

[0737] < / insdqualifier>

[0738] < / INSDFeature_quals>

[0739] < / insdfeature>

[0740] < / INSDSeq_feature-table>

[0741] <INSDSeq_sequence> EIVMTQSPATLSVSPGERATLSCRASQSVSNDVAWYQQKPGQAPRLLIY

[0742] YASNRYTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDYSSPWTFGQG < / INSDSeq_sequence

[0743] >

[0744] < / insdseq>

[0745] < / sequencedata>

[0746] <sequencedata sequenceidnumber="14">

[0747] <insdseq>

[0748] <INSDSeq_length>109< / INSDSeq_length>

[0749] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0750] <INSDSeq_division>PAT< / INSDSeq_division>

[0751] <INSDSeq_feature-table>

[0752] <insdfeature>

[0753] <INSDFeature_key>source< / INSDFeature_key>

[0754] <INSDFeature_location>1..109< / INSDFeature_location>

[0755] <INSDFeature_quals>

[0756] <insdqualifier>

[0757] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0758] <INSDQualifier_value>protein< / INSDQualifier_value>

[0759] < / insdqualifier>

[0760] <insdqualifier id="q27">

[0761] <INSDQualifier_name>organism< / INSDQualifier_name>

[0762] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0763] < / insdqualifier>

[0764] < / INSDFeature_quals>

[0765] < / insdfeature>

[0766] <insdfeature>

[0767] <INSDFeature_key>REGION< / INSDFeature_key>

[0768] <INSDFeature_location>1..109< / INSDFeature_location>

[0769] <INSDFeature_quals>

[0770] <insdqualifier id="q28">

[0771] <INSDQualifier_name>note< / INSDQualifier_name>

[0772] <INSDQualifier_value>VH aa sequence of humanized variant of

[0773] 4C11< / INSDQualifier_value>

[0774] < / insdqualifier>

[0775] < / INSDFeature_quals>

[0776] < / insdfeature>

[0777] < / INSDSeq_feature-table>

[0778] <INSDSeq_sequence>QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQATGQGLEWMG

[0779] AIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGTGFAYWGQG< / INSDSeq_

[0780] sequence>

[0781] < / insdseq>

[0782] < / sequencedata>

[0783] <sequencedata sequenceidnumber="15">

[0784] <insdseq>

[0785] <INSDSeq_length>109< / INSDSeq_length>

[0786] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0787] <INSDSeq_division>PAT< / INSDSeq_division>

[0788] <INSDSeq_feature-table>

[0789] <insdfeature>

[0790] <INSDFeature_key>source< / INSDFeature_key>

[0791] <INSDFeature_location>1..109< / INSDFeature_location>

[0792] <INSDFeature_quals>

[0793] <insdqualifier>

[0794] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0795] <INSDQualifier_value>protein< / INSDQualifier_value>

[0796] < / insdqualifier>

[0797] <insdqualifier id="q29">

[0798] <INSDQualifier_name>organism< / INSDQualifier_name>

[0799] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0800] < / insdqualifier>

[0801] < / INSDFeature_quals>

[0802] < / insdfeature>

[0803] <insdfeature>

[0804] <INSDFeature_key>REGION< / INSDFeature_key>

[0805] <INSDFeature_location>1..109< / INSDFeature_location>

[0806] <INSDFeature_quals>

[0807] <insdqualifier id="q30">

[0808] <INSDQualifier_name>note< / INSDQualifier_name>

[0809] <INSDQualifier_value>VH aa sequence of humanized variant of

[0810] 4C11< / INSDQualifier_value>

[0811] < / insdqualifier>

[0812] < / INSDFeature_quals>

[0813] < / insdfeature>

[0814] < / INSDSeq_feature-table>

[0815] <INSDSeq_sequence>QVQLQQSGPGLVKPSQTLSLTCAISGYTFTSYNMHWIRQPPGKGLEWIG

[0816] AIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGTGFAYWGQG< / INSDSeq_

[0817] sequence>

[0818] < / insdseq>

[0819] < / sequencedata>

[0820] <sequencedata sequenceidnumber="16">

[0821] <insdseq>

[0822] <INSDSeq_length>109< / INSDSeq_length>

[0823] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0824] <INSDSeq_division>PAT< / INSDSeq_division>

[0825] <INSDSeq_feature-table>

[0826] <insdfeature>

[0827] <INSDFeature_key>source< / INSDFeature_key>

[0828] <INSDFeature_location>1..109< / INSDFeature_location>

[0829] <INSDFeature_quals>

[0830] <insdqualifier>

[0831] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0832] <INSDQualifier_value>protein< / INSDQualifier_value>

[0833] < / insdqualifier>

[0834] <insdqualifier id="q31">

[0835] <INSDQualifier_name>organism< / INSDQualifier_name>

[0836] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0837] < / insdqualifier>

[0838] < / INSDFeature_quals>

[0839] < / insdfeature>

[0840] <insdfeature>

[0841] <INSDFeature_key>REGION< / INSDFeature_key>

[0842] <INSDFeature_location>1..109< / INSDFeature_location>

[0843] <INSDFeature_quals>

[0844] <insdqualifier id="q32">

[0845] <INSDQualifier_name>note< / INSDQualifier_name>

[0846] <INSDQualifier_value>VH aa sequence of humanized variant of

[0847] 4C11< / INSDQualifier_value>

[0848] < / insdqualifier>

[0849] < / INSDFeature_quals>

[0850] < / insdfeature>

[0851] < / INSDSeq_feature-table>

[0852] <INSDSeq_sequence>QVQLQQSGPGLVKPSQTLSLTCAISGYTFTSYNMHWVRQATGQGLEWMG

[0853] AIYPGNGDTSYNQKFKGRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGTGFAYWGQG< / INSDSeq_

[0854] sequence>

[0855] < / insdseq>

[0856] < / sequencedata>

[0857] <sequencedata sequenceidnumber="17">

[0858] <insdseq>

[0859] <INSDSeq_length>109< / INSDSeq_length>

[0860] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0861] <INSDSeq_division>PAT< / INSDSeq_division>

[0862] <INSDSeq_feature-table>

[0863] <insdfeature>

[0864] <INSDFeature_key>source< / INSDFeature_key>

[0865] <INSDFeature_location>1..109< / INSDFeature_location>

[0866] <INSDFeature_quals>

[0867] <insdqualifier>

[0868] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0869] <INSDQualifier_value>protein< / INSDQualifier_value>

[0870] < / insdqualifier>

[0871] <insdqualifier id="q33">

[0872] <INSDQualifier_name>organism< / INSDQualifier_name>

[0873] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0874] < / insdqualifier>

[0875] < / INSDFeature_quals>

[0876] < / insdfeature>

[0877] <insdfeature>

[0878] <INSDFeature_key>REGION< / INSDFeature_key>

[0879] <INSDFeature_location>1..109< / INSDFeature_location>

[0880] <INSDFeature_quals>

[0881] <insdqualifier id="q34">

[0882] <INSDQualifier_name>note< / INSDQualifier_name>

[0883] <INSDQualifier_value>VH aa sequence of humanized variant of

[0884] 4C11< / INSDQualifier_value>

[0885] < / insdqualifier>

[0886] < / INSDFeature_quals>

[0887] < / insdfeature>

[0888] < / INSDSeq_feature-table>

[0889] <INSDSeq_sequence>EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYNMHWVRQATGQGLEWMG

[0890] AIYPGNGDTSYNQKFKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARGGTGFAYWGQG< / INSDSeq_

[0891] sequence>

[0892] < / insdseq>

[0893] < / sequencedata>

[0894] <sequencedata sequenceidnumber="18">

[0895] <insdseq>

[0896] <INSDSeq_length>109< / INSDSeq_length>

[0897] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0898] <INSDSeq_division>PAT< / INSDSeq_division>

[0899] <INSDSeq_feature-table>

[0900] <insdfeature>

[0901] <INSDFeature_key>source< / INSDFeature_key>

[0902] <INSDFeature_location>1..109< / INSDFeature_location>

[0903] <INSDFeature_quals>

[0904] <insdqualifier>

[0905] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0906] <INSDQualifier_value>protein< / INSDQualifier_value>

[0907] < / insdqualifier>

[0908] <insdqualifier id="q35">

[0909] <INSDQualifier_name>organism< / INSDQualifier_name>

[0910] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0911] < / insdqualifier>

[0912] < / INSDFeature_quals>

[0913] < / insdfeature>

[0914] <insdfeature>

[0915] <INSDFeature_key>REGION< / INSDFeature_key>

[0916] <INSDFeature_location>1..109< / INSDFeature_location>

[0917] <INSDFeature_quals>

[0918] <insdqualifier id="q36">

[0919] <INSDQualifier_name>note< / INSDQualifier_name>

[0920] <INSDQualifier_value>VH aa sequence of humanized variant of

[0921] 4C11< / INSDQualifier_value>

[0922] < / insdqualifier>

[0923] < / INSDFeature_quals>

[0924] < / insdfeature>

[0925] < / INSDSeq_feature-table>

[0926] <INSDSeq_sequence>QVQLQESGPGLVKPSQTLSLTCTVSGYTFTSYNMHWVRQAPGQGLEWMG

[0927] AIYPGNGDTSYNQKFKGRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGTGFAYWGQG< / INSDSeq_

[0928] sequence>

[0929] < / insdseq>

[0930] < / sequencedata>

[0931] <sequencedata sequenceidnumber="19">

[0932] <insdseq>

[0933] <INSDSeq_length>109< / INSDSeq_length>

[0934] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0935] <INSDSeq_division>PAT< / INSDSeq_division>

[0936] <INSDSeq_feature-table>

[0937] <insdfeature>

[0938] <INSDFeature_key>source< / INSDFeature_key>

[0939] <INSDFeature_location>1..109< / INSDFeature_location>

[0940] <INSDFeature_quals>

[0941] <insdqualifier>

[0942] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0943] <INSDQualifier_value>protein< / INSDQualifier_value>

[0944] < / insdqualifier>

[0945] <insdqualifier id="q37">

[0946] <INSDQualifier_name>organism< / INSDQualifier_name>

[0947] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0948] < / insdqualifier>

[0949] < / INSDFeature_quals>

[0950] < / insdfeature>

[0951] <insdfeature>

[0952] <INSDFeature_key>REGION< / INSDFeature_key>

[0953] <INSDFeature_location>1..109< / INSDFeature_location>

[0954] <INSDFeature_quals>

[0955] <insdqualifier id="q38">

[0956] <INSDQualifier_name>note< / INSDQualifier_name>

[0957] <INSDQualifier_value>VH aa sequence of humanized variant of

[0958] 4C11< / INSDQualifier_value>

[0959] < / insdqualifier>

[0960] < / INSDFeature_quals>

[0961] < / insdfeature>

[0962] < / INSDSeq_feature-table>

[0963] <INSDSeq_sequence>QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQATGQGLEWMG

[0964] AIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGTGFAYWGQG< / INSDSeq_

[0965] sequence>

[0966] < / insdseq>

[0967] < / sequencedata>

[0968] <sequencedata sequenceidnumber="20">

[0969] <insdseq>

[0970] <INSDSeq_length>109< / INSDSeq_length>

[0971] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0972] <INSDSeq_division>PAT< / INSDSeq_division>

[0973] <INSDSeq_feature-table>

[0974] <insdfeature>

[0975] <INSDFeature_key>source< / INSDFeature_key>

[0976] <INSDFeature_location>1..109< / INSDFeature_location>

[0977] <INSDFeature_quals>

[0978] <insdqualifier>

[0979] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0980] <INSDQualifier_value>protein< / INSDQualifier_value>

[0981] < / insdqualifier>

[0982] <insdqualifier id="q39">

[0983] <INSDQualifier_name>organism< / INSDQualifier_name>

[0984] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0985] < / insdqualifier>

[0986] < / INSDFeature_quals>

[0987] < / insdfeature>

[0988] <insdfeature>

[0989] <INSDFeature_key>REGION< / INSDFeature_key>

[0990] <INSDFeature_location>1..109< / INSDFeature_location>

[0991] <INSDFeature_quals>

[0992] <insdqualifier id="q40">

[0993] <INSDQualifier_name>note< / INSDQualifier_name>

[0994] <INSDQualifier_value>VH aa sequence of humanized variant of

[0995] 4C11< / INSDQualifier_value>

[0996] < / insdqualifier>

[0997] < / INSDFeature_quals>

[0998] < / insdfeature>

[0999] < / INSDSeq_feature-table>

[1000] <INSDSeq_sequence>QVQLQQSGPGLVKPSQTLSLTCAISGYTFTSYNMHWVRQATGQGLEWMG

[1001] AIYPGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGTGFAYWGQG< / INSDSeq_

[1002] sequence>

[1003] < / insdseq>

[1004] < / sequencedata>

[1005] <sequencedata sequenceidnumber="21">

[1006] <insdseq>

[1007] <INSDSeq_length>108< / INSDSeq_length>

[1008] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1009] <INSDSeq_division>PAT< / INSDSeq_division>

[1010] <INSDSeq_feature-table>

[1011] <insdfeature>

[1012] <INSDFeature_key>source< / INSDFeature_key>

[1013] <INSDFeature_location>1..108< / INSDFeature_location>

[1014] <INSDFeature_quals>

[1015] <insdqualifier>

[1016] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1017] <INSDQualifier_value>protein< / INSDQualifier_value>

[1018] < / insdqualifier>

[1019] <insdqualifier id="q41">

[1020] <INSDQualifier_name>organism< / INSDQualifier_name>

[1021] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1022] < / insdqualifier>

[1023] < / INSDFeature_quals>

[1024] < / insdfeature>

[1025] <insdfeature>

[1026] <INSDFeature_key>REGION< / INSDFeature_key>

[1027] <INSDFeature_location>1..108< / INSDFeature_location>

[1028] <INSDFeature_quals>

[1029] <insdqualifier id="q42">

[1030] <INSDQualifier_name>note< / INSDQualifier_name>

[1031] <INSDQualifier_value>VH aa sequence of humanized variant of

[1032] 4C11< / INSDQualifier_value>

[1033] < / insdqualifier>

[1034] < / INSDFeature_quals>

[1035] < / insdfeature>

[1036] < / INSDSeq_feature-table>

[1037] <INSDSeq_sequence>QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMG

[1038] AIYPGNGDTSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGTGFAYWGQ< / INSDSeq_s

[1039] equence>

[1040] < / insdseq>

[1041] < / sequencedata>

[1042] <sequencedata sequenceidnumber="22">

[1043] <insdseq>

[1044] <INSDSeq_length> 5< / INSDSeq_length>

[1045] <INSDSeq_moltype> SECOND< / INSDSeq_moltype>

[1046] <INSDSeq_division> PAT< / INSDSeq_division>

[1047] <INSDSeq_feature-table>

[1048] <insdfeature>

[1049] <INSDFeature_key>source< / INSDFeature_key>

[1050] <INSDFeature_location>1..5< / INSDFeature_location>

[1051] <INSDFeature_quals>

[1052] <insdqualifier>

[1053] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1054] <INSDQualifier_value>protein< / INSDQualifier_value>

[1055] < / insdqualifier>

[1056] <insdqualifier id="q43">

[1057] <INSDQualifier_name>organism< / INSDQualifier_name>

[1058] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1059] < / insdqualifier>

[1060] < / INSDFeature_quals>

[1061] < / insdfeature>

[1062] <insdfeature>

[1063] <INSDFeature_key>REGION< / INSDFeature_key>

[1064] <INSDFeature_location>1..5< / INSDFeature_location>

[1065] <INSDFeature_quals>

[1066] <insdqualifier id="q44">

[1067] <INSDQualifier_name>note< / INSDQualifier_name>

[1068] <INSDQualifier_value>aa seq. of CDR1-H

[1069] (Kabat)< / INSDQualifier_value>

[1070] < / insdqualifier>

[1071] < / INSDFeature_quals>

[1072] < / insdfeature>

[1073] < / INSDSeq_feature-table>

[1074] <INSDSeq_sequence> SYNMH< / INSDSeq_sequence>

[1075] < / insdseq>

[1076] < / sequencedata>

[1077] <sequencedata sequenceidnumber="23">

[1078] <insdseq>

[1079] <INSDSeq_length> 17< / INSDSeq_length>

[1080] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1081] <INSDSeq_division> PAT< / INSDSeq_division>

[1082] <INSDSeq_feature-table>

[1083] <insdfeature>

[1084] <INSDFeature_key>source< / INSDFeature_key>

[1085] <INSDFeature_location>1..17< / INSDFeature_location>

[1086] <INSDFeature_quals>

[1087] <insdqualifier>

[1088] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1089] <INSDQualifier_value>protein< / INSDQualifier_value>

[1090] < / insdqualifier>

[1091] <insdqualifier id="q45">

[1092] <INSDQualifier_name>organism< / INSDQualifier_name>

[1093] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1094] < / insdqualifier>

[1095] < / INSDFeature_quals>

[1096] < / insdfeature>

[1097] <insdfeature>

[1098] <INSDFeature_key>REGION< / INSDFeature_key>

[1099] <INSDFeature_location>1..17< / INSDFeature_location>

[1100] <INSDFeature_quals>

[1101] <insdqualifier id="q46">

[1102] <INSDQualifier_name>note< / INSDQualifier_name>

[1103] <INSDQualifier_value>aa seq. of CDR2-H

[1104] (Kabat)< / INSDQualifier_value>

[1105] < / insdqualifier>

[1106] < / INSDFeature_quals>

[1107] < / insdfeature>

[1108] < / INSDSeq_feature-table>

[1109] <INSDSeq_sequence> AIYPGNGDTSYNQKFKG< / INSDSeq_sequence>

[1110] < / insdseq>

[1111] < / sequencedata>

[1112] <sequencedata sequenceidnumber="24">

[1113] <insdseq>

[1114] <INSDSeq_length> 7< / INSDSeq_length>

[1115] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1116] <INSDSeq_division> PAT< / INSDSeq_division>

[1117] <INSDSeq_feature-table>

[1118] <insdfeature>

[1119] <INSDFeature_key>source< / INSDFeature_key>

[1120] <INSDFeature_location>1..7< / INSDFeature_location>

[1121] <INSDFeature_quals>

[1122] <insdqualifier>

[1123] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1124] <INSDQualifier_value>protein< / INSDQualifier_value>

[1125] < / insdqualifier>

[1126] <insdqualifier id="q47">

[1127] <INSDQualifier_name>organism< / INSDQualifier_name>

[1128] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1129] < / insdqualifier>

[1130] < / INSDFeature_quals>

[1131] < / insdfeature>

[1132] <insdfeature>

[1133] <INSDFeature_key>REGION< / INSDFeature_key>

[1134] <INSDFeature_location>1..7< / INSDFeature_location>

[1135] <INSDFeature_quals>

[1136] <insdqualifier id="q48">

[1137] <INSDQualifier_name>note< / INSDQualifier_name>

[1138] <INSDQualifier_value>aa seq. of CDR3-H

[1139] (Kabat)< / INSDQualifier_value>

[1140] < / insdqualifier>

[1141] < / INSDFeature_quals>

[1142] < / insdfeature>

[1143] < / INSDSeq_feature-table>

[1144] <INSDSeq_sequence> GGTGFAY< / INSDSeq_sequence>

[1145] < / insdseq>

[1146] < / sequencedata>

[1147] <sequencedata sequenceidnumber="25">

[1148] <insdseq>

[1149] <INSDSeq_length> 11< / INSDSeq_length>

[1150] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1151] <INSDSeq_division> PAT< / INSDSeq_division>

[1152] <INSDSeq_feature-table>

[1153] <insdfeature>

[1154] <INSDFeature_key>source< / INSDFeature_key>

[1155] <INSDFeature_location>1..11< / INSDFeature_location>

[1156] <INSDFeature_quals>

[1157] <insdqualifier>

[1158] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1159] <INSDQualifier_value>protein< / INSDQualifier_value>

[1160] < / insdqualifier>

[1161] <insdqualifier id="q49">

[1162] <INSDQualifier_name>organism< / INSDQualifier_name>

[1163] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1164] < / insdqualifier>

[1165] < / INSDFeature_quals>

[1166] < / insdfeature>

[1167] <insdfeature>

[1168] <INSDFeature_key>REGION< / INSDFeature_key>

[1169] <INSDFeature_location>1..11< / INSDFeature_location>

[1170] <INSDFeature_quals>

[1171] <insdqualifier id="q50">

[1172] <INSDQualifier_name>note< / INSDQualifier_name>

[1173] <INSDQualifier_value>aa seq. of CDR1-L

[1174] (Kabat)< / INSDQualifier_value>

[1175] < / insdqualifier>

[1176] < / INSDFeature_quals>

[1177] < / insdfeature>

[1178] < / INSDSeq_feature-table>

[1179] <INSDSeq_sequence> KASQSVSNDVA< / INSDSeq_sequence>

[1180] < / insdseq>

[1181] < / sequencedata>

[1182] <sequencedata sequenceidnumber="26">

[1183] <insdseq>

[1184] <INSDSeq_length> 7< / INSDSeq_length>

[1185] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1186] <INSDSeq_division> PAT< / INSDSeq_division>

[1187] <INSDSeq_feature-table>

[1188] <insdfeature>

[1189] <INSDFeature_key>REGION< / INSDFeature_key>

[1190] <INSDFeature_location>1..7< / INSDFeature_location>

[1191] <INSDFeature_quals>

[1192] <insdqualifier id="q51">

[1193] <INSDQualifier_name>note< / INSDQualifier_name>

[1194] <INSDQualifier_value>aa seq. of CDR2-L

[1195] (Kabat)< / INSDQualifier_value>

[1196] < / insdqualifier>

[1197] < / INSDFeature_quals>

[1198] < / insdfeature>

[1199] <insdfeature>

[1200] <INSDFeature_key>source< / INSDFeature_key>

[1201] <INSDFeature_location>1..7< / INSDFeature_location>

[1202] <INSDFeature_quals>

[1203] <insdqualifier>

[1204] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1205] <INSDQualifier_value>protein< / INSDQualifier_value>

[1206] < / insdqualifier>

[1207] <insdqualifier id="q52">

[1208] <INSDQualifier_name>organism< / INSDQualifier_name>

[1209] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1210] < / insdqualifier>

[1211] < / INSDFeature_quals>

[1212] < / insdfeature>

[1213] < / INSDSeq_feature-table>

[1214] <INSDSeq_sequence> YASNRYT< / INSDSeq_sequence>

[1215] < / insdseq>

[1216] < / sequencedata>

[1217] < / st26sequencelisting>

[1218] <---

Claims

1. A netrin-1 binding compound comprising: an antibody against netrin-1 or its antigen-binding fragment and a chelating group associated with a given antibody or fragment, wherein the chelating group is linked to a radioisotope, and the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising: variable domain VH, including: – H-CDR1 with the sequence given in SEQ ID NO: 1; – H-CDR2 with the sequence given in SEQ ID NO: 2; – H-CDR3 with the sequence given in SEQ ID NO: 3; variable domain VL, including: – L-CDR1 with the sequence given in SEQ ID NO: 4; – L-CDR2 with YAS sequence; – L-CDR3 with the sequence given in SEQ ID NO: 5; or variable domain VH, including: – H-CDR1 with the sequence given in SEQ ID NO: 22; – H-CDR2 with the sequence given in SEQ ID NO: 23; – H-CDR3 with the sequence given in SEQ ID NO: 24; variable domain VL, including: – L-CDR1 with the sequence given in SEQ ID NO: 25; – L-CDR2 with the sequence given in SEQ ID NO: 26; – L-CDR3 with the sequence given in SEQ ID NO:

5.

2. The compound of claim 1, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof comprising the sequences of a VH and VL pair selected from the following pairs: SEQ ID NOs: 21 and 13, SEQ ID NOs: 14 and 8, SEQ ID NOs: 15 and 9, SEQ ID NOs: 16 and 10, SEQ ID NOs: 17 and 11, SEQ ID NOs: 18 and 11, SEQ ID NOs: 19 and 10, SEQ ID NOs: 20 and 11, SEQ ID NOs: 16 and 11, SEQ ID NOs: 19 and 12, SEQ ID NOs: 15 and 10.

3. The compound according to claim 2, wherein the antibody further comprises a constant region of the heavy chain (CH) of human IgG1 and / or a constant region of the light chain (CL) of human IgG1.

4. The compound of any one of claims 1 to 3, wherein the chelating group comprises NODAGA, NODAGA-NHS, DOTA, DOTA-NHS, p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, diethylenetriamine-pentaacetic acid (DTPA) or 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).

5. A compound according to any one of paragraphs 1-3, wherein the radioisotope is 68 Ga , 64 Cu, 89 Zr, 186 Re, 188 Re, 153 Sm, 111 In, 99m Tc, 123 I, 177 Lu, 90 Y, 131 I, 213 Bi, 212 Bi, 211 At or 225 Ac.

6. The compound of any one of claims 1 to 3, wherein the chelating group comprises NODAGA, NODAGA-NHS, DOTA, DOTA-NHS, p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, diethylenetriaminepentaacetic acid (DTPA) or 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), and the radioisotope is 68 Ga, 64 Cu, 89 Zr, 186 Re, 188 Re, 153 Sm, 111 In, 99m Tc, 123 I, 177 Lu, 90 Y, 131 I, 213 Bi, 212 Bi, 211 At or 225 Ac.

7. A method for visualizing the presence or localization of netrin-1 in a subject, comprising: a) administering to the subject a compound according to any one of paragraphs 1-5, comprising: an antibody against netrin-1 or its antigen-binding fragment, a chelating group associated with a given antibody or fragment, And a radioisotope bound to a chelating group, b) waiting 4 to 172 hours for the compound to bind to netrin-1 sequestered in the tumor extracellular matrix; c) detection or localization of the bound compound by in vivo imaging.

8. The method of claim 7, wherein localizing in step b) comprises detecting the presence or accumulation of the compound in at least one part of the body, such as an organ or tissue.

9. The method according to any one of paragraphs 7, 8, wherein the radioisotope is selected from the group consisting of 68 Ga, 64 Cu, 89 Zr, 186 Re, 188 Re, 153 Sm, 111 In, 99m Tc and 123 I.

10. Use of a compound comprising: an antibody against netrin-1 or its antigen-binding fragment, a chelating group associated with a given antibody or fragment, And a radioisotope bound to a chelating group, wherein the compound according to any one of claims 1 to 3, in the treatment of cancer expressing netrin-1 by internal radiation therapy.

11. The use according to paragraph 10, wherein the radioisotope is 177 Lu, 90 Y, 131 I, 213 Bi, 212 Bi, 211 At or 225 Ac.

12. A method of internal radiation therapy for treating a netrin-1 expressing cancer in a patient having such cancer, comprising administering a sufficient amount of a compound according to any one of claims 1-3.

13. The method according to claim 12, wherein the compound comprises a radioisotope selected from the group consisting of 177 Lu, 90 Y, 131 I, 213 Bi, 212 Bi, 211 At and 225 Ac.

14. A method for identifying and treating patients with netrin-1 expressing cancer, comprising: a) administering to the subject a compound according to any one of paragraphs 1-3, b) waiting from 4 to 172 hours for the compound to bind to netrin-1 sequestered in the tumor extracellular matrix, c) detection of the compound by in vivo imaging, d) visualization of the localization of the presence or accumulation of netrin-1, e) treatment of this patient for visualized cancer.

15. The method according to claim 14, wherein the compound that is introduced in step a) is a compound according to any one of claims 1-5, which is associated with a radioisotope selected from the group consisting of 68 Ga, 64 Cu, 89 Zr, 186 Re, 188 Re, 153 Sm, 111 In, 99m Tc and 123 I.

16. The method of claim 14, wherein treating the patient in step e) comprises administering to the patient an effective amount of a compound of any one of claims 1-5, which is associated with a radioisotope selected from the group consisting of 177 Lu, 90 Y, 131 I, 213 Bi,212 Bi, 211 At and 225 Ac.

17. The method of claim 14, wherein treating the patient in step e) comprises administering to the patient an effective amount of an antibody against netrin-1 or an antigen-binding fragment thereof as described in any one of claims 1-3.

Images