Pre-targeting

Abstract

The present invention relates to the field of VHHs that can be used in pretargeting combinations comprising a VHH and a labeling compound. Depending on the application, the labeling compound may comprise a radionuclide. The pretargeting combination is preferably for use in the treatment and / or diagnosis of cancer.

Description

Technical Field

[0001] The present invention relates to the field of VHHs that can be used in pretargeting combinations comprising a VHH and a labeling compound. Depending on the application, the labeling compound may comprise a radionuclide. The pretargeting combination is preferably for use in the treatment and / or diagnosis of cancer.

Background Art

[0002] Cancer is one of the main causes of morbidity and mortality worldwide. There is a continuing need for improved therapies to combat cancer while minimizing side effects. As such therapies, radiolabeled heavy chain variable domains derived from heavy chain antibodies (VHHs) have been proposed. However, some of these therapies have problems of high nephrotoxicity and low therapeutic index (Parihar, Ashwin Singh, Sejal Chopra, and Vikas Prasad. “Nephrotoxicity after radionuclide therapies.” Translational Oncology 15.1 (2022): 101295.). Therefore, there is a need for VHH radiotherapy for cancer with low nephrotoxicity and high therapeutic index.

Brief Description of the Drawings

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[0004] VHH or a fragment thereof In a first aspect of the present invention, there is provided a heavy-chain variable domain derived from a heavy-chain antibody (VHH) containing a click group or a fragment thereof for use as a medicament, preferably for the treatment and / or diagnosis of cancer. Preferably, the click group does not cause a click reaction when the medicament is administered to a patient.

[0005] In a second aspect of the present invention, there is provided a heavy chain variable domain derived from a heavy chain antibody (VHH) or a fragment thereof comprising a Click group, said VHH or a fragment thereof comprising 10 -1 s -1 Below 10, preferably -2 s -1 Less than 10, more preferably -3 s -1 Below, most preferably 10 -4 s -1 The following dissociation constant (k off ) capable of specifically binding to an antigen, preferably for use as a medicament, more preferably for use in the treatment and / or diagnosis of a cancer associated with said antigen. Preferably, said click group does not undergo a click reaction when said medicament is administered to a patient.

[0006] In the present application, whenever a heavy chain antibody (VHH) or a fragment thereof is mentioned, it refers to a heavy chain antibody (VHH) or a fragment thereof comprising a Click group as defined in the first or second aspect, unless expressly stated otherwise. Such a VHH or a fragment thereof may also be referred to as a VHH or a fragment thereof according to the invention. It is understood herein that a VHH or a fragment thereof according to the invention is preferably for use in the treatment and / or diagnosis of cancer.

[0007] In some embodiments, the VHHs disclosed herein are -1 s -1 ~10 -5 s -1 in the range of 10 -2 s -1 ~10 -5 s -1 More preferably, in the range of 10 -3 s -1 ~10 -5 s -1 , most preferably in the range of 10 -4 s -1 ~10 -5 s -1 The dissociation constant (k off) can specifically bind to an antigen. Example 7 shows that a limited k off rate is suitable for the pre-targeting approach.

[0008] The heavy-chain variable domain derived from the heavy-chain antibody (VHH) or a fragment thereof disclosed herein consists of a single polypeptide chain. More specifically, the VHH is derived from a protein of the innate immune system or the adaptive immune system, preferably the innate immune system or the adaptive immune system. Even more specifically, the VHH disclosed herein may include four framework regions (FRs) and three complementarity-determining regions (CDRs) or any suitable fragment thereof (usually including at least some of the amino acid residues forming at least one of the CDRs). In particular, the VHH disclosed herein is preferably easy to produce in high yields in a microbial recombinant expression system and is subsequently convenient for isolation and / or purification.

[0009] The VHH disclosed in the present invention may include the CDR (complementarity-determining region) sequences of an antibody (or may be based on and / or derived from such CDR sequences as further described herein), and they are generally also referred to herein as "CDR sequences" (i.e., CDR1 sequence, CDR2 sequence, and CDR3 sequence, respectively). In one embodiment, the VHH disclosed herein includes at least one amino acid sequence selected from the group consisting of the CDR1 sequence, CDR2 sequence, and CDR3 sequence described herein.

[0010] In one embodiment, the VHH disclosed herein has the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and has.

[0011] In connection with the present invention, using the IMGT nomenclature, the FR (framework region) FR1, FR2, FR3 and FR4 and the corresponding CDR regions CDR1, CDR2 and CDR3 are defined. The definitions of the IMGT nomenclature used are described later in the general part for the definitions of the present invention in particular.

[0012] The fragment of VHH preferably exhibits at least to some extent the activity of VHH. "To some extent" means at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more. The preferred activity of the fragment of VHH is preferably 10 -1 s -1 Hereinafter, preferably 10 -2 s -1 Hereinafter, more preferably 10 -3 s -1 Hereinafter, most preferably 10 -4 s -1 The following dissociation constant (k off ) is to specifically bind to the antigen. Preferred antigens are described below. A more preferred activity of the fragment of VHH is 10 -1 s -1 ~10 -5 s -1 in the range of, preferably 10 -2 s -1 ~10 -5 s -1 in the range of, more preferably 10 -3 s -1 ~10 -5 s -1 in the range of, most preferably 10 -4 s -1 ~10 -5 s -1 and to specifically bind to the antigen with a dissociation constant (k off ) in the range of.

[0013] The VHH fragment preferably has a length of 20 - 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 amino acids or 50 - 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 140 or 150 amino acids.

[0014] In a plurality of embodiments, the VHH or fragment thereof according to the present invention contains 1 - 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 click groups. In a plurality of embodiments, the VHH or fragment thereof according to the present invention contains two or more click groups. In a plurality of embodiments, the VHH or fragment thereof according to the present invention contains 2 - 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 click groups. In a plurality of embodiments, the VHH or fragment thereof according to the present invention contains 3 - 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 click groups. In a plurality of embodiments, the VHH or fragment thereof according to the present invention contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 click groups.

[0015] In a plurality of embodiments, all of the click groups contained in the VHH or fragment thereof according to the present invention are of the same type. According to a preferred definition, two click groups are of the same type if they are conformational isomers, tautomers, isotope substituents or isotopomers of each other.

[0016] The first aspect of the present invention relates to "a heavy chain antibody (VHH) or fragment thereof containing a click group". It should be understood that "containing a click group" refers to whether the click group is conjugated to the VHH or, in other ways, incorporated into the VHH by, for example, a modified amino acid.

[0017] In multiple embodiments, the click group contained in the VHH or its fragment according to the present invention is conjugated to the VHH.

[0018] In multiple embodiments, the click group contained in the VHH or its fragment according to the present invention is conjugated to the VHH at a lysine residue, cysteine residue, sugar chain, N-terminus or C-terminus contained in the VHH, preferably at a lysine residue, N-terminus or C-terminus contained in the VHH.

[0019] In multiple embodiments, the above click group is conjugated to the VHH or its fragment according to the present invention by reacting a corresponding VHH not conjugated to the click group with a compound containing a first group capable of conjugating with a lysine residue, cysteine residue, sugar chain, N-terminus or C-terminus, preferably a lysine residue, N-terminus or C-terminus contained in the VHH, and one of the click groups. Preferably, the reaction is carried out in an aqueous buffer, more preferably in a basic aqueous buffer. Preferably, the first group is N-hydroxysuccinimide (NHS), and more preferably, the first group is N-hydroxysuccinimide (NHS) and each of the click groups is TCO.

[0020] Combination In one aspect of the present invention, - a heavy chain variable domain derived from a heavy chain antibody (VHH) or its fragment according to the present invention, - a labeled compound containing a label and a click group A combination for use in the treatment and / or diagnosis of cancer is provided, The VHH or its fragment is 10 -1 s -1 or less, preferably 10 -2 s -1 or less, more preferably 10 -3 s -1 or less, most preferably 10 -4 s -1With the following dissociation constant (koff), it can specifically bind to the antigen associated with the cancer, The use includes the first administration of the VHH or its fragment to a subject in need thereof and subsequent administration of the labeled compound, The click group contained in the VHH or its fragment can undergo a click reaction with the click group contained in the labeled compound in the subject.

[0021] Such a combination may be referred to as the combination according to the present invention, and it is understood that the combination is for use in the treatment and / or diagnosis of cancer.

[0022] In a plurality of embodiments, the VHH included in the combination according to the present invention has a dissociation constant (k -1 s -1 ~10 -5 s -1 in the range of, preferably 10 -2 s -1 ~10 -5 s -1 in the range of, more preferably 10 -3 s -1 ~10 -5 s -1 in the range of, most preferably 10 -4 s -1 ~10 -5 s -1 ) and can specifically bind to the antigen. off ) and can specifically bind to the antigen.

[0023] Without wishing to be bound by this theory, the use of the combination according to the present invention achieves selective or specific delivery of the label to cells, tissues or organs that (over)express the antigen.

[0024] It is understood that any VHH or its fragment according to the present invention disclosed in this application or any labeled compound disclosed in this application can be included in the combination according to the present invention. Therefore, all of the following preferred embodiments can also be considered as the combination according to the present invention.

[0025] In multiple embodiments, the combination according to the invention is a kit or a kit of parts, wherein the VHH or its fragment and the labeling fragment contained therein are present as separate parts or components. In this context, separate parts or components preferably mean, as will be understood by those skilled in the art, that the VHH (or its fragment) and the labeling fragment are not present in a single chemical composition. This means that the above components are designed for separate administrations (i.e., the first administration and subsequent administrations as described above). In other words, since the composition means a single chemical composition, the combination should not be confused with the composition.

[0026] Conventional radioimmunotherapy and diagnosis (for which detailed protocols are readily available to experts) (Cancer Radiotherapy::Methods and Protocols (Methods in Molecular Medicine), Huddart RA Ed., Human Press 2002) involve labeling the targeting moiety (such as a VHH or its fragment) ex vivo and then administering the resulting labeled-targeting moiety conjugate in a single dose. In this case, the label can be either a diagnostic compound or a therapeutic compound, as described below. In other words, specific binding to a clinical target (such as an antigen associated with cancer) is performed simultaneously with the delivery of a substance (label) effective for treatment or diagnosis.

[0027] On the other hand, the use of the combination according to the invention in the treatment and / or diagnosis of cancer involves two subsequent (and thus separate) administration steps, where the first administration is the administration of the VHH or its fragment (resulting in specific binding to the antigen), and the second administration is the labeled compound containing the label (resulting in a click reaction between the VHH or fragment and the labeled compound). In other words, specific binding and the delivery of a substance effective for treatment or diagnosis are separated. Thus, the use of the combination according to the invention may be referred to as "pretargeting", and in the context of the present invention, the combination may be referred to as a "pretargeting combination".

[0028] Preferably, the click reaction between the click group contained in the VHH or its fragment and the click group contained in the labeling compound is preferably a specific reaction that does not cause an undesirable side reaction with biomolecules. Thus, without being bound by this theory, the specific binding of the VHH or fragment, which is the result of the first administration, is interpreted as the specific delivery of the labeling compound in subsequent administrations.

[0029] Without being bound by this theory, in the treatment or diagnosis of cancer, the advantages of the pretargeting use of the combination according to the present invention compared to the use of radiolabeled VHH or its fragment using conventional radioimmunotherapy and diagnosis characterized by a single administration are - a reduction in the retention of the label in the kidney, preferably at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%; - an increase in the therapeutic index of the label, preferably at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500% or one or more of them.

[0030] Preferably, as described in Example 3, the above advantages can be obtained while maintaining the tumor targeting ability.

[0031] In a plurality of embodiments, the time between the first administration and the subsequent administration is around 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day before, and preferably, around X days means from 0.8 times X days to 1.2 times X days.

[0032] In a plurality of embodiments, the time between the first administration and the subsequent administration is around 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour before, and preferably, around X hours means from 0.8 times X hours to 1.2 times X days.

[0033] In a plurality of embodiments, the time between the first administration and the subsequent administration is around 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 minutes before, and preferably, around X minutes means from 0.8 times X minutes to 1.2 times X minutes.

[0034] In a plurality of embodiments, the time between the first administration and the subsequent administration is within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, and preferably, the time between the first administration and the subsequent administration is at least 1 day, more preferably at least 2 days.

[0035] In multiple embodiments, the time between the first administration and subsequent administrations is within 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour, and preferably, the time between the first administration and subsequent administrations is at least 1 hour, more preferably at least 2 hours.

[0036] In multiple embodiments, the first administration and subsequent administrations are within 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 minutes, and preferably, the time between the first administration and subsequent administrations is at least 10 minutes.

[0037] In multiple embodiments, the time between the first administration and subsequent administrations is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, and preferably, the time between the first administration and subsequent administrations is less than 30 days.

[0038] In multiple embodiments, the time between the first administration and subsequent administrations is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours, and the time between the first administration and subsequent administrations is less than 72 hours.

[0039] In multiple embodiments, the time between the first administration and subsequent administrations is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 minutes, and preferably, the time between the first administration and subsequent administrations is less than 240 minutes.

[0040] In connection with the present invention, the time between the first administration and subsequent administrations may be referred to as the lag time.

[0041] In Example 1, the lag time optimization of pretargeting was performed using TCO-VHH1 (first administration) and 177 Lu-DOTA-PEG 7 -Tz (second administration). The same protocol can be implemented for other applications.

[0042] The use of a pretargeting combination comprising a monoclonal antibody containing a click moiety and a labeling compound has already been described in the literature. However, in the case of monoclonal antibodies, the use of the pretargeting combination addresses the problem of high toxicity due to long blood circulation. Monoclonal antibodies generally have a long circulation time, so the corresponding radiolabeled counterparts remain in circulation for a long time after direct administration, causing toxicity problems. The pretargeting approach first administers an unlabeled monoclonal antibody, then a scavenger to effectively remove from circulation the unlabeled monoclonal antibody that is not specifically bound to its target, and then a labeling compound that can specifically bind to any monoclonal antibody specifically bound to the target (i.e., the tumor). By doing so, this problem can be solved. Generally, since the labeling compound does not remain in circulation for a long time, this pretargeting approach solves the toxicity problem.

[0043] In contrast, the use of the pretargeting combination according to the present invention comprises a heavy-chain antibody (VHH) or a fragment thereof. These targeting compounds are not characterized by a long circulation time. In fact, they are rapidly excreted from the bloodstream compared to monoclonal antibodies. Therefore, they are less hampered by the general toxicity problems associated with radiolabeled monoclonal antibodies. As a result, one of ordinary skill in the art would not consider using the pretargeting approach with VHH or a fragment thereof because it is unlikely to add any value or solve specific problems associated with its use. In this case, the pretargeting approach addresses the problem of nephrotoxicity associated with the rapid clearance of VHH or a fragment thereof, which is an effect not apparent from the prior art. In other words, based on the generally available knowledge regarding pretargeting of monoclonal antibodies, it is not obvious for one of ordinary skill in the art to use the pretargeting approach with VHH or a fragment thereof.

[0044] Furthermore, the use of the pretargeting combinations according to the invention usually does not involve the use of a blood purifying agent. This is because this agent only serves its purpose in relation to monoclonal antibodies with a long circulation time. In fact, not administering an additional agent can be an advantage of using the pretargeting combination according to the invention. Clearly, the possibility of omitting the blood purifying agent would not be obvious to a person skilled in the art based on the generally available knowledge regarding the pretargeting of monoclonal antibodies.

[0045] In a plurality of embodiments, the use of the combination according to the invention does not include the administration of a blood purifying agent to the subject between the first administration and the subsequent administration. As used herein, a blood purifying agent is an agent that can remove or excrete unbound VHH from the subject's blood circulation and / or is the agent of interest, and is typically administered between the first administration and the subsequent administration in relation to pretargeting. Examples of blood purifying agents include, but are not limited to, avidin, galactose, biotinylated N-acetylgalactosamine and binding agents, such as antibodies targeted to VHH and fragments thereof. Without wishing to be bound by this theory, there may be a fraction of VHH or a fragment thereof that is not specifically bound to the antigen present in the subject's blood circulation after the first administration. A fraction may be, for example, from 0% to 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the administered VHH or a fragment thereof. A preferred advantage of the use of the combination according to the invention is that it is not necessary to administer a blood purifying agent between the first administration and the subsequent administration in order to achieve a therapeutic or diagnostic effect as described below.

[0046] In a plurality of embodiments, the use of the combination according to the invention does not include the administration of biotinylated N-acetylgalactosamine.

[0047] As described below, it is understood that any preferred VHH or fragment thereof according to the invention should also be understood as a preferred combination according to the invention comprising the preferred VHH or fragment thereof. In this application, whenever reference is made to the administration of a combination according to the invention, it always refers to both the first and subsequent administrations.

[0048] Click group and pair Click chemistry is a chemical approach introduced by Sharpless in 2001 and is well known to those skilled in the art. According to a preferred definition, a click (chemistry) reaction involves binding a small molecule modular group (i.e., part of the reactant) that can bind to a large moiety such as a biomolecule with high speed, yield, and specificity (e.g., stereospecificity and / or regioselectivity). More preferably, only limited amounts of unwanted by-products are formed and they can be removed from the resulting reaction mixture with limited effort, preferably without using chromatography. More preferably, click reactions are relatively insensitive to solvent parameters and the presence of oxygen and / or water. Most preferably, click reactions are biocompatible, i.e., they do not cause unwanted side reactions with biomolecules and / or do not result in the formation of toxic and / or non-biocompatible compounds.

[0049] Examples of click reactions are described in Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395). Click reactions include, but are not limited to, the formation of esters, thioesters, and amides from activated acids or acyl halides (e.g., peptide coupling, etc.), nucleophilic substitution reactions (e.g., nucleophilic substitution of halides or ring opening of strained ring systems, etc.), azide-alkyne Huisgen cycloaddition reactions (e.g., 1,3-dipolar cycloaddition reaction between azide and alkyne to form a 1,2,3-triazole linker), thiol-ene addition reactions, imine formation, Diels-Alder reactions between tetrazine and trans-cyclooctene (TCO), and Michael addition reactions (such as maleimide addition reactions).

[0050] The click chemistry reaction between an alkyne and an azide usually requires the addition of a copper catalyst to promote the 1,3-addition cycloaddition reaction and is known as the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. However, the click chemistry reaction between a cyclooctyne or a cyclooctyne derivative and an azide usually does not require the addition of a copper catalyst and instead proceeds via a strain-promoted azide-alkyne cycloaddition reaction (SPAAC).

[0051] As used herein, a "click group" refers to a moiety that can participate as a module group, i.e., a part of a reactant, in a click chemistry reaction. Click groups are rarely found in naturally occurring biomolecules and can be chemically inert sites with respect to biomolecules. However, when reacted with, for example, an azide-reactive group or an alkyne-reactive group, they can efficiently react under biologically relevant conditions, such as cell culture conditions in the absence of excessive heat or harsh reactants. Preferably, click groups are biocompatible.

[0052] In this section, preferred click groups are described. It is understood that these selections apply to the click groups contained in the VHH or fragments thereof according to the invention and to the click groups contained in the labeling compounds in the combinations according to the invention.

[0053] In a plurality of embodiments, the click group is an alkene, diene, alkyne, azide, nitrone, tetrazine or sydnone.

[0054] In a plurality of embodiments, a VHH or a fragment thereof according to the invention is provided, wherein the click group is an alkene, alkyne, azide, nitrone or tetrazine, preferably the alkene is trans-cyclooctene.

[0055] In a plurality of embodiments, a combination according to the invention is provided, wherein the click group contained in the labeling compound is an alkene, alkyne, azide, nitrone or tetrazine, preferably the alkene is trans-cyclooctene, and more preferably the click group contained in the labeling compound is tetrazine.

[0056] As used herein, the term "alkene" refers to an unsaturated hydrocarbon moiety or molecule containing a carbon-carbon double bond. Preferred alkenes are trans-cyclooctene (TCO). Preferred alkenes have 2 to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 carbon atoms.

[0057] As used herein, the term "diene" refers to a moiety or molecule having two double bonds, where these double bonds are conjugated at the 1,3-positions. The double bonds of the diene can be either cis- or trans-type. Preferred dienes have 1, 2, 3, 4, 5, or 6 nitrogen atoms. Preferred alkenes have 1 to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 carbon atoms. Preferred dienes contain 1, 2, 3, 4, or 4 covalent nitrogen-nitrogen bonds. A preferred diene is tetrazine.

[0058] As used herein, the term "alkyne" refers to a moiety or molecule containing a carbon-carbon triple bond. Alkyne moieties include terminal alkynes and cyclic alkynes, preferably terminal alkynes and cyclic alkynes that are reactive with azide groups. A terminal alkyne has at least one hydrogen atom bonded to the triple-bonded carbon atom. A cyclic alkyne is a cycloalkyl ring containing one or more triple bonds. Preferred alkenes are cyclic alkenes. Preferred cyclic alkynes are cyclooctyne and cyclooctyne derivatives, such as bicyclononyne (BCN), biarylazacyclooctynone (BARAC), and dibenzoazacyclooctyne (DIBAC).

[0059] As used herein, the term "azide" refers to a moiety or molecule containing an -N 3 functional group. Preferred azides have 1 to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 carbon atoms. A preferred azide is NHS-azide.

[0060] As used herein, the term "nitrone" refers to a >C=N + (-O - )-functional group, preferably -C=N + (-O- )-H refers to a moiety or molecule containing it. Preferred nitrones have 1 to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 carbon atoms.

[0061] As used herein, the term "tetrazine" refers to a 6-membered aromatic ring containing exactly 4 nitrogen atoms. Preferred tetrazines have 2 to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 carbon atoms. A preferred tetrazine is 1,2,4,5-tetrazine.

[0062] A click reaction requires at least two click groups that can react with each other. In this context, click groups that can react with each other are sometimes referred to as a click (chemistry) pair. The click groups that make up a click pair are sometimes referred to as click pair partners. In the context of this application, "being able to undergo a click reaction" and similar expressions are synonymous with "forming a click pair" and similar expressions.

[0063] An open click group is a click group that has not undergone a click reaction. In other words, an open click group is a click group that can undergo a click reaction with another click group.

[0064] A closed click group is a click group that has undergone a click reaction. In other words, a closed click group is the moiety that is bonded, typically covalently, to its click pair partner.

[0065] In this section, preferred click pairs are described. The selection of a click pair consisting of a click group A and a click group B includes preferred combinations according to the invention in which the VHH or a fragment thereof of the invention comprises one or more click groups A and the labeling compound comprises a click group B, and preferred combinations according to the invention in which the VHH or a fragment thereof of the invention comprises one or more click groups B and the labeling compound comprises a click group A.

[0066] In some embodiments, the click pair consists of an azide and an alkyne, preferably a cyclic alkyne such as BCN, BARAC or DIBAC.

[0067] In some embodiments, the click pair consists of a tetrazine and an alkene or alkyne, preferably a cyclic alkene or alkyne. In this context, the cyclic alkene is preferably trans-cyclooctene. In this context, the cyclic alkyne is preferably BCN, BARAC or DIBAC.

[0068] Label The combination according to the invention comprises a labeling compound comprising a label and a click group. Preferred labels are provided below. It is understood that these selections include the preferred corresponding combinations according to the invention.

[0069] In a plurality of embodiments, the label is a therapeutic or diagnostic moiety, preferably useful for the treatment or diagnosis of cancer as discussed herein.

[0070] In a plurality of embodiments, the label is a small molecule. Preferably, the small molecule is defined as an organic molecule or corresponding moiety having a molecular weight of up to 2000 Da, preferably up to 1500 Da, more preferably up to 1000 Da, for example up to 900 Da.

[0071] Radionuclide In a plurality of embodiments, the label is a radionuclide. In this context, the corresponding labeling compound comprising a label that is a radionuclide may be referred to as a radiolabeled compound.

[0072] In multiple embodiments, the label is a radionuclide suitable for therapeutic use, preferably a radionuclide selected from the group consisting of alpha-emitting radioisotopes and beta-emitting radioisotopes, including but not limited to actinium-225, astatine-211, bismuth-212, bismuth-213, cesium-137, chromium-51, cobalt-60, copper-67, dysprosium-165, erbium-169, fermium-255, gold-198, holmium-166, iodine-125, iodine-131, iridium-192, iron-59, lead-212, lutetium-177, molybdenum-99, palladium-103, phosphorus-32, potassium-42, rhenium-186, rhenium-188, samarium-153, radium-223, radium-224, ruthenium-106, scandium-47, sodium-24, strontium-89, terbium-149, terbium-161, terbium-149, thorium-227, xenon-133, ytterbium-169, ytterbium-177, and yttrium-90. In a more preferred embodiment, the label is iodine-131. In a preferred embodiment, the label is selected from the group consisting of actinium-225, bismuth-213, iodine-125, iodine-131, lutetium-177, yttrium-90, copper-67, rhenium-186, rhenium-188, terbium-149, terbium-161, astatine-211, or fluorine-18. In a more preferred embodiment, the label is astatine-211 or fluorine-18. In a more preferred embodiment, the label is actinium-225 or bismuth-213, most preferably actinium-225. In a more preferred embodiment, the label is lutetium-177 or terbium-161, most preferably lutetium-177.

[0073] In multiple embodiments, the label is a radionuclide suitable for diagnostic use, preferably a radionuclide selected from the group consisting of positron-emitting radioisotopes (PET) or gamma-emitting radioisotopes (SPECT), including but not limited to iodine-131, yttrium-90, iodine-125, lutetium-177, lead-203, rhenium-186, rhenium-188, scandium-43, scandium-44, technetium-99m, terbium-161, terbium-149, indium-111, xenon-133, thallium-201, fluorine-18, gallium-68, gallium-67, copper-67, iodine-123, iodine-124, zirconium-89, and copper-64.

[0074] In a more preferred embodiment, the label is iodine-131. In a more preferred embodiment, the label is actinium-225 or bismuth-213, most preferably actinium-225. In a more preferred embodiment, the label is lutetium-177 or terbium-161, most preferably lutetium-177.

[0075] In multiple embodiments, the label is a radionuclide that can form a coordination bond non-covalently with a chelating agent, as described below, and is selected from the group consisting of actinium-225, bismuth-212, bismuth-213, cesium-137, chromium-51, cobalt-60, copper-67, erbium-169, fermium-255, gold-198, iron-59, lead-212, lutetium-177, holmium-166, potassium-42, rhenium-186, rhenium-188, samarium-153, radium-223, radium-224, scandium-47, sodium-24, terbium-149, terbium-161, and terbium-149.

[0076] In multiple embodiments, the label is a radionuclide that can form a coordinate bond with DOTA through non-covalent bonding, as described below, and is selected from the group consisting of actinium-225, bismuth-212, bismuth-213, copper-67, erbium-169, iron-59, lead-212, lutetium-177, samarium-153, radium-223, radium-224, scandium-47, terbium-149, terbium-161, thorium-227, ytterbium-169, ytterbium-177, and yttrium-90.

[0077] In multiple embodiments, the label is a radionuclide that can covalently bond with a linker, as described below, and is selected from the group consisting of astatine-211, dysprosium-165, iodine-125, iodine-131, iridium-192, molybdenum-99, palladium-103, phosphorus-32, ruthenium-106, strontium-89, and xenon-133.

[0078] Other labels In multiple embodiments, the label is a non-radioactive label. In one embodiment, such a non-radioactive label is a fluorescent label. Such labels and the corresponding combinations according to the present invention can be used for diagnostic uses as defined herein. Other uses include image-guided surgery or photodynamic therapy. Examples of fluorescent labels suitable for diagnostic uses include Alexa fluor variants, Cy3, Cy5, FITC (fluorescein), coumarin, Texas red, Oregon green, Pacific blue, Pacific green, Pacific orange, PE-Cyanine7, PerCP-Cyanine5.5, TRITC (tetramethylrhodamine). Examples of fluorescent labels suitable for image-guided surgery include IRDye800CW, IRDye680-RD, ZW800-1, FNIR (e.g., Pieterjan Debie et al, Front Pharmacology, 2019; 10:510, doi:10.3389 / fphar.2019.00510, PMCID: PMC6527780, PMID: 31139085). Examples of fluorescent labels suitable for photodynamic therapy include IRDye700DX. Most labels can be obtained from ThermoFisher or Licor.

[0079] In multiple embodiments, the label is a molecule to be delivered to cells, tissues, organs that express human and / or mouse FAP. Any moiety, molecule or pharmaceutical known to act on cells, tissues, organs that express FAP is potentially encompassed in these embodiments. The molecule can be a peptide, a small molecule or a nucleic acid. The peptide can be a cytokine. The small molecule can be a chemotherapeutic agent. The entity can be a cell such as a CAR-T cell, a CAR-NK cell, a BITE or a LITE.

[0080] In multiple embodiments, the label is AcTakine (target-activated cytokine) or AcTaferon (IFNα-based AcTakine), preferably AcTakine or AcTaferon as described in WO 2017 / 077382 A1, WO 2017 / 134301 A1, WO 2017 / 194783 A1, WO 2017 / 194782 A2, WO 2018 / 077893 A1, WO 2018 / 141964 A1, WO 2018 / 144999 A1, WO 2019 / 032661 A1, WO 2019 / 032663 A1, WO 2019 / 032662 A1, WO 2019 / 148089 A1, WO 2019 / 191519 A1 or WO 2020 / 033646 A1. In the above embodiments, the label is preferably a pharmaceutical for cancer.

[0081] In multiple embodiments, the label is a pyrrolobenzodiazepine, preferably a pyrrolobenzodiazepine dimer such as those described in WO 2014 / 057074 A1, WO 2015 / 052322 A1, WO 2014 / 140174 A1, WO 2015 / 052321 A1, WO 2017 / 186894 A1, WO 2017 / 137555 A1, WO 2017 / 137553 A1, WO 2016 / 038383 A1 or WO 2018 / 192944 A1. In the present specification, more preferably, the pyrrolobenzodiazepine dimer is selected from the group consisting of: - (11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaoctatriacontanamide)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamide)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate, - (S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-(2-(2-(4-(((8-methoxy-2-(6-methoxynaphthalen-2-yl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)propanamide, - (S)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-(2-(2-(4-(((2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)propanamide, - 1-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)-N-(3-(((S)-8-((5-(((S)-7-Methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide, - 3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-(2-(2-(4-((((S)-8-((5-(((S)-7-Methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)propanamide, - 1-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)-N-((S)-1-(((S)-1-((4-((S)-7-Methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide, - 6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(3-Fluorophenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide, - (R)-2-((3-Nitropyridin-2-yl)disulfanyl)propyl (11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate, - 4-((2S,5S)-37-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaoctatriacontanamide)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-carboxylate, and - 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaoctatriacontanamide)benzyl (11S,11aS)-11-hydroxy-8-(3-(((11S,11aS)-11-hydroxy-10-(((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amide)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate. In these embodiments, the label is preferably a pharmaceutical for cancer.

[0082] In a plurality of embodiments, the label is an octacoordinate thallium chelating agent such as those described in WO 2017211809A1. In these embodiments, the label is preferably a pharmaceutical for cancer.

[0083] In a plurality of embodiments, the label is dolastatin or auristatin as described in WO 2015162293A1. In these embodiments, the label is preferably a pharmaceutical for cancer.

[0084] In a plurality of embodiments, the label is a cytolysin or Nigrin-b A chain such as those described in WO 2015118030A2. In these embodiments, the label is preferably a pharmaceutical for cancer.

[0085] In multiple embodiments, the label is 2-propyltriazolo[4,5-c]quinolin-4-amine, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine, 4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol, 1-(4-amino-2-ethylaminomethylimidazo-[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol, N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl-]methanesulfonamide, 4-amino-2-ethoxymethyl-αα-dimethyl-6,7,8,9-tetrahydro-1h-imidazo[4,5-c]quinoline-1-ethanol, 4-amino-αα-dimethyl-2-methoxyethyl-1h-imidazo[4,5-c]quinoline-1-ethanol, 1-{2-[3-(benzyloxy)propoxy]ethyl}-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine, N-[4-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)butyl]-N'-butylurea, N1-[2-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)ethyl]-2-amino-4-methylpentanamide, N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-n'-phenylurea, 1-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine, 1-{4-[(3,5-dichlorophenyl)sulfonyl]butyl}-2-ethyl-1H-imidazo[4,5-c]quinolin-4-amine, N-(2-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-N'-cyclohexylurea, N-{3-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]propyl}n'-(3-cyanophenyl)thiourea, N-[3-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2,2-Dimethylpropyl]benzamide, 2-Butyl-1-[3-(methylsulfonyl)propyl]-1H-imidazo[4,5-c]quinolin-4-amine, N-{2-[4-Amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}-2-ethoxyacetamide, 1-[4-Amino-2-ethoxymethyl-7-(pyridin-4-yl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol, 1-[4-Amino-2-(ethoxymethyl)-7-(pyridin-3-yl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol, N-{3-[4-Amino-1-(2-hydroxy-2-methylpropyl)-2-(methoxyethyl)-1H-imidazo[4,5-c]quinolin-7-yl]phenyl}methanesulfonamide, 1-[4-Amino-7-(5-hydroxymethylpyridin-3-yl)-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol, 3-[4-Amino-2-(ethoxymethyl)-7-(pyridin-3-yl)-1H-imidazo[4,5-c]quinolin-1-yl]propane-1,2-diol, 1-[2-(4-Amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)-1,1-dimethylethyl]-3-propylurea, 1-[2-(4-Amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)-1,1-dimethylethyl]-3-cyclopentylurea, 1-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]-2-(ethoxymethyl)-7-(4-hydroxymethylphenyl)-1H-imidazo[4,5-c]quinolin-4-amine, 4-[4-Amino-2-ethoxymethyl-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-7-yl]-N-methoxy-N-methylbenzamide, 2-Ethoxymethyl-N1-isopropyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1,4-diamine, 1-[4-Amino-2-ethyl-7-(pyridin-4-yl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol, N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide or N-[4-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)butyl]-N'-cyclohexylurea. In these embodiments, the label is preferably a pharmaceutical for cancer.,

[0086] In a plurality of embodiments, the label is a Pseudomonas exotoxin such as that described in WO 2015 / 051199 A2. In these embodiments, the label is preferably a pharmaceutical for cancer.

[0087] International Publication No. WO 2017 / 137553 A1 pamphlet, International Publication No. WO 2016 / 038383 A1 pamphlet, International Publication No. WO 2018 / 192944 A1 pamphlet, International Publication No. WO 2015 / 051199 A2 pamphlet, International Publication No. WO 2017 / 211809 A1 pamphlet, International Publication No. WO 2014 / 057074 A1 pamphlet, International Publication No. WO 2015 / 052322 A1 pamphlet, International Publication No. WO 2014 / 140174 A1 pamphlet, International Publication No. WO 2015 / 052321 A1 pamphlet, International Publication No. WO 2017 / 186894 A1 pamphlet, International Publication No. WO 2017 / 137555 A1 pamphlet, International Publication No. WO 2015 / 162293 A1 pamphlet, International Publication No. WO 2015 / 118030 A2 pamphlet, International Publication No. WO 2015 / 103990 A1 pamphlet, International Publication No. WO 2017 / 077382 A1 pamphlet, International Publication No. WO 2017 / 134301 A1 pamphlet, International Publication No. WO 2017 / 194783 A1 pamphlet, International Publication No. WO 2017 / 194782 A2 pamphlet, International Publication No. WO 2018 / 077893 A1 pamphlet, International Publication No. WO 2018 / 141964 A1 pamphlet, International Publication No. WO 2018 / 144999 A1 pamphlet, International Publication No. WO 2019 / 032661 A1 pamphlet, International Publication No. WO 2019 / 032663 A1 pamphlet, International Publication No. WO 2019 / 032662 A1 pamphlet, International Publication No. WO 2019 / 148089 A1 pamphlet, International Publication No. WO 2019 / 191519 A1 pamphlet and International Publication No. WO 2020 / 033646 A1 pamphlet are incorporated herein in their entirety, and all compounds disclosed therein may be relevant to this application as labels.

[0088] In the embodiment, the label is a prodrug of adrenomedullin described in WO 2013 / 064508 A1, an autotaxin inhibitor described in WO 2014 / 097151 A2, a pyrimido[4,5-b]quinoline-4,5(3H,10H)-dione derivative described in WO 2014 / 091446 A1, an amiloride derivative described in WO 2013 / 064450 A1, a pyrrolo[2,3-d]pyrimidine derivative described in WO 2014 / 177527 A1, a pyrazolopyridine derivative or a pyrazolopyrimidine derivative described in WO 2015 / 173683 A1, a piperidino-dihydrothienopyrimidine sulfoxide derivative described in WO 2013 / 26797 A1, a 2-[1,4]pyridin-3-yl]-2,3-dihydro-benzo[1,4]dioxine derivative described in WO 2016 / 061161 A1, a pyridine derivative described in WO 2014 / 086705 A1, a pyridine derivative or a pyrazine derivative described in WO 2011 / 113894 A1, an aminopyrimidinyl derivative described in WO 2016 / 027195 A1, a carboxamide derivative described in WO 2015 / 175796 A1, an oxazole-substituted indazole derivative described in WO 2010 / 125082 A1, a bisphenylbutanoic acid derivative described in WO 2014 / 126979 A1, a pyrazine derivative described in WO 2012 / 035158 A1, an oxazolidin-2-one-pyrimidine derivative described in WO 2014 / 072956 A1, a pyrazolopyridinamine derivative described in WO 2016 / 096721 A1, an N-(hetero)aryl, 2-(hetero)aryl-substituted acetamide derivative described in WO 2010 / 101849 A1, a 3-azabicyclo[3.1.0]hexane derivative described in WO 2017 / 115205 A1, a phenoxyacetamide derivative described in WO 2017 / 028927 A1,The N-(5-(4-acetylpiperazin-1-yl)pyridin-2-yl)-2-(2'-fluoro-3-methyl-2,4'-bipyridin-5-yl)acetamide derivative or 2-(2',3-dimethyl-2,4'-bipyridin-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide derivative described in International Publication No. WO2017221142A1, the xanthine derivative described in International Publication No. WO2013171166A1, the xanthine derivative described in International Publication No. WO2013174768A1, the heterocyclylmethyl-thienouracil derivative described in International Publication No. WO2016150901A1, the n,2-diarylquinoline-4-carboxamide derivative described in International Publication No. WO201637954A1, the pyridine-2-carboxamide derivative described in International Publication No. WO2012168350A1, the benzamide derivative acting as a modulator of cell adhesion described in International Publication No. WO2005044817A1, the 3-amino-pyridine derivative described in International Publication No. WO2012117000A1, the Nampt or rock inhibitor described in International Publication No. WO201267965A1, the oxetane derivative described in International Publication No. WO201616242A1, the 1,1,1-trifluoro-3-hydroxypropan-2-ylcarbamate derivative described in International Publication No. WO2018134695A1, the pyrazole derivative described in International Publication No. WO2014135507A1, the indole derivative described in International Publication No. WO2009156462A1, the [1,2,3]triazolo[4,5-d]pyrimidine derivative described in International Publication No. WO201815088A1, the sgc stimulant described in International Publication No. WO2016177660A1, the CFTR protein described in International Publication No. WO201760879A1, the 6-carboxylic acid of benzimidazole or 4-aza-, 5-aza-, 7-aza- or 4,7-diaza-benzimidazole described in International Publication No. WO2018109607A1, the benzamide derivative described in International Publication No. WO201728926A1The 8-azabicyclo[3.2.1]octane derivative described in International Publication No. WO2012 / 087519A1, the triazolo[4,5-d]pyrimidine derivative described in International Publication No. WO2016 / 071375A1, the aryl sultam derivative described in International Publication No. WO2015 / 104354A1, the 2-(azaindol-2-yl)benzimidazole derivative described in International Publication No. WO2014 / 015905A1, or the quinuclidine or isoquinuclidine derivative described in International Publication No. WO2005 / 104745A1. Preferably, the label is a pharmaceutical for treating fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis, and / or other inflammatory and fibrotic diseases. International Publication No. WO2013 / 064508A1, WO2014 / 097151A2, WO2014 / 091446A1, WO2013 / 064450A1, WO2014 / 177527A1, WO2015 / 173683A1, WO2013 / 26797A1, WO2016 / 061161A1, WO2014 / 086705A1, WO2011 / 113894A1, WO2016 / 027195A1, WO2015 / 175796A1, WO2010 / 125082A1, WO2014 / 126979A1, WO2012 / 035158A1, WO2014 / 072956A1, WO2016 / 096721A1, WO2010 / 101849A1, WO2017 / 115205A1, WO2017 / 028927A1, WO2017 / 221142A1, WO2013 / 171166A1, WO2013 / 174768A1, WO2016 / 150901A1, WO2016 / 037954A1, WO2012 / 168350A1, WO2005 / 044817A1, WO2012 / 117000A1, WO2012 / 067965A1, WO2016 / 016242A1, WO2018 / 134695A1,The entire contents of the pamphlets numbered 2014135507A1, 2009156462A1, 201815088A1, 2016177660A1, 201760879A1, 2018109607A1, 201728926A1, 201287519A1, 201671375A1, 2015104354A1, 201415905A1 and 2005104745A1 are incorporated, and all compounds disclosed therein may be labels in relation to the present application.

[0089] In a plurality of embodiments, the label is a molecule to be delivered to cells, tissues, organs that express human FOLR1. Any moiety, molecule or pharmaceutical known to act on cells, tissues, organs that express FOLR1 may be included in these embodiments.

[0090] In a plurality of embodiments, the label is a molecule to be delivered to the central nervous system (CNS). More preferably, the molecule is a pharmaceutical that acts on the CNS, preferably the brain. Even more preferably, the molecule passes through the blood-brain barrier (BBB) and / or the blood-cerebrospinal fluid barrier (BCSFB) by transport via human FOLR1 (a mechanism called receptor-mediated transcytosis (RMT)). Any moiety, molecule or pharmaceutical known to act on the CNS or the brain may be a label in relation to the present invention. Such moiety, molecule or pharmaceutical may be any molecule or pharmaceutical that does not pass through the blood-brain barrier (BBB) by itself. The molecule may be a peptide, a small molecule or a nucleic acid. The peptide may be a cytokine. The small molecule may be a chemotherapeutic agent. The entity may be a cell such as a CAR-T cell, a CAR-NK cell, a BITE or a LITE.

[0091] In multiple embodiments, the pharmaceutical agent acting on the brain is a pharmaceutical agent for preventing and / or treating choroid plexus papilloma and / or hydrocephalus. Folic acid receptor α (FOLR1) is overexpressed in some human cancers including choroid plexus papilloma or tumors. An increase in the expression of folic acid receptor α (FOLR1) in the brain is associated with hydrocephalus.

[0092] In multiple embodiments, the label is an aziridinyl-epothilone as described in WO 2007140297A2 pamphlet, preferably, the label is 7,11-dihydroxy-17-[2-hydroxyethyl]-8,8,10,12-tetramethyl-3-[1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-4-oxa-17-azabicyclo[14.1.0]heptadecane-5,9-dione or a derivative thereof or a pharmaceutically acceptable salt thereof, more preferably, the label is a compound for the treatment and / or prevention of choroid plexus papilloma, and most preferably, the label is contained in the combination according to the present invention for the treatment and / or prevention of choroid plexus papilloma.

[0093] In multiple embodiments, the label is a 1H-pyrrolo[3,2-b]pyridine derivative as described in WO 2014 / 145051 A1 pamphlet. Preferably, the label is 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(1-(pyridin-2-yl)ethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid, or 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(phenyl(pyridin-2-yl)methyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid, or methyl 2-(4-(6-(3,5-dimethylisoxazol-4-yl)-1-(1-(pyridin-2-yl)ethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)phenyl)acetate, or methyl 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(pyridazin-3-ylmethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzoate, or N-cyclopropyl-4-(6-(3,5-dimethylisoxazol-4-yl)-1-(1-(pyridin-2-yl)ethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzenesulfonamide, or (4-(6-(3,5-dimethylisoxazol-4-yl)-1-(phenyl(pyridin-2-yl)methyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)phenyl)methanol, or 3,5-dimethyl-4-(1-(pyridazin-3-ylmethyl)-3-(1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-b]pyridin-6-yl)isoxazole, or 3,5-dimethyl-4-(3-(1-methyl-1H-pyrazol-4-yl)-1-(1-(pyridin-2-yl)ethyl)-1H-pyrrolo[3,2-b]pyridin-6-yl)isoxazole or a derivative thereof or a pharmaceutically acceptable salt thereof. More preferably, the label is a compound for the treatment and / or prevention of choroid plexus papilloma. Most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of choroid plexus papilloma.

[0094] In multiple embodiments, the label is a 1H-pyrrolo[3,2-b]pyridine derivative as described in WO 2017 / 053243 A1 pamphlet. Preferably, the label is 4-(1-(1,1-di(pyridin-2-yl)ethyl)-6-(3,5-dimethylisoxazol-4-yl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid, or 4-(1-(cyanodipyridin-2-ylmethyl)-6-(3,5-dimethylisoxazol-4-yl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid, or 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(fluorodipyridin-2-ylmethyl)-1H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid or its derivative or its pharmaceutically acceptable salt. More preferably, the label is a compound for the treatment and / or prevention of choroid plexus papilloma. Most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of choroid plexus papilloma.

[0095] In multiple embodiments, the label is a pyrrolidine sulfonamide TRPC4 antagonist as described in WO 2018 / 055524 A1 pamphlet. Preferably, the label is 2-(((3R,4S)-4-(4-chlorophenoxy)-3-hydroxy-3-(hydroxymethyl)pyrrolidin-1-yl)sulfonyl)-5-(trifluoromethyl)benzonitrile, or 4-(((3S,4S)-1-((2-cyano-4-(trifluoromethyl)phenyl)sulfonyl)-4-hydroxy-4-((S)-1-hydroxyethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile or its derivative or its pharmaceutically acceptable salt. More preferably, the label is a compound for the treatment and / or prevention of hydrocephalus. Most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of hydrocephalus.

[0096] In multiple embodiments, the label is a sulfonpyrazole or sulfonylpyrazoline carboxamidine 5-HT6 antagonist as described in WO 2008 / 034863 A2 pamphlet. Preferably, the label is N'-(1-acetylindoline-5-ylsulfonyl)-N,4-diethyl-4,5-dihydro-1H-pyrazole-1-carboximidamide, or N'-(4-(1H-pyrazole-1-yl)phenylsulfonyl)-N,4-diethyl-4,5-dihydro-1H-pyrazole-1-carboximidamide, or N-((5-(N-((4-ethyl-4,5-dihydro-1H-pyrazole-1-yl)(ethylamino)methylene)sulfamoyl)thiophen-2-yl)methyl)benzamide, or N'-(5-(5-(chloromethyl)-1,2,4-oxadiazol-3-yl)thiophen-2-ylsulfonyl)-N,4-diethyl-4,5-dihydro-1H-pyrazole-1-carboximidamide or its derivative or its pharmaceutically acceptable salt. More preferably, the label is a compound for the treatment and / or prevention of hydrocephalus. Most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of hydrocephalus.

[0097] In multiple embodiments, the label is an arylsulfonylpyrazoline carboxamidine 5-HT6 antagonist as described in WO 2009 / 115515 A1, and preferably, the label is N'-(4-amino-3-chlorophenylsulfonyl)-N-ethyl-2,3,8-triazaspiro[4.5]dec-3-ene-2-carboximidamide, or N'-(4-amino-3-chlorophenylsulfonyl)-N-ethyl-2,3-diazaspiro[4.4]non-3-ene-2-carboximidamide, or N'-(4-amino-3-chlorophenylsulfonyl)-N-ethyl-4,4-dimethyl-4,5-dihydro-1H-pyrazole-1-carboximidamide, or N'-(4-aminophenylsulfonyl)-2,3-diazaspiro[4.4]non-3-ene-2-carboximidamide, or N'-(4-aminophenylsulfonyl)-8-oxa-2,3-diazaspiro[4.5]dec-3-ene-2-carboximidamide, or N'-(4-aminophenylsulfonyl)-N,4-diethyl-4,5-dihydro-1H-pyrazole-1-carboximidamide, or N'-(4-aminophenylsulfonyl)-N-ethyl-5-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide, or N'-(4-aminophenylsulfonyl)-N-methyl-8-oxa-2,3-diazaspiro[4.5]dec-3-ene-2-carboximidamide or a derivative thereof or a pharmaceutically acceptable salt thereof.

[0098] In one embodiment, the label is a cyclodextrin-API conjugate as described in WO 2013 / 116200 A1, more preferably, the label is a compound for the treatment and / or prevention of hydrocephalus, and most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of hydrocephalus.

[0099] In multiple embodiments, the label is a pyrrolidine sulfonamide TRPV4 antagonist as described in WO 2018 / 055527 A1. Preferably, the label is 4-(((3S,4R)-1-((2, dichlorophenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)methyl)-2-fluorobenzonitrile, or 4-(((3S,4R)-1-((2-chloro-4-(trifluoromethyl)phenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)methyl)benzonitrile, or 4-(((3S,4R)-1-((5-chloropyridin-2-yl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)methyl)-3-(2,2,2-trifluoroethoxy)benzonitrile, or 4-((3S,4S)-1-((2,4-dichlorophenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)methyl)-2-fluorobenzonitrile, or 4-((3S,4R)-1-((2-cyano-4-(trifluoromethyl)phenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)methyl)-2-fluorobenzonitrile, or 4-(((3S,4R)-1-((2-chloro-4-cyanophenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)methyl)-2-fluorobenzonitrile, or 4-(((3S,4S)-4-(aminomethyl)-1-((5-chloropyridin-2-yl)sulfonyl)-4-hydroxypyrrolidin-3-yl)methyl)-2-fluorobenzonitrile or a derivative thereof or a pharmaceutically acceptable salt thereof. More preferably, the label is a compound for the treatment and / or prevention of hydrocephalus. Most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of hydrocephalus.

[0100] In multiple embodiments, the label is [5-(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-pyridin-2-yl]-(6-trifluoromethyl-pyridin-3-ylmethyl)-amine or a derivative thereof or a pharmaceutically acceptable salt thereof as described in WO 2016 / 179415 A1, preferably, the label is a compound for the treatment and / or prevention of hydrocephalus, and most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of hydrocephalus.

[0101] In multiple embodiments, the label is an N-(pyridin-3-ylmethyl) substituted 1H-pyrrolo[2,3-b]pyridine derivative as described in WO 2013 / 142427 A1 or a 1H-pyrrolo[2,3-b]pyridine derivative or a derivative thereof or a pharmaceutically acceptable salt thereof as described in WO 2017 / 100201 A1, preferably, the label is a compound for the treatment and / or prevention of hydrocephalus, and most preferably, the label is included in the combination according to the present invention for the treatment and / or prevention of hydrocephalus.

[0102] WO 2007 / 140297 A2, WO 2014 / 145051 A1, WO 2017 / 053243 A1, WO 2018 / 055524 A1, WO 2008 / 034863 A2, WO 2009 / 115515 A1, WO 2013 / 116200 A1, WO 2018 / 055527 A1, WO 2016 / 179415 A1, WO 2013 / 142427 A1 and WO 2017 / 100201 A1 are incorporated in their entirety and all compounds disclosed therein can be labels in connection with the present application.

[0103] Linker The combination according to the present invention comprises a labeled compound. Preferred labeled compounds are provided below. It is understood that these selections encompass the preferred corresponding combinations according to the present invention.

[0104] In multiple embodiments, the labeled compound is CL-L 1 -L 2 -R * and can be represented by, where CL is a click group contained in the labeled compound, L 1 is a first linker or bond, L 2 is a second linker, and R * is a label contained in the labeled compound. Preferably, R * is the radionuclide described above.

[0105] In multiple embodiments, L 1 is a bond, and thus the labeled compound can be represented by CL-L 2 -R * and can be represented by.

[0106] In multiple embodiments, L 1 is a linker. Any suitable linker known to those skilled in the art from the perspective of the present disclosure can be used in the present invention. The linker can be, for example, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a polyethylene glycol (PEG) linker, a hydrazone, a maleoyl-caproyl polymer, a glucuronide (polymer), a succinimide-thioether, a peptide linker (such as a dipeptide linker), a sugar-based linker, or a cleavable linker, such as a disulfide bond or a protease cleavage site like valine-citrulline, valine-citrulline-PAB, or a PAB polymer. In this context, the term polymer may be interpreted as an oligomer.

[0107] In multiple embodiments, L 1 is an oligomer having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues. In multiple embodiments, L 1 is an oligomer having 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues. In multiple embodiments, L1 is an oligomer having from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues. In these embodiments, R * is preferably iodine-131, actinium-225, bismuth-213, lutetium-177 or terbium-161, more preferably iodine-131, actinium-225 or lutetium-177. In this regard, an oligomer having 1 residue is a monomer.

[0108] In a plurality of embodiments, L 1 is PEG n and n is an integer from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In a plurality of embodiments, L 1 is PEG n and n is an integer from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In a plurality of embodiments, L 1 is PEG n and n is an integer from 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In a plurality of embodiments, L 1 is PEG n and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and most preferably, L 1 is PEG 7 In these embodiments, R * is preferably iodine-131, actinium-225, bismuth-213, lutetium-177 or terbium-161, more preferably iodine-131, actinium-225 or lutetium-177.

[0109] The type of the second linker L 2 depends on the identity of R * and in particular R * is a radionuclide as described above. Without limitation, radioisotopes of iodine (preferred R* ) has the ability to be incorporated directly into a molecule by electrophilic or nucleophilic substitution or indirectly through conjugation, and L 2 and R * suggests a covalent bond between them. On the other hand, since the radioactive metal is labeled through complex formation with a chelating agent, it suggests that the bond between L 2 and R * is a non-covalent bond.

[0110] In a plurality of embodiments, L 2 is a chelating agent, and preferably, the bond L 2 -R * is a non-covalent coordination bond.

[0111] In a plurality of embodiments, L 2 is a chelating agent, and R * is a non-covalently bound radionuclide, preferably actinium-225, bismuth-212, bismuth-213, cesium-137, chromium-51, cobalt-60, copper-67, erbium-169, fermium-255, gold-198, iron-59, lead-212, lutetium-177, holmium-166, potassium-42, rhenium-186, rhenium-188, samarium-153, radium-223, radium-224, scandium-47, sodium-24, terbium-149, terbium-161 or terbium-149, more preferably actinium-225, bismuth-212, bismuth-213, copper-67, erbium-169, iron-59, lead-212, lutetium-177, samarium-153, radium-223, radium-224, scandium-47, terbium-149, terbium-161, thorium-227, ytterbium-169, ytterbium-177 or yttrium-90, most preferably, R * is actinium-225, bismuth-213, lutetium-177 or terbium-161, and most preferably, actinium-225 or lutetium-177.

[0112] As used herein, the term "chelating agent" refers to a chemical group through which a metal (such as actinium-225 or lutetium-177) can be chelated via a coordination bond. Any chelating agent known to those skilled in the art from the perspective of the present disclosure can be used in the present invention.

[0113] In a plurality of embodiments, the chelating agent includes a macrocycle. Preferred chelating agents that include a macrocycle include, but are not limited to, the following: 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N’,N’’,N’’’,N’’’’-hexaacetic acid (HEHA), 1,4,7,10,13-pentaazacyclopentadecane-N’,N’’,N’’’,N’’’’-pentaacetic acid (PEPA), N,N’-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (Macropa) (Thiele et al., An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy. Angew Chem Int Ed Engl. 2017 Nov 13;56(46):p.14712-14717), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid (TETPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP), 3,6,9,15-tetraazabicyclo[9.3.1]pentadec-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), and 2,2’,2’’,2’’’-(1,10-dioxa-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid (Crown) (Yang, Hua, et al. “Synthesis and Evaluation of a Macrocyclic Actinium-225 Chelator, Quality Control and In Vivo Evaluation of 225Ac-crown-αMSH Peptide”. Chemistry-A European Journal 26.50(2020):11435-11440).Preferably, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), Macropa, PCTA or Crown.

[0114] In multiple embodiments, the labeled compound is 225 Ac-DOTA-PEG n -CL, 225 Ac-HEHA-PEG n -CL, 225 Ac-PEPA-PEG n -CL, 225 Ac-Macropa-PEG n -CL, 225 Ac-TETA-PEG n -CL, 225 Ac-DOTPA-PEG n -CL, 225 Ac-TETPA-PEG n -CL or 225 Ac-DOTMP-PEG n -CL, 177 Lu-DOTA-PEG n -CL, 177 Lu-HEHA-PEG n -CL, 177 Lu-PEPA-PEG n -CL, 177 Lu-Macropa-PEG n -CL, 177 Lu-TETA-PEG n -CL, 177 Lu-DOTPA-PEG n -CL, 177 Lu-TETPA-PEG n -CL or 177It can be represented by Lu-DOTMP-PEGn-CL. Preferably, n is an integer from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is an integer from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Most preferably, L 1 is PEG 7 . Preferably, CL is tetrazine (Tz).

[0115] In multiple embodiments, the labeled compound is 225 Ac-DOTA-PEG 7 -Tz or 177 Lu-DOTA-PEG 7 -Tz.

[0116] In multiple embodiments, the chelating agent includes an open-chain ligand. Preferred chelating agents containing an open-chain ligand include, but are not limited to, deferoxamine (DFO), ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetic acid (DTPA).

[0117] In multiple embodiments, the labeled compound is 225 Ac-DFO-PEG n -Tz, 225 Ac-EDTA-PEG n -Tz, 225 Ac-DTPA-PEG n -Tz, 177 Lu-DFO-PEG n -Tz, 177 Lu-EDTA-PEG n -Tz or 177 Lu-DTPA-PEG n-Tz, and preferably, n is an integer from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is an integer from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and most preferably, L 1 is PEG 7 . Preferably, CL is tetrazine.

[0118] In multiple embodiments, the labeled compound may be 225 Ac-EDTA-PEG-PEG 7 -Tz or 177 Lu-EDTA-PEG 7 -Tz.

[0119] In multiple embodiments, L 2 is a group covalently bonded to the label. In other words, L 2 -R * is a covalent bond. Preferably, R * is astatine-211, dysprosium-165, iodine-125, iodine-131, iridium-192, molybdenum-99, palladium-103, phosphorus-32, ruthenium-106, strontium-89 or xenon-133.

[0120] In multiple embodiments, L 2 is a benzoic acid linker, preferably 3-pyridinecarboxyl, benzoate or guanidinomethyl benzoate, more preferably guanidinomethyl benzoate, and most preferably 4-guanidinomethyl benzoate. These active esters exhibit high stability in vivo.

[0121] In multiple embodiments, the labeled compound may be represented by R * -L 2 -PEG n -CL, where L 2is a benzoic acid linker, more preferably 4-guanidinomethyl benzoic acid, and even more preferably, R * is iodine-131, and most preferably, n is 7.

[0122] In multiple embodiments, the labeled compound can be represented by [I-131]GMIB-PEG n -CL, where [I-131]GMIB is 4-guanidinomethyl-3-[I-131] iodobenzoic acid, and preferably, n is an integer from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is an integer from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and most preferably, L 1 is PEG 7 is. In such a labeled compound, the linker L 2 is guanidinomethyl benzoic acid, and R * is understood to be iodine-131.

[0123] [I-131]GMIB-PEG n The labeled compound represented by -CL can also be represented by the following structure.

Chemical formula

[0124] In multiple embodiments, the labeled compound can be represented by [I-131]GMIB-CL as defined above. The labeled compound according to these embodiments can also be represented by the following structure.

Chemical formula

[0125] In multiple embodiments, the labeled compound can be [I-131]GMIB-PEG as defined above n-Tz, and [I-131]GMIB can be represented by 4-guanidinomethyl-3-[I-131] iodobenzoic acid. Preferably, n is an integer from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is an integer from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and most preferably, L 1 is PEG 7 . In such a labeled compound, the linker L 2 is guanidinomethyl benzoate, and R * is understood to be iodine-131.

[0126] In multiple embodiments, the labeled compound can be represented by [I-131]GMIB-Tz as defined above.

[0127] Antigen Overview In multiple embodiments, the VHH or fragment thereof according to the present invention can specifically bind to an antigen, the antigen is contained in a target, and the binding of the VHH or fragment thereof to the antigen causes a significant structural change in the target.

[0128] In multiple embodiments, the VHH or fragment thereof according to the present invention has a dissociation constant (k -1 s -1 ), preferably 10 -2 s -1 or less, more preferably 10 -3 s -1 or less, and most preferably 10 -4 s -1 or less, and can specifically bind to an antigen. off )

[0129] In multiple embodiments, the VHH or fragment thereof according to the present invention has a dissociation constant of 10 -1 s -1 to 10-5 s -1 in the range of, preferably 10 -2 s -1 ~10 -5 s -1 in the range of, more preferably 10 -3 s -1 ~10 -5 s -1 in the range of, most preferably 10 -4 s -1 ~10 -5 s -1 dissociation constant (k off ) can specifically bind to the antigen.

[0130] In multiple embodiments, the VHH or fragment thereof according to the present invention binds to an antigen (the antigen is present on the surface of tumor cells or in the tumor microenvironment), more preferably with a dissociation constant (k -1 s -1 or less, preferably 10 -2 s -1 or less, more preferably 10 -3 s -1 or less, most preferably 10 -4 s -1 dissociation constant (k off ) and can specifically bind.

[0131] In multiple embodiments, the VHH or fragment thereof according to the present invention binds to an antigen (the antigen is present on the surface of tumor cells or in the tumor microenvironment), more preferably with a dissociation constant (k -1 s -1 ~10 -5 s -1 in the range of, preferably 10 -2 s -1 ~10 -5 s -1 in the range of, more preferably 10 -3 s -1 ~10 -5 s -1 in the range of, most preferably 10 -4 s -1 ~10 -5 s -1 dissociation constant (k off ) and can specifically bind.

[0132] In multiple embodiments, the VHH or fragment thereof according to the present invention binds to an antigen (wherein the antigen is a cancer cell and / or the antigen is expressed on the surface of a cancer cell or within the tumor microenvironment), more preferably with a dissociation constant (k -1 s -1 In the following, preferably with a dissociation constant (k -2 s -1 In the following, more preferably with a dissociation constant (k -3 s -1 In the following, most preferably with a dissociation constant (k -4 s -1 ) that is able to specifically bind at a dissociation constant (k off ) as follows.

[0133] In multiple embodiments, the VHH or fragment thereof according to the present invention binds to an antigen (wherein the antigen is a cancer cell and / or the antigen is expressed on the surface of a cancer cell or within the tumor microenvironment), more preferably with a dissociation constant (k -1 s -1 ~10 -5 s -1 in the range of, preferably 10 -2 s -1 ~10 -5 s -1 in the range of, more preferably 10 -3 s -1 ~10 -5 s -1 in the range of, most preferably 10 -4 s -1 ~10 -5 s -1 ) that is able to specifically bind at a dissociation constant (k off ) as follows.

[0134] In multiple embodiments, the antigen is CEA, PSMA, 4Ig-B7-H3, A33, a5β1 integrin, AGS-16, AGS-8, avβ3 integrin, CAIX / MN, CCR-2 / CCL-2, CD40, CD44v6, CK19, CTLA-4, d9-E-cadherin, DNA / histone H1, ED-B, fibronectin, EFGL7, EGFR, EGP-1, endoglin, EpCAM, FAP, FZD10, GC128, GD2, GD3, HER-2, HER-3, HPV-16 E6, IGF-1R, IL-6, L1-CAM, Lewis y , MAGE-A3, melanin, mesothelin, MET / HGF, MUC1, NCAM, neuropilin-1, PD-1 / PD-L1, PDGFR, PSCA, TAG-72, TEM-1, tenascin-C, thrombomodulin, TRAIL-R, Tweak-R, VE cadherin, VEGFR / VEGF or VEGFR1. Preferably, the antigen is an oncogene and / or is expressed on cancer cells or within the tumor microenvironment.

[0135] In multiple embodiments, the VHH or fragment thereof according to the present invention can specifically bind to human fibroblast activation protein (FAP), human folate receptor alpha (FOLR1) or human epidermal growth factor receptor 2 (HER2), preferably human fibroblast activation protein (FAP) or human folate receptor alpha (FOLR1).

[0136] Fibroblast activation protein (FAP) In multiple embodiments, the VHH or fragment thereof according to the present invention is particularly suitable for binding to human and / or mouse FAP. In one embodiment, the VHH or fragment thereof specifically binds to human and mouse FAP. In one embodiment, the VHH or fragment thereof binds to a part of the extracellular domain of human and / or mouse FAP.

[0137] Examples 3, 5 and 6 demonstrate the usefulness of the pretargeting approach of FAP-binding VHH for treating cancer. Example 3 used the pretargeting strategy 177It shows the long-term biodistribution and tumor targeting of Lu-VHH1. Example 5 shows that, without related signs of acute toxicity, pretargeted 225 the therapeutic potential of Ac-VHH1 is demonstrated. In Example 6, the long-term toxicity of healthy female C57Bl / 6 mice treated with pretargeted 225 Ac-VHH1 was examined, and it was revealed that there were no signs of acute or long-term toxicity.

[0138] The VHH or its fragment according to the present invention and the combination according to the present invention are preferably used for treating cancers associated with the expression of human FAP in CAF cells and / or cancer cells.

[0139] In another use, the above part can be a pharmaceutical for treating fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis and other inflammatory and fibrotic diseases. In such uses, the antibody fragment of the present invention is used to target the pharmaceutical to the site of the described disease for treating the disease.

[0140] Human FAP is specifically expressed, and more specifically overexpressed, in cancer-associated fibroblasts (CAFs) with tumor-forming functions, so it is very attractive as a target (Pure et al 2018, Oncogene Aug;37(32):4343 - 4357). It is also expressed in some cancer cells (e.g., leukemia, bone, uterus, pancreas, skin, muscle, brain, breast, colorectal, esophagus, stomach, liver, lung, ovary, parathyroid, kidney cancers, etc. disclosed later herein), and is not highly expressed in normal cells. Therefore, since human FAP can be regarded as a tumor antigen or cancer cell antigen, it can be used as a diagnostic target and / or a therapeutic target.

[0141] However, other uses (diagnostic and therapeutic) of the VHH or fragments thereof of the present invention are also encompassed by the present invention. Such other diagnostic and / or therapeutic uses may be related to fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis, and other inflammatory and fibrotic diseases, but not related to cancer. In other words, the VHH or fragments thereof of the present invention can be used for diagnostic and / or therapeutic uses for diagnosing and / or treating fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis, and other inflammatory and fibrotic diseases. More detailed explanations will be described later in this specification.

[0142] As used herein, human FAP is considered as "cancer cell-specific antigen", "cancer-specific antigen", "cancer antigen", "target protein present on ~", "target protein expressed in ~" and / or "specific to cancer cells", "cancer cell-specific target (protein)", "cancer (cell)-associated antigen", which are used interchangeably herein and refer to the fact that human FAP is mainly present (or mainly expressed) on cancer cells and in the vicinity of tumors and / or metastases. FAP is specifically expressed, more specifically overexpressed, in cancer-associated fibroblasts (CAF) having tumor-forming functions. This is also expressed in some cancer cells (e.g., cancers of leukemia, bone, uterus, pancreas, skin, muscle, brain, breast, colorectal, esophagus, stomach, liver, lung, ovary, parathyroid, kidney, etc.) (as disclosed later herein, Pure et al 2018, Oncogene Aug;37(32):4343-4357). FAP is hardly expressed in healthy cells. Therefore, since human FAP can be regarded as a tumor antigen or cancer cell antigen, it can be used as a diagnostic target and / or a therapeutic target. For example, human FAP is expressed in cancer-associated fibroblasts. As used herein, the terms "FAP-positive", or "expressing FAP", or "overexpressing FAP" may refer to cancerous or malignant human cells and / or cancer-associated fibroblasts or tissues characterized by overexpression of the FAP protein, and thus, this has an abnormally high level of FAP gene and / or FAP protein compared to normal healthy cells. In this regard, "overexpression" may mean that the expression is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than the expression in a control cell line. The control cell line can be a healthy cell or a non-diseased cell.

[0143] In one embodiment, the VHH or its fragment according to the present invention specifically binds to human and / or mouse FAP and satisfies at least one of the following: a. The epitope is contained within amino acids 26 to 760 of SEQ ID NO: 5, and preferably, the epitope is contained within (or includes) amino acids 65 to 90 and / or 101 to 140 of SEQ ID NO: 5. b. At least amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5 interact with the antibody fragment. c. The VHH or a fragment thereof is represented by an amino acid sequence comprising an amino acid sequence having at least 80% sequence identity with at least one or a part of SEQ ID NOs: 1, 2, 3, 4.

[0144] In a plurality of embodiments, the VHH or a fragment thereof according to the present invention specifically binds to human and / or mouse FAP and satisfies at least one of the following: a. The epitope is contained within amino acids 26 to 760 of SEQ ID NO: 5, and preferably, the epitope is contained within (or includes) amino acids 65 to 90 and / or 101 to 140 of SEQ ID NO: 5. b. The VHH or a fragment thereof is the following amino acids of SEQ ID NO: 5: 1) At least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and / or G159, and / or 6) R175, and / or 7) D457 and / or Y458 specifically binds to c. The VHH or its fragment is represented by an amino acid sequence having at least 80% sequence identity with at least one or a part of SEQ ID NOs: 1, 2, 3, 4.

[0145] In one embodiment, the VHH or its fragment according to the present invention specifically binds to human and / or mouse FAP, and its epitope is included within the range of amino acids 26 to 760 of SEQ ID NO: 5, and the following amino acids of SEQ ID NO: 5: 1) At least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and / or G159, and 6) R175, and 7) D457 and / or Y458 specifically binds to.

[0146] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to human and / or mouse FAP, and its epitope is included within the range of amino acids 65 to 90 and / or 101 to 140 of SEQ ID NO: 5 (or has an epitope thereof including these), and the following amino acids of SEQ ID NO: 5: 1) At least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and / or G159, and 6) R175, and 7) D457 and / or Y458 specifically binds to.

[0147] Throughout this application, FAP is synonymous with FAP alpha and corresponds to the prolyl endopeptidase FAP of the polypeptide, which is also referred to as fibroblast activation protein alpha (FAP alpha). The gene encoding this protein is called FAP.

[0148]

Table 1

[0149] Binding to human and / or mouse FAP can be evaluated with a homogeneous mixture of different antigens. In certain embodiments, the specific binding interaction discriminates between the desired and undesired antigens in the sample, in some embodiments by about 10-fold to 100-fold or more (e.g., more than about 1000-fold or more than 10,000-fold).

[0150] In one embodiment, binding can be evaluated in vitro using cells that express human and / or mouse FAP, optionally in vivo or ex vivo as defined hereinabove. These cells can be human cells and can express endogenous human and / or mouse FAP. Alternatively, these cells can overexpress human and / or mouse FAP. Cells that overexpress human and / or mouse FAP can be human or non-human cells. Preferred cells are fibroblasts that express human and / or mouse FAP. A preferred transfected cell line is HEK293. Preferred cell lines that express FAP are GM05389 or U-87 MG. A preferred cancer cell line that expresses human FAP is U-87MG.

[0151] In one embodiment, a VHH or fragment thereof according to the invention specifically binds to human and / or mouse FAP. This evaluation is preferably performed using ELISA, surface plasmon resonance or biolayer interferometry.

[0152] It is also contemplated that a VHH or fragment thereof according to the invention binds to some naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of human and / or mouse FAP.

[0153] In one embodiment, a VHH or fragment thereof according to the invention will specifically bind to at least those analogs, variants, mutants, alleles, naturally occurring synthetic analogs, parts and fragments of human and / or mouse FAP that (still) contain the epitope of the (native / wild-type) antigen to which the VHH or fragment thereof binds.

[0154] In one embodiment, the epitope of the VHH or its fragment according to the present invention for human FAP is included within the range of amino acids 26 to 760 of SEQ ID NO: 5.

[0155] In one embodiment, the epitope of the VHH or its fragment according to the present invention is included within amino acids 65 to 90 and / or 101 to 140 of SEQ ID NO: 5.

[0156] In one embodiment, the epitope of the VHH or its fragment according to the present invention comprises an amino acid stretch or region of 65 to 90 and / or 101 to 140 of SEQ ID NO: 5.

[0157] In one embodiment, the epitope of the VHH or its fragment according to the present invention comprises a combination of amino acid stretches or regions of 65 to 90 and 101 to 140 of SEQ ID NO: 5.

[0158] In one embodiment, the epitope of the VHH or its fragment according to the present invention is included within a combination of amino acid stretches or regions of 65 to 90 and 101 to 140 of SEQ ID NO: 5.

[0159] In one embodiment, at least one of the following amino acids of SEQ ID NO: 5: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457, and Y458 binds to, contacts, or interacts with the VHH or its fragment according to the present invention.

[0160] In one embodiment, the VHH or its fragment according to the present invention is the following amino acid of SEQ ID NO: 5: 1) at least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and / or G159, and / or 6) R175, and / or 7) D457 and / or Y458 Specifically binds to.

[0161] In one embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 5: 1) At least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and / or G159, and 6) R175, and 7) D457 and / or Y458 Specifically binds to.

[0162] In one embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 5: 1) at least two or at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) at least two or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) at least two or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) at least two or at least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and G159, and / or 6) R175, and / or 7) D457 and Y458 that specifically binds thereto.

[0163] In one embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 5: 1) at least two or at least three amino acids selected from I62, S63, G64, Q65, E66, and 2) at least two or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) at least two or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) at least two or at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and G159, and 6) R175, and 7) D457 and Y458 that specifically binds thereto.

[0164] In one embodiment, the VHH or fragment thereof according to the present invention comprises the following amino acids of SEQ ID NO: 5: 1) at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) at least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and G159, and / or 6) R175, and 7) D457 and Y458 and specifically binds thereto.

[0165] In one embodiment, the VHH or fragment thereof according to the present invention comprises the following amino acids of SEQ ID NO: 5: 1) at least three amino acids selected from I62, S63, G64, Q65, E66, and 2) at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and G159, and 6) R175, and 7) D457 and Y458 and specifically binds thereto.

[0166] In one embodiment, the following amino acids of SEQ ID NO: 5: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and Y458 bind to, contact with, or interact with a VHH according to the present invention or a fragment thereof. In this connection, the human amino acids of FAP can be bound by a VHH or a fragment thereof if the amino acids belong to an epitope of the VHH or a fragment thereof.

[0167] A VHH or a fragment thereof according to the present invention is said to be "specific" for a first target antigen of interest when it binds to the first target antigen of interest with an affinity that is at least 5-fold, for example at least 10-fold, for example at least 100-fold, preferably at least 1000-fold higher than the affinity with which the VHH or a fragment thereof binds to a second molecule, in contrast to, for example, one of the closest homologs of FAP (i.e., DPP IV, dipeptidyl aminopeptidase IV, Juillerat-Jeanneret, L. et al (2017), Expert Opinion on therapeutic targets, 21: 977-991). The amino acid sequence of DPP IV has 52% identity with the amino acid sequence of FAP, yet the VHH or a fragment thereof can distinguish between the two related prolyl-specific serine proteases. Thus, in certain embodiments, when a VHH or a fragment thereof as disclosed herein is said to be "specific" for a first target antigen of interest in contrast to a second molecule, this means that it can specifically bind to the first target antigen of interest (as defined herein), but cannot bind to the second molecule.

[0168] The terms "competition", "cross-blocking", "cross-linking" and "cross-inhibition" used interchangeably herein generally refer to a VHH according to the invention or a fragment thereof that, when measured using a suitable in vitro or in vivo assay, is capable of interfering with the binding of another VHH according to the invention or a fragment thereof or another molecule to human and / or mouse FAP. Preferred cells used to test for in vitro binding to human and / or mouse FAP are cells that express human and / or mouse FAP. Preferred cell lines can be GM05389, U-87MG or transfected HEK293 as previously defined herein. Thus, more specifically, "competition (with)", "cross-blocking", "cross-linking" and "cross-inhibition" with a VHH or a fragment thereof according to the invention means interfering with or competing with the binding of another VHH or a fragment thereof according to the invention to human FAP and / or mouse FAP, whereby the binding, when measured using a suitable in vitro, cellular or in vivo assay, is reduced by at least 10%, but preferably at least 20%, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, compared to the binding of another VHH or a fragment thereof according to the invention to human and / or mouse FAP without using the "cross-blocking" VHH or a fragment thereof according to the invention. In one embodiment, a VHH or a fragment thereof according to the invention does not compete with a ligand of FAP for binding to that ligand. As a result, it is also contemplated that a VHH or a fragment thereof according to the invention does not interfere with the natural function of this receptor. This means that, in one embodiment, a VHH or a fragment thereof according to the invention does not compete with the natural ligand of human and / or mouse FAP and thus does not inhibit binding to human FAP-expressing cells and / or mouse FAP-expressing cells in an in vitro or in vivo or ex vivo environment.

[0169] In one embodiment, the VHH or fragment thereof according to the present invention does not substantially modify FAP activity, which means that it preferably does not substantially inhibit FAP activity. In the context of the present invention, "substantially" means that at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the FAP activity is still detectable compared to the same FAP activity in the absence of the VHH or fragment thereof. In the context of the present invention, the FAP activity can be exopeptidase activity and / or endopeptidase activity. This exopeptidase activity can be FAP dipeptidyl peptidase activity. In one embodiment, the VHH or fragment thereof according to the present invention does not substantially inhibit FAP dipeptidyl peptidase activity. This endopeptidase activity can be gelatinase and / or collagenase activity. In one embodiment, the VHH or fragment thereof according to the present invention does not substantially inhibit the FAP gelatinase activity and / or collagenase activity of FAP. Thus, in one embodiment, the VHH or fragment thereof according to the present invention is not a modulator of human and / or mouse FAP. In one embodiment, it is not an inhibitor and it is not an activator of human and / or mouse FAP. Any of the FAP activities can be evaluated as exemplified in Juillerat-Jeanneret L.et al, (2017), Expert Opinion on Therapeutic Targets (http: / / dx.doi.org / 10.1080 / 14728222.2017.1370455). The FAP dipeptidyl peptidase activity can be evaluated using techniques well known to those skilled in the art. Briefly, the human FAP enzyme activity can be measured using the fluorogenic substrate benzyloxycarbonyl-Gly-Pro-7-amido-4-methylcoumarin (Z-Gly-Pro-AMC; Bachem).

[0170] A VHH or a fragment thereof according to the present invention is said to exhibit "cross-reactivity" with respect to these different target proteins of interest if it is specific (as defined herein) for both of two different target proteins of interest. In one embodiment, the two different target proteins of interest can be human FAP and mouse FAP.

[0171] Hereinafter, the inventors describe some preferred structural features (i.e., the first, second, third, and fourth structural features) of the VHH or a fragment thereof according to the present invention. The VHH or a fragment thereof can be characterized by the presence of at least one or all of these four structural features: - The third and fourth structural features, - The first and second structural features, - The first, second, and third structural features - The third and fourth structural features, - The third structural feature, - The fourth structural feature, - The first, third, and fourth structural features, - The second, third, and fourth structural features, - The first and third structural features, - The first and fourth structural features, - The second and third structural features, - The second and fourth structural features - The first, second, third, and fourth structural features.

[0172] The first structural feature of the fragment according to the present invention: Based on the contact region of FAP. The first structural feature is that the fragment according to the present invention contacts, binds to, specifically binds to, or interacts with the region of human FAP contained within the range of amino acids 26 to 760 of SEQ ID NO: 5. The region within amino acids 26 to 760 of SEQ ID NO: 5 that is specifically bound or targeted by the VHH or its fragment can be a linear region (i.e., a linear epitope or a continuous epitope) within the primary amino acid sequence. Alternatively, the region may not be linear and may correspond to a structural epitope. Usually, a linear epitope contains a linear sequence of amino acids having a length of 5 to 30 amino acids, i.e., it can have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Usually, a structural epitope is characterized by several non - contiguous amino acids within amino acids 26 to 760 of SEQ ID NO: 5 that come together in the three - dimensional tertiary structure of the protein and contact the VHH or its fragment.

[0173] In the following paragraphs regarding the second structural feature of the VHH or its fragment, the linear epitope and the structural epitope of the VHH or its fragment are defined.

[0174] The second structural feature of the VHH or its fragment according to the present invention: Based on the epitope of the VHH or its fragment.

[0175] The VHH or its fragment according to the present invention that specifically binds to the epitope of human and / or mouse FAP can be further defined, alternatively or in combination with the first structural feature defined above, by the second structural feature defined below.

[0176] The second structural feature is that the VHH or its fragment according to the present invention contacts, binds to, or specifically binds to some of the amino acids within amino acids 26 to 760 of SEQ ID NO: 5. These specific amino acids within amino acids 26 to 760 of SEQ ID NO: 5 are further defined below.

[0177] In the first embodiment of this second structural feature, the VHH or a fragment thereof according to the present invention contacts, binds to, or specifically binds to at least one of the amino acids contained within positions 65 - 90 and / or 101 - 140 of SEQ ID NO: 5. Accordingly, each combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66 amino acids from the identified 66 amino acids is included for the VHH or a fragment thereof to contact.

[0178] In the second embodiment of this second structural feature, the VHH or a fragment thereof according to the present invention contacts, binds to, or specifically binds to at least one of the amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and Y458 of SEQ ID NO: 5. Accordingly, each combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 amino acids from the identified 37 amino acids is included for the VHH or a fragment thereof to contact.

[0179] In this third embodiment of the second structural feature, the stretch of amino acids 65 to 90 of SEQ ID NO: 1 defines a first region of hFAP that contacts, binds, or specifically binds to a VHH or a fragment thereof. Each amino acid within this stretch or region may not contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of this stretch or region contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, this first stretch or region is an epitope of the VHH or a fragment thereof. In one embodiment, the epitope of the VHH or a fragment thereof is contained within this first stretch or region.

[0180] Preferably, within this first stretch, at least one of Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90 contacts, binds, or specifically binds to the VHH or a fragment thereof.

[0181] More preferably, within this first stretch, at least two of Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90 contact, bind, or specifically bind to the VHH or a fragment thereof.

[0182] Even more preferably, within this first stretch, at least three of Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, and L90 contact, bind, or specifically bind to the VHH or a fragment thereof.

[0183] Even more preferably, and preferably within this first stretch, all of Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90 contact, bind to, or specifically bind to the VHH or a fragment thereof.

[0184] Most preferably, within this first stretch, all of Q65, E66, I76, V77, L78, N80, I81, E82, T83, Q85, S86, Y87, T88, I89, L90 contact, bind to, or specifically bind to the VHH or a fragment thereof.

[0185] In a fourth embodiment of this second structural feature, the stretch of amino acids 101 - 140 of SEQ ID NO: 5 defines a second region of hFAP that contacts, binds to, or specifically binds to the VHH or a fragment thereof. Each amino acid within this stretch or region need not contact, bind to, or specifically bind to the VHH or a fragment thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids of this stretch or region contact, bind to, or specifically bind to the VHH or a fragment thereof. In one embodiment, this second stretch or region is an epitope of the VHH or a fragment thereof. In one embodiment, the epitope of the VHH or a fragment thereof is contained within this second stretch or region.

[0186] Preferably, within this second stretch, at least one of L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137 contacts, binds to, or specifically binds to the VHH or a fragment thereof.

[0187] More preferably, within this second stretch, at least two of L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137 contact, bind to, or specifically bind to the VHH or a fragment thereof.

[0188] Even more preferably, within this second stretch, at least three of L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137 contact, bind to, or specifically bind to the VHH or a fragment thereof.

[0189] Most preferably, within this second stretch, all of L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137 contact, bind to, or specifically bind to the VHH or a fragment thereof.

[0190] In a fifth embodiment of this second structural feature, the VHH or a fragment thereof further contacts additional amino acids such as I62, S63, G64, S91, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5.

[0191] In this sixth embodiment of the second structural feature, each of the first stretch and the second extension or region defined above contacts, binds to, or specifically binds to a VHH or a fragment thereof. In one embodiment, the combination of these two stretches defines a structural epitope of the VHH or a fragment thereof. In one embodiment, the structural epitope is included within the combination of these two stretches. Each amino acid within each of these stretches or regions need not contact, bind to, or specifically bind to the VHH or a fragment thereof. In one embodiment, 1, 2, 3, 4, 5, or 6 amino acids of each of the stretches or regions (or more depending on the length of each stretch) contact, bind to, or specifically bind to the VHH or a fragment thereof. In one embodiment, the VHH or a fragment thereof further contacts additional amino acids such as I62, S63, G64, S91, V158, G159, R175, D457, and / or Y458 of SEQ ID NO: 5.

[0192] In one embodiment, provided is a VHH or a fragment thereof according to the invention that specifically binds to an epitope of human FAP, the epitope being included within amino acid stretch or region 65 - 90 and / or 101 - 140 of SEQ ID NO: 5. Optionally, the VHH or a fragment thereof further contacts additional amino acids such as I62, S63, G64, S91, V158, G159, R175, D457, and / or Y458 of SEQ ID NO: 5.

[0193] In one embodiment, provided is a VHH or a fragment thereof according to the invention that specifically binds to an epitope of human FAP, the epitope being included within the combination of amino acid stretches or regions 65 - 90 and 101 - 140 of SEQ ID NO: 5. Optionally, the VHH or a fragment thereof further contacts additional amino acids such as I62, S63, G64, S91, V158, G159, R175, D457, and / or Y458 of SEQ ID NO: 5.

[0194] In one embodiment, the VHH or a fragment thereof according to the invention is the following amino acids of SEQ ID NO: 5: 1) At least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and / or G159, and / or 6) R175, and / or 7) D457 and / or Y458 contact with them, or bind to them, or specifically bind to them.

[0195] In one embodiment, the VHH or fragment thereof according to the present invention comprises the following amino acids of SEQ ID NO: 5: 1) At least one, or at least two, or at least three amino acids selected from I62, S63, G64, Q65, E66, and 2) At least one, or at least two, or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) At least one, or at least two, or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) At least one, or at least two, or at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and / or G159, and 6) R175, and 7) D457 and / or Y458 contact with, bind to, or specifically bind to them.

[0196] In one embodiment, the VHH or a fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 5: 1) at least two or at least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) at least two or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) at least two or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) at least two or at least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and G159, and / or 6) R175, and / or 7) D457 and Y458 contact with, bind to, or specifically bind to them.

[0197] In one embodiment, the VHH or a fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 5: 1) at least two or at least three amino acids selected from I62, S63, G64, Q65, E66, and 2) at least two or at least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) at least two or at least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) At least two or at least three amino acids selected from D134, L135, S136, N137, and 5) V158 and G159, and 6) R175, and 7) D457 and Y458 contact with them, or bind to them, or specifically bind to them.

[0198] In one embodiment, the VHH or fragment thereof according to the invention is the following amino acids of SEQ ID NO: 5: 1) At least three amino acids selected from I62, S63, G64, Q65, E66, and / or 2) At least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and / or 3) At least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and / or 4) At least three amino acids selected from D134, L135, S136, N137, and / or 5) V158 and G159, and / or 6) R175, and / or 7) D457 and Y458 contact with them, or bind to them, or specifically bind to them.

[0199] In one embodiment, the VHH or fragment thereof according to the invention is the following amino acids of SEQ ID NO: 5: 1) At least three amino acids selected from I62, S63, G64, Q65, E66, and 2) At least three amino acids selected from I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, and 3) At least three amino acids selected from L105, S106, P107, D108, R109, Q110, F111, and 4) At least three amino acids selected from D134, L135, S136, N137, and 5) V158 and G159, and 6) R175, and 7) D457 and Y458 contact with, bind to, or specifically bind to them.

[0200] In one embodiment, the following amino acids of SEQ ID NO: 5: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and Y458 bind to, contact, or interact with the VHH or a fragment thereof according to the present invention.

[0201] In one embodiment, the following amino acids of SEQ ID NO: 5: I62, S63, G64, Q65, E66, I76, V77, L78, N80, I81, E82, T83, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and Y458 bind to, contact, or interact with the VHH or a fragment thereof according to the present invention.

[0202] The third structural feature of the VHH or a fragment thereof according to the present invention: based on the full-length sequence The third structural feature means that the VHH or a fragment thereof according to the present invention relates to the full-length amino acid sequence. The present invention discloses a family of VHHs that are structurally closely related, represented by an amino acid sequence comprising, consisting of, or essentially consisting of SEQ ID NO: 4 or a fragment thereof. VHH VHH1 is represented by SEQ ID NO: 4 (see the following table).

[0203] In one embodiment, the VHH or fragment thereof according to the present invention can be defined by its first structural feature defined above and its third structural feature further defined below.

[0204] In one embodiment, the VHH or fragment thereof according to the present invention can be defined by its second structural feature defined above and its third structural feature further defined below.

[0205] In a first embodiment of this third structural feature, the VHH or fragment thereof according to the present invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4 or a part thereof. In one embodiment, the sequence identity with this sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0206] In one embodiment of this third structural feature, the VHH or fragment thereof according to the present invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 81% sequence similarity with SEQ ID NO: 4 or a part thereof. In one embodiment, the sequence similarity with this sequence is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0207] In a second embodiment of this third structural feature, the VHH or fragment thereof according to the present invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4 or a part thereof, and has a length ranging from the exact length of SEQ ID NO: 4 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.

[0208] In one embodiment of this third structural feature, the VHH or fragment thereof according to the invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 81% sequence similarity with SEQ ID NO: 4 or a part thereof, and having a length ranging from the exact length of SEQ ID NO: 4 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.

[0209] For example, tags such as His-tag can be added to the VHH or fragment thereof according to the invention. Usually, the His-tag contains 4, 5, 6, 7, 8, 9, 10 histidines. Alternative tags can be hemagglutinin tag (HA-tag): YPYDVPDYA (SEQ ID NO: 11); YPYDVPDYGS (SEQ ID NO: 12) or cysteine tag (Cys-tag). The cysteine tag is a tag containing one or several cysteines. Non-limiting examples of cysteine tags are C; GGC; SPSTPPTPSPSTPPC (SEQ ID NO: 13).

[0210] The methods by which identity and similarity are evaluated are described in detail in the part for the purpose of the definitions at the end of the description. Usually, when identity is defined by reference to a SEQ ID NO, the identity is evaluated over the entire SEQ ID NO. However, it is also encompassed by the present invention that identity (or similarity) is evaluated over a part (or fragment) of said sequence. In this context, a part can mean at least 50%, 60%, 70%, 80%, 90%, 95% of the length of the SEQ ID NO. The length of the sequence encompassed may still be longer than the length of the SEQ ID NO used to evaluate the identity (or similarity) (i.e., the same as that of the SEQ ID NO even if the identity (or similarity) is evaluated over a part of this SEQ ID NO, a length of at least 50%, 60%, 70%, 80%, 90% of the length of the SEQ ID NO or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of the SEQ ID NO).

[0211] In this third embodiment of the third structural feature, the length of the VHH or its fragment is 110 to 130 amino acids or 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. This length does not include the length of tags such as His tags that can be added to the sequence of the VHH or its fragment.

[0212] In one embodiment, the VHH or its fragment according to the present invention has a length ranging from the exact length of SEQ ID NO: 4 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.

[0213] In one embodiment, the VHH or its fragment according to the present invention has a length ranging from 110 to 130 amino acids or 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids and includes SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0214] In a fourth embodiment of this third structural feature, the VHH or a fragment thereof according to the invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 80% sequence identity with at least one of SEQ ID NO: 4 or a part thereof, and the length of the VHH or a fragment thereof is 80 to 150 amino acids or 90 to 140, or 100 to 130, or 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity with at least one of these sequences is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0215] In one embodiment of this third structural feature, the VHH or a fragment thereof according to the invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 81% sequence similarity with at least one of SEQ ID NO: 4 or a part thereof, and the length of the VHH or a fragment thereof is 80 to 150 amino acids or 90 to 140, or 100 to 130, or 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence similarity with at least one of these sequences is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0216] In one embodiment, the VHH or a fragment thereof according to the present invention contacts, binds to, or specifically binds to at least one (preferably both) of the stretches or regions of amino acids of SEQ ID NO: 5 (i.e., amino acid stretches or regions 65-90 and / or 101-140 of SEQ ID NO: 5) defined previously herein. In one embodiment, the epitope of said VHH or a fragment thereof is contained within these stretches or regions of amino acids of SEQ ID NO: 5.

[0217] Furthermore, in one embodiment, the VHH or a fragment thereof according to the present invention has, for a structural epitope, a combination of stretches or regions of amino acids of SEQ ID NO: 5 (i.e., amino acid stretches or regions 65-90 and / or 101-140 of SEQ ID NO: 5) defined previously herein. In one embodiment, the structural epitope of said VHH or a fragment thereof is contained within these stretches or regions of amino acids of SEQ ID NO: 5.

[0218] These epitopes define a family of VHH or a fragment thereof. This family of VHH or a fragment thereof shares at least one of these epitopes, linear epitopes, and / or this structural epitope.

[0219] In one embodiment, the VHH or a fragment thereof according to the present invention - is represented by an amino acid sequence that includes or consists essentially of an amino acid sequence having at least 80% sequence identity (or similarity) with SEQ ID NO: 4 or a part thereof (the third structural feature), - which has, for an epitope, an amino acid stretch or region contained within 65-90 and / or 101-140 of SEQ ID NO: 5 (the second structural feature).

[0220] In one embodiment, the sequence identity (or similarity) with this sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0221] In one embodiment, the VHH or fragment thereof according to the invention has a length ranging from the exact length of SEQ ID NO: 4 or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.

[0222] In one embodiment, the VHH or fragment thereof according to the invention - is represented by or consists essentially of an amino acid sequence having at least 80% sequence identity (or similarity) with SEQ ID NO: 4 or a part thereof (the third structural feature), - contacts, binds to, or specifically binds to at least one of amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5. (the second structural feature).

[0223] In one embodiment, the sequence identity (or similarity) with this sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0224] In one embodiment, the VHH or fragment thereof according to the invention has a length ranging from the exact length of SEQ ID NO: 4 or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.

[0225] Each of the other embodiments of the second structural feature can be combined with each embodiment of the third structural feature.

[0226] Fourth Structural Feature: CDR / CDR Grafting In the fourth structural feature, the VHH or fragment thereof according to the invention is represented by an amino acid sequence comprising at least one combination of CDR sequences selected from the group consisting of a CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. One or two amino acids of said CDR1, CDR2 and / or CDR3 can be substituted with another amino acid without substantially changing the activity of the resulting VHH or fragment thereof. The same applies to any framework region of the VHH or fragment thereof. Each of these variants is also encompassed by the present invention. The activity of said variant is the specific binding activity as defined hereinbefore. In the context of the present invention, "substantially" may mean that at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of said binding activity is still detectable as compared to the activity of a VHH or fragment thereof having the initial CDR and / or FR regions.

[0227] Thus, in certain embodiments, the present invention provides a VHH or fragment thereof according to the invention, comprising a heavy chain antibody having or derived from a (generic) structure. FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, as further defined herein (see Table 2 listing the amino acid sequences of the heavy chain variable domains raised against human and / or mouse FAP).

[0228] [Table 2]

[0229] The present invention is not limited with respect to the origin of the VHH or fragment thereof (or nucleotide sequence for expressing them) according to the present invention, nor is it limited with respect to the method by which the VHH or fragment thereof or the nucleotide sequences disclosed herein are generated or obtained (or have been obtained). Thus, the VHH or fragment thereof according to the present invention can be a naturally occurring amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence. Methods for isolating VHH or fragments thereof and methods for generating VHH or fragments thereof, as well as nucleic acid molecules encoding VHH or fragments thereof, constructs containing these nucleic acid molecules, and cells containing these constructs are disclosed in detail in the definition section at the end of the description.

[0230] In a specific but non-limiting aspect of the present invention, the amino acid sequence of the VHH or fragment thereof is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, and as such, includes, but is not limited to, "humanized" immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized VHH sequences), "camelized" immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity maturation, CDR grafting, veneering, combinations of fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those skilled in the art (starting from, for example, synthetic, random, or naturally occurring immunoglobulin sequences), or any suitable combination of any of the foregoing. The VHH or fragment thereof according to the present invention can also be suitably humanized as described in more detail herein to provide one or more additional (partial or complete) humanized amino acid sequences of the present invention.

[0231] In one embodiment, the VHH or fragment thereof according to the present invention is obtained from the aforementioned VHH or fragment thereof using CDR grafting.

[0232] Preferred VHHs or fragments thereof according to the present invention comprise: - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1 and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. The CDR2 region is derived from another VHH or fragment thereof. - A CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2 and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. The CDR1 region is derived from another VHH or fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1 and a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. The CDR3 region is derived from another VHH or fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, which comprises CDR2 and CDR3 regions derived from another VHH or fragment thereof and can have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. - A CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, which may include CDR1 and CDR3 regions from another VHH or a fragment thereof, and may have the FRs identified in Table 2. Alternatively, the FR regions may be derived from another VHH or a fragment thereof. - A CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3, which may include CDR1 and CDR2 regions from another VHH or a fragment thereof, and may have the FRs identified in Table 2. Alternatively, the FR regions may be derived from another VHH or a fragment thereof.

[0233] Similarly, when the amino acid sequence includes a synthetic or semi-synthetic sequence (such as a partially humanized sequence), the sequence may optionally be further preferably humanized as described herein again to provide one or more additional (partial or complete) humanized amino acid sequences as disclosed herein. At the end of the description, more detailed definitions of "agonist", "antagonist", "variant", and "post-translational structural property evaluation" are provided.

[0234] In particular, the humanized VHH or a fragment thereof according to the invention may be represented by an amino acid sequence in which there is at least one amino acid residue (in particular in at least one framework residue) that is a humanized substitution and / or corresponds to a humanized substitution. Additionally or alternatively, the sequence of the framework region of the naturally occurring VHH sequence is replaced with one or more closely related human V HBy comparing with the corresponding framework arrays of the arrays, other potentially useful humanized substitutions can be identified, and then one or more (or combinations thereof) of the potentially useful humanized substitutions thus determined can be introduced into the VHH array (in any manner known per se as further described herein), and the resulting humanized VHH array or functional fragment thereof can be tested for affinity for the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited amount of trial and error, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art.

[0235] In one embodiment, a VHH or fragment thereof according to the invention can be defined by its first structural feature as defined above and its fourth structural feature as further defined herein. In one embodiment, a VHH or fragment thereof according to the invention can be defined by its second structural feature as defined above and its fourth structural feature as further defined herein. In one embodiment, a VHH or fragment thereof according to the invention can be defined by its second structural feature as defined above and its third and fourth structural features as further defined herein.

[0236] In one embodiment, a VHH or fragment thereof according to the invention contacts, binds to, or specifically binds to at least one (preferably both) of the stretches or regions of amino acids of SEQ ID NO: 5 (i.e., amino acid stretches or regions 65-90 and / or 101-140 of SEQ ID NO: 5) previously defined herein. In one embodiment, the epitope of the VHH or fragment thereof is contained within these stretches or regions of amino acids of SEQ ID NO: 5.

[0237] Furthermore, the VHH or fragment thereof according to the present invention has, with respect to the structural epitope, a stretch or combination of regions of the amino acids of SEQ ID NO: 5 as previously defined herein (i.e., amino acid stretches or regions 65-90 and / or 101-140 of SEQ ID NO: 5). In one embodiment, the structural epitope of said VHH or fragment thereof is contained within this combination of stretches or regions of the amino acids of SEQ ID NO: 5.

[0238] These epitopes define a family of VHH or fragments thereof. This family shares at least one of these epitopes, linear epitopes, and / or this structural epitope.

[0239] Preferred VHH or fragments thereof according to the present invention include the following: - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1 and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. The CDR2 region is derived from another VHH or fragment thereof. - A CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2 and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or fragment thereof. The CDR1 region is derived from another VHH or fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1 and a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. The CDR3 region is derived from another VHH or a fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, which comprises CDR2 and CDR3 regions derived from another VHH or a fragment thereof and may have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. - A CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, which comprises CDR1 and CDR3 regions derived from another VHH or a fragment thereof and may have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. - A CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3, which comprises CDR1 and CDR2 regions derived from another VHH or a fragment thereof and may have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof (the fourth structural feature). Also, preferred antibody fragments are - Having an epitope contained within an amino acid stretch or region contained within positions 65 - 90 and / or 101 - 140 of SEQ ID NO: 5 (the second structural feature).

[0240] Preferred VHHs or fragments thereof according to the present invention comprise the following: - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. The CDR2 region is derived from another VHH or a fragment thereof. - A CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, and a CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. The CDR1 region is derived from another VHH or a fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, and a CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2. The FR regions can be as identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. The CDR3 region is derived from another VHH or a fragment thereof. - A CDR1 region comprising, consisting of, or essentially consisting of SEQ ID NO: 1, which includes CDR2 and CDR3 regions derived from another VHH or a fragment thereof, and may have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. - A CDR2 region comprising, consisting of, or essentially consisting of SEQ ID NO: 2, which includes CDR1 and CDR3 regions derived from another VHH or a fragment thereof, and may have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof. - A CDR3 region comprising, consisting of, or essentially consisting of SEQ ID NO: 3, which includes CDR1 and CDR2 regions derived from another VHH or a fragment thereof, and may have the FRs identified in Table 2. Alternatively, the FR regions can be derived from another VHH or a fragment thereof (the fourth structural feature). Also, preferred antibody fragments are - Contact, bind to, or specifically bind to at least one of amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457, and / or Y458 of SEQ ID NO: 5 (second structural feature).

[0241] Each of the other embodiments of the second structural feature can be combined with each embodiment of the fourth structural feature.

[0242] In one embodiment, the VHH or fragment thereof according to the invention is represented by the first structural feature and / or the second structural feature and / or the third structural feature and / or the fourth structural feature identified herein. Alternatively or in combination with said structural features, said VHH or fragment thereof is characterized by at least one of the following functional features: - Specifically bind to human and / or mouse FAP, preferably specifically bind to human FAP and mouse FAP, and - Do not modulate FAP activity.

[0243] Specific binding is described earlier herein. Most preferably, the VHH or fragment thereof according to the invention has a K in the range of 10 -9 ~10 -12 mol / liter and / or a k in the range of 10 D ~10 -2 ~10 -5 seconds -1 as evaluated preferably using biolayer interferometry, more preferably a K in the range of 10 off ~10 -9 ~10 -12 mol / liter and a k in the range of 10 D ~10 -2 ~10 -5 seconds -1 as evaluated preferably using biolayer interferometry. offIt specifically binds to human and / or mouse FAP.

[0244] A second functional feature regarding the fact that the VHH or its fragment may not be a modulator (i.e., not an inhibitor and not an activator of human and / or mouse FAP) is also described in detail herein.

[0245] Thus, in one embodiment, the VHH or its fragment according to the invention should satisfy at least one structural feature and / or at least one functional feature.

[0246] In a plurality of embodiments, the combination according to the invention comprises: - A VHH or its fragment according to the invention that specifically binds to human and / or mouse FAP, wherein the epitope is contained within the amino acid stretch of SEQ ID NO: 5 or regions 65 - 90 and / or 101 - 140, and a labeled compound comprising iodine - 131 and GMIB, - A VHH or its fragment according to the invention that specifically binds to human and / or mouse FAP, wherein the epitope is contained within the amino acid stretch of SEQ ID NO: 5 or regions 65 - 90 and / or 101 - 140, and a labeled compound comprising DOTA or DTPA and lutetium - 177, preferably, the labeled compound is 177 Lu - DOTA - PEG 7 -Tz or 177 Lu - DTPA - PEG 7 -Tz, which can be represented by the labeled compound, - A VHH or its fragment according to the invention that specifically binds to human and / or mouse FAP, wherein the epitope is contained within the amino acid stretch of SEQ ID NO: 5 or regions 65 - 90 and / or 101 - 140, and a labeled compound comprising DOTA and actinium - 225, preferably, the labeled compound is 225 Ac - DOTA - PEG 7 -Tz, which can be represented by the labeled compound, or - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the epitope is contained within the amino acid stretch of SEQ ID NO: 5 or regions 65-90 and / or 101-140, and technetium-99m.

[0247] In multiple embodiments, the combination according to the present invention includes: - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the structural epitope is contained within the combination of the amino acid stretch of SEQ ID NO: 5 or regions 65-90 and 101-140, and GMIB and iodine-131. - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the structural epitope is contained within the combination of the amino acid stretch of SEQ ID NO: 5 or regions 65-90 and 101-140, and DOTA or DTPA and lutetium-177, preferably the labeled compound is 177 Lu-DOTA-PEG 7 -Tz or 177 Lu-DTPA-PEG 7 -Tz, represented by the labeled compound. - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the structural epitope is contained within the combination of the amino acid stretch of SEQ ID NO: 5 or regions 65-90 and 101-140, and DOTA and actinium-225, preferably the labeled compound is 225 Ac-DOTA-PEG 7 -Tz, represented by the labeled compound, or - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the structural epitope is contained within the amino acid stretch of SEQ ID NO: 5 or the combination of regions 65-90 and 101-140, and technetium-99m.

[0248] In multiple embodiments, the combination according to the present invention comprises: - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein at least one of the amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5 interacts with the VHH or fragment, and the VHH or fragment and a labeled compound comprising GMIB and iodine-131 - A labeled compound comprising a VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein at least one of the amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5 interacts with the VHH or fragment, and the VHH or fragment and a labeled compound comprising DOTA or DTPA and lutetium-177, preferably, the labeled compound is 177 Lu-DOTA-PEG 7 -Tz or 177 Lu-DTPA-PEG 7 A labeled compound that can be represented by -Tz - A VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein at least one of the amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5 interacts with the VHH or fragment, a labeled compound comprising the VHH or a fragment thereof, DOTA and actinium-225, preferably, the labeled compound is 225 Ac-DOTA-PEG 7 - represented by -Tz, a labeled compound, or - A VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein at least one of the amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, I81, E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91, L105, S106, P107, D108, R109, Q110, F111, D134, L135, S136, N137, V158, G159, R175, D457 and / or Y458 of SEQ ID NO: 5 interacts with the VHH or fragment, a labeled compound comprising the VHH or a fragment thereof and technetium-99m.

[0249] In multiple embodiments, the combination according to the present invention comprises: - A VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the VHH or fragment is represented by an amino acid sequence comprising an amino acid sequence having at least 80% sequence identity (or similarity) with at least one or a part of SEQ ID NOs: 1, 2, 3, 4, a labeled compound comprising the VHH or a fragment thereof, GMIB and iodine-131, - A VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the VHH or fragment comprises an amino acid sequence having at least 80% sequence identity (or similarity) with at least one or a part of SEQ ID NOs: 1, 2, 3, 4, and a labeled compound comprising DOTA or DTPA and lutetium-177, preferably, the labeled compound is 177 Lu-DOTA-PEG 7 -Tz or 177 Lu-DTPA-PEG 7 -Tz, and can be represented by - A VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the VHH or fragment comprises an amino acid sequence having at least 80% sequence identity (or similarity) with at least one or a part of SEQ ID NOs: 1, 2, 3, 4, and a labeled compound comprising DOTA and actinium-225, preferably, the labeled compound is 225 Ac-DOTA-PEG 7 -Tz, and can be represented by, or - A VHH or a fragment thereof according to the present invention that specifically binds to human and / or mouse FAP, wherein the VHH or fragment comprises an amino acid sequence having at least 80% sequence identity (or similarity) with at least one or a part of SEQ ID NOs: 1, 2, 3, 4, and a labeled compound comprising technetium-99m and the VHH or a fragment thereof.

[0250] Human folate receptor alpha (FOLR1) In multiple embodiments, the VHH or a fragment thereof according to the present invention is particularly suitable for binding to human folate receptor alpha (FOLR1).

[0251] Example 8 demonstrates that the TCO conjugate FOLR1-binding VHH can be used for pretargeting applications.

[0252] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to human folate receptor α (FOLR1, represented by SEQ ID NO: 14), but does not specifically bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3), and is further characterized by additional (structural) features (such as the first, second, third, and / or fourth structural features) defined herein.

[0253] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to human folate receptor α (FOLR1, represented by SEQ ID NO: 14), but does not specifically bind to mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3), and is further characterized by additional (structural) features (such as the first, second, third, and / or fourth structural features) defined herein.

[0254] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to human folate receptor α (FOLR1, represented by SEQ ID NO: 14), but does not specifically bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3) (or the VHH or fragment thereof specifically binds to human FOLR1, but does not specifically bind to either human FOLR2 or human FOLR3), and satisfies at least one of the following: d. The epitope is contained within amino acids 25 - 233 of SEQ ID NO: 14. e. At least amino acids C89, G90, E91, M92, A93, P94, E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181, F182, P183, and / or T184 of SEQ ID NO: 14 interact with the VHH or fragment thereof. The f.VHH or a fragment thereof is represented by an amino acid sequence having at least 60% sequence identity with at least one or a part of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 or 22.

[0255] In one embodiment, provided is a VHH or a fragment thereof according to the present invention that specifically binds to human folate receptor α (FOLR1) but does not specifically bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or the VHH or a fragment thereof specifically binds to human FOLR1 but does not specifically bind to any of human FOLR2 or human FOLR3), and the VHH or a fragment thereof satisfies a) and / or b): a. The epitope is contained within amino acids 25 to 233 of SEQ ID NO: 14, and the VHH or a fragment thereof is the following amino acids of SEQ ID NO: 14: 1) at least one, at least two or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) at least one, at least two or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or 3) at least one, at least two or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 and specifically binds thereto, b. The VHH or a fragment thereof is represented by an amino acid sequence containing at least one of SEQ ID NOs: 21, 22, 16, 17, 18, 19 or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0256] In one embodiment, provided is a VHH or a fragment thereof according to the present invention that specifically binds to human folate receptor α (FOLR1), but does not specifically bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or the VHH or the fragment thereof specifically binds to human FOLR1, but does not specifically bind to any of human FOLR2 or human FOLR3), wherein the epitope is contained within amino acids 25 to 233 of SEQ ID NO: 14, and the VHH or the fragment thereof is the following amino acids of SEQ ID NO: 14: 1) at least one, or at least two, or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) at least one, or at least two, or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and 3) at least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 to which it specifically binds.

[0257] In one embodiment, provided is a VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3), and the VHH or the fragment thereof is represented by an amino acid sequence comprising at least one of SEQ ID NOs: 21, 22, 15, 16, 17, 18, 19 or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0258] In one embodiment, provided is a VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3), and the VHH or the fragment thereof is represented by an amino acid sequence comprising at least one of SEQ ID NO: 21, 22, 15, 16, 17, 18, 19 or 20 and having at least 63% sequence identity over the full length of the sequence or over at least 50% of the length of the sequence.

[0259]

Table 3

[0260] In connection with the present invention, folate receptor α, also known as folate receptor 1 or folate-binding protein, is denoted by the abbreviations FOLR1 or FR-α, both at the protein level and at the gene level. Officially, FR-α is the protein name recommended by Uniprot, and FOLR1 is used as the gene name.

[0261] Binding to human FOLR1 can be evaluated in a homogeneous mixture of different antigens. In certain embodiments, the specific binding interaction discriminates between the desired antigen and the undesired antigen in the sample, and in some embodiments, discriminates by more than about 10 to 100-fold (e.g., more than about 1000-fold or 10000-fold).

[0262] In one embodiment, binding can be evaluated in vitro using cells that express human FOLR1, and optionally, can also be evaluated in vivo or ex vivo as defined previously herein. These cells can be human cells and can express endogenous human FOLR1. Alternatively, these cells can overexpress human FOLR1. Cells that overexpress human FOLR1 can be human cells or non-human cells. Preferred cells are SKOV3 and OVCAR3.

[0263] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to human FOLR1 and does not specifically bind to mouse FOLR1 or human FOLR2 or human FOLR3. This evaluation is preferably performed using ELISA or SPR.

[0264] The VHH or fragment thereof is also expected to bind to some naturally occurring or synthetic analogs, variants, alleles, parts, and fragments of human FOLR1.

[0265] In one embodiment, the VHH or fragment thereof according to the present invention will specifically bind to at least those analogs, variants, mutants, alleles, naturally occurring analogs, synthetic analogs, parts, and fragments of human FOLR1 that (still) contain the epitope of the (natural / wild-type) antigen to which the VHH or fragment thereof binds. The epitope of human FOLR1 of the VHH or fragment thereof of the present invention is contained within amino acids 25 to 233 of SEQ ID NO: 14.

[0266] In one embodiment, at least one of the following amino acids of this portion of SEQ ID NO: 14: C89, G90, E91, M92, A93, P94, E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181, F182, P183 and / or T184 is bound by the VHH or fragment thereof.

[0267] In another embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 14: 1) at least one, or at least two, or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) At least one, or at least two, or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or 3) At least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 that specifically binds thereto.

[0268] In another embodiment, the VHH or fragment thereof according to the invention comprises the following amino acids of SEQ ID NO: 14: 1) At least one, or at least two, or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and 2) At least one, or at least two, or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and 3) At least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 that specifically binds thereto.

[0269] In another embodiment, the VHH or fragment thereof according to the invention comprises the following amino acids of SEQ ID NO: 14: 1) At least two or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) At least two or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or 3) At least two or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 bind specifically to

[0270] In another embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 14: 1) At least two or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and 2) At least two or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and 3) At least two or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 bind specifically to

[0271] In another embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 14: 1) At least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) At least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or 3) Or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 bind specifically to

[0272] In one embodiment, the VHH or fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 14: 1) At least three amino acids selected from C89, G90, E91, M92, A93 or P94, and 2) At least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and 3) At least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 bind specifically to.

[0273] In another embodiment, the following amino acids of amino acids 25 - 233 of SEQ ID NO: 14: C89, G90, E91, M92, A93, P94, E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181, F182, P183 and T184 are bound to the VHH or fragment thereof according to the present invention.

[0274] In this connection, one amino acid of human FOLR1 can be bound to the VHH or fragment thereof when the amino acid belongs to the epitope of the VHH or fragment thereof.

[0275] The first structural feature of the VHH or fragment thereof The first structural feature is that the VHH or fragment thereof according to the present invention contacts, binds to, or specifically binds to the region of human FOLR1 contained within amino acids 25 to 233 of SEQ ID NO: 14. SEQ ID NO: 14 is the human amino acid sequence of human FOLR1. Amino acids 25 to 233 (R25 to M233) of SEQ ID No. 14 are defined as the extracellular domain of human FOLR1 (excluding the signal peptide M1 to T24, and the GPI anchor S234 and propeptide G235 to S257) (UniProt Knowledgebase: entry P15328). The region within amino acids 25 to 233 of SEQ ID NO: 14 that is specifically bound or targeted by the VHH or fragment thereof can be a linear region (i.e., a linear epitope or continuous epitope) within the primary amino acid sequence. Alternatively, the region may not be linear and may correspond to a structural epitope. Typically, a linear epitope comprises a linear sequence of amino acids having a length of 5 to 30 amino acids, i.e., it can have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Typically, a structural epitope is characterized by several non - contiguous amino acids within amino acids 25 to 233 of SEQ ID NO: 14 that come together in the three - dimensional tertiary structure of the protein and contact the VHH or fragment thereof.

[0276] In the following paragraphs regarding the second structural feature of the VHH or fragment thereof, the linear epitope and the structural epitope of the VHH or fragment thereof are defined.

[0277] The second structural feature of the VHH or fragment thereof The VHH or fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) can be further defined, alternatively or in combination, by a second structural feature.

[0278] The VHH or fragment thereof according to the invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) can alternatively or in combination be further defined by a second structural feature.

[0279] The second structural feature is that the VHH or fragment thereof according to the invention contacts, binds to, or specifically binds to some of the amino acids within amino acids 25 - 233 of SEQ ID NO: 14. These specific amino acids within amino acids 25 - 233 of SEQ ID NO: 14 are further defined below.

[0280] In one embodiment, the VHH or fragment thereof according to the invention contacts, binds to, or specifically binds to at least one of the amino acids C89, G90, E91, M92, A93, P94, E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181, F182, P183 and / or T184 of SEQ ID NO: 14. Accordingly, each combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acids from the identified 26 amino acids is included for the VHH or fragment thereof to contact.

[0281] In one embodiment, the linear stretch of amino acids 89 - 94 of SEQ ID NO: 14 defines a first linear region of hFOLR1 that contacts, binds to, or specifically binds to the VHH or fragment thereof. Each amino acid within this stretch or region does not necessarily contact, bind to, or specifically bind to the VHH or fragment thereof. In one embodiment, 1, 2, 3, 4, 5 or 6 amino acids of this linear stretch or region contact, bind to, or specifically bind to the VHH or fragment thereof. In one embodiment, this linear stretch or region is an epitope of the VHH or fragment thereof.

[0282] In one embodiment, the linear stretch of amino acids 140 - 150 of SEQ ID NO: 14 defines a second linear region of hFOLR1 that contacts, binds, or specifically binds to a VHH or a fragment thereof. Each amino acid within this stretch or region need not contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids of this linear stretch or region contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, this second linear stretch or region is a second epitope of the VHH or a fragment thereof.

[0283] In one embodiment, the linear stretch of amino acids 176 - 184 of SEQ ID NO: 14 defines a third linear region of hFOLR1 that contacts, binds, or specifically binds to a VHH or a fragment thereof. Each amino acid within this stretch or region need not contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of this linear stretch or region contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, this third linear stretch or region is a third epitope of the VHH or a fragment thereof.

[0284] In one embodiment, each of the first, second, and third linear stretches or regions defined above contacts, binds, or specifically binds to a VHH or a fragment thereof. The combination of these three stretches defines a structural epitope of the VHH or a fragment thereof. Each amino acid within each of these stretches or regions need not contact, bind, or specifically bind to the VHH or a fragment thereof. In one embodiment, 1, 2, 3, 4, 5, or 6 amino acids (or more depending on the length of each stretch) of each linear stretch or region contact, bind, or specifically bind to the VHH or a fragment thereof.

[0285] In one embodiment, there is provided a VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3), wherein the epitope comprises a linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150, and / or 176-184.

[0286] In one embodiment, there is provided a VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of human folate receptor β (FOLR2) or human folate receptor γ (FOLR3), wherein the epitope comprises a linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150, and / or 176-184.

[0287] In one embodiment, there is provided a VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3), wherein the epitope comprises a combination of linear amino acid stretches of regions 89-94, 140-150, and 176-184 of SEQ ID NO: 14.

[0288] In one embodiment, there is provided a VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of human folate receptor β (FOLR2) or human folate receptor γ (FOLR3), wherein the epitope comprises a combination of linear amino acid stretches of regions 89-94, 140-150, and 176-184 of SEQ ID NO: 14.

[0289] In one embodiment, the VHH or a fragment thereof according to the present invention is the following amino acids of SEQ ID NO: 14: - at least one, or at least two, or at least three amino acids selected from C89, G90, E91, M92, A93, or P94, and / or At least one, or at least two, or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or at least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact with, or bind to, or specifically bind to.

[0290] In another embodiment, the VHH or fragment thereof according to the invention comprises the following amino acids of SEQ ID NO: 14: - at least one, or at least two, or at least three, or at least four, or at least five, or all six amino acids selected from C89, G90, E91, M92, A93 or P94, and - at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or all eleven amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and - at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or all nine amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact with, or bind to, or specifically bind to.

[0291] In another embodiment, the VHH or fragment thereof according to the invention comprises the following amino acids of SEQ ID NO: 14: 1) at least two or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) at least two or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or 3) at least two or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact, bind or specifically bind to.

[0292] In another embodiment, the VHH or fragment thereof according to the invention is the following amino acids of SEQ ID NO: 14: 1) at least two or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and 2) at least two or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and 3) at least two or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact, bind or specifically bind to.

[0293] In another embodiment, the VHH or fragment thereof according to the invention is the following amino acids of SEQ ID NO: 14: 1) at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or 2) at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or 3) at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact, bind or specifically bind to.

[0294] In another embodiment, the VHH or a fragment thereof according to the present invention comprises the following amino acids of SEQ ID NO: 14: 1) at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and 2) at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and 3) or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact, or bind, or specifically bind to.

[0295] In one embodiment, the VHH or a fragment thereof according to the present invention contacts, or binds, or specifically binds to the following amino acids of SEQ ID NO: 14: C89, G90, E91, M92, A93 and / or P94.

[0296] In one embodiment, the VHH or a fragment thereof according to the present invention contacts, or binds, or specifically binds to the following amino acids of SEQ ID NO: 14: E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 and / or Y150.

[0297] In one embodiment, the VHH or a fragment thereof according to the present invention contacts, or binds, or specifically binds to the following amino acids of SEQ ID NO: 14: Q176, P177, F178, H179, F180, Y181, F182, P183 and / or T184.

[0298] In one embodiment, the following amino acids of amino acids 25 to 233 of SEQ ID NO: 14: C89, G90, E91, M92, A93, P94, E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181, F182, P183 and T184 contact, bind or specifically bind to the VHH or a fragment thereof according to the present invention.

[0299] The third structural feature of the VHH or a fragment thereof The VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) can alternatively or in combination be further defined by a third structural feature.

[0300] The VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) can alternatively or in combination be further defined by a third structural feature.

[0301] The third structural feature relates to an (entire-length) amino acid sequence that represents a way of defining a family of the VHH or a fragment thereof according to the present invention. The present invention discloses a family of structurally closely related VHH or a fragment thereof, represented by an amino acid sequence comprising, consisting of or essentially consisting of SEQ ID NO: 21 or 22. VHH VHH2 is represented by SEQ ID NO: 21, and VHH3 is represented by SEQ ID NO: 22 (see Table 4 below).

[0302] Each of SEQ ID NOs: 15, 17 and 18 is part of VHH2 that is conserved among the family of the VHH or a fragment thereof (see Table 4 below).

[0303] Each of Sequence Nos. 16, 19 and 20 is part of VHH3 that is conserved among families of VHH or its fragments (see Table 4 below).

[0304] Both VHH, VHH2 and VHH, VH3 contact, bind or specifically bind to each of the linear stretches or regions of the amino acids of Sequence No. 14 as defined earlier herein (i.e., the linear amino acid stretches or regions 89-94, 140-150 and / or 176-184 of Sequence No. 14). Further, both VHH2 and VHH3 have, as a structural epitope, a combination of stretches or regions of the amino acids of Sequence No. 14 as defined earlier herein (i.e., the linear amino acid stretches or regions 89-94, 140-150 and / or 176-184 of Sequence No. 14). VHH2 and VHH3 are two members of the family of VHH or its fragments according to the present invention. This family of VHH or its fragments shares at least one of these linear epitopes and / or this structural epitope. This family of VHH or its fragments has excellent kinetic properties and / or excellent tissue distribution when used in the combinations according to the present invention.

[0305]

Table 4

[0306] This third structural feature of the VHH or its fragment according to the present invention can be defined in several ways.

[0307] In the first embodiment of this third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with at least one or a part of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 or 22. In one embodiment, the sequence identity (or similarity) with at least one of these sequences is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0308] In the second embodiment of this third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with at least one or a part of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 or 22, and has a length in the range of exactly the length of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 or 22 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 or 22. For example, a tag such as a His tag can be added to the VHH of the present invention or a fragment thereof. Usually, the His tag contains 4, 5, 6, 7, 8, 9, 10 histidines. Another tag can be a hemagglutinin tag (HA tag): YPYDVPDYA (SEQ ID NO: 37); YPYDVPDYGS (SEQ ID NO: 38) or a cysteine tag (Cys tag). The cysteine tag is a tag containing one or several cysteines. Non-limiting examples of the cysteine tag are C; GGC; SPSTPPTPSPSTPPC (SEQ ID NO: 39).

[0309] The methods by which identity and similarity are evaluated are described in detail in the portion for the purpose of the definitions at the end of the description. Usually, when identity is defined by reference to a sequence number, the identity is evaluated over the entire sequence number. However, it is also encompassed by the present invention that identity (or similarity) is evaluated over a part (or fragment) of the sequence. In this regard, a part may mean at least 50%, 60%, 70%, 80%, 90%, 95% of the length of the sequence number. The length of the sequence encompassed may be longer than the length of the sequence number used to evaluate identity (or similarity) (i.e., the length is at least 50%, 60%, 70%, 80%, 90% of the length of the sequence number, which is the same as that of the sequence number even if identity (or similarity) is evaluated over a part of this sequence number, or the length is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of sequence numbers 15, 16, 17, 18, 19, 20, 21 or 22).

[0310] In the third embodiment of this third structural feature, the length of the VHH or a fragment thereof is 110 to 130 amino acids or 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. This length does not include the length of tags such as His tags that may be added to the sequence of the VHH or a fragment thereof.

[0311] In this fourth embodiment of the third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22 or a part thereof, and the length of the VHH or a fragment thereof is 80 to 150 amino acids or 90 to 140, or 100 to 130, or 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity (or similarity) with at least one of these sequences is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0312] In this fifth embodiment of the third structural feature, the VHH or fragment thereof of the present invention is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 15 or a part thereof. In one embodiment, the VHH or fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 15 or a part thereof, and has a length of 78 to 130 amino acids, or in the range of 78 to 130 or 90 to 120, or 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity (or similarity) is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A part thereof has been already defined herein.

[0313] In this sixth embodiment of the third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 17 and / or 18 or a part thereof. In one embodiment, the VHH or a fragment thereof is represented by an amino acid sequence having at least 60% sequence identity (or similarity) with any one of SEQ ID NO: 17 and / or 18 or a part thereof, and has a length of 14 to 130 amino acids, or in the range of 20 to 120 or 30 to 110, or 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity (or similarity) is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A part thereof has been already defined herein.

[0314] In this seventh embodiment of the third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 16 or a part thereof. In one embodiment, the VHH or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with either SEQ ID NO: 16 or a part thereof, and has a length of 76 to 130 amino acids, or in the range of 76 to 120 or 90 to 110, or 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity (or similarity) is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A part thereof has been already defined herein.

[0315] In the eighth embodiment of this third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 19 and / or 20 or a part thereof. In one embodiment, the VHH or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 19 and / or 20 or a part thereof, and has a length of 13 to 150 amino acids, or in the range of 13 to 140 or 30 to 130, or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity (or similarity) is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A part thereof has been already defined herein.

[0316] In the ninth embodiment of this third structural feature, the VHH of the present invention or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 21 and / or 22 or a part thereof. In one embodiment, the VHH or a fragment thereof is represented by an amino acid sequence comprising or consisting essentially of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 21 and / or 22 or a part thereof, and has a length of 110 to 130 amino acids or in the range of 100 to 130 or 110 to 120 or 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In one embodiment, the sequence identity (or similarity) is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A part thereof has been defined hereinbefore.

[0317] In the tenth embodiment of this third structural feature, the VHH or a fragment thereof, preferably the VHH or a functional fragment thereof, is represented by an amino acid sequence comprising any one of SEQ ID NO: 15, 16, 17, 18, 19 and / or 20. In one embodiment, it has a length in the range of 13 to 130 amino acids or 20 to 120 or 30 to 121 amino acids. More preferably, it is represented by an amino acid sequence comprising SEQ ID NO: 21 or 22. Even more preferably, it has a length of 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.

[0318] In yet another embodiment, the VHH of the present invention or a fragment thereof conforms to the ninth embodiment of this third structural feature, which is the following amino acids of SEQ ID NO: 14: - at least one, or at least two, or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or - at least one, or at least two, or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or - at least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact, or bind, or specifically bind to.

[0319] In yet another embodiment, the VHH of the present invention or a fragment thereof conforms to the tenth embodiment of this third structural feature, which is the following amino acids of SEQ ID NO: 14: - at least one, or at least two, or at least three amino acids selected from C89, G90, E91, M92, A93 or P94, and / or - at least one, or at least two, or at least three amino acids selected from E140, Q141, W142, W143, E144, D145, C146, R147, T148, S149 or Y150, and / or - at least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181, F182, P183 or T184 contact, or bind, or specifically bind to.

[0320] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3). The VHH or fragment thereof is represented by an amino acid sequence comprising at least one of SEQ ID NOs: 21, 22, 15, 16, 17, 18, 19, or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0321] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of human folate receptor β (FOLR2) or human folate receptor γ (FOLR3). The VHH or fragment thereof is represented by an amino acid sequence comprising at least one of SEQ ID NOs: 21, 22, 15, 16, 17, 18, 19, or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0322] In one embodiment, the sequence identity (or similarity) is at least 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0323] In one embodiment, the VHH or a fragment thereof according to the present invention specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3). The epitope includes the linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150, and / or 176-184. The VHH or a fragment thereof is represented by an amino acid sequence comprising at least one of SEQ ID NO: 21, 22, 15, 16, 17, 18, 19, or 20 and an amino acid sequence having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0324] In one embodiment, the VHH or a fragment thereof according to the present invention specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3). The epitope includes the linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150, and / or 176-184. The VHH or a fragment thereof is represented by an amino acid sequence comprising at least one of SEQ ID NO: 21, 22, 15, 16, 17, 18, 19, or 20 and an amino acid sequence having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0325] In one embodiment, the sequence identity (or similarity) is at least 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0326] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3). The above epitope includes a linear amino acid stretch of SEQ ID NO: 14 or a combination of regions 89-94, 140-150, and 176-184. The VHH or fragment thereof is represented by an amino acid sequence including at least one of SEQ ID NO: 21, 22, 15, 16, 17, 18, 19, or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0327] In one embodiment, the VHH or fragment thereof according to the present invention specifically binds to an epitope of human folate receptor α (FOLR1), but does not bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3). The above epitope includes a linear amino acid stretch of SEQ ID NO: 14 or a combination of regions 89-94, 140-150, and 176-184. The VHH or fragment thereof is represented by an amino acid sequence including at least one of SEQ ID NO: 21, 22, 15, 16, 17, 18, 19, or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence.

[0328] In one embodiment, the sequence identity (or similarity) is at least 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0329] The fourth structural feature The VHH or fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) can alternatively or in combination be further defined by a fourth structural feature.

[0330] The VHH or fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) can alternatively or in combination be further defined by a fourth structural feature.

[0331] In the fourth structural feature, the VHH or fragment thereof of the present invention is a CDR1 region having SEQ ID NO: 23, a CDR2 region having SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 25, and / or a CDR1 region having SEQ ID NO: 30, a CDR2 region having SEQ ID NO: 31, and a CDR3 region having SEQ ID NO: 32 (see Table 5) represented by an amino acid sequence comprising at least one combination of CDR sequences selected from the group consisting of.

[0332] Thus, in certain embodiments, the present invention provides a heavy chain variable domain comprising a heavy chain antibody having a (general) structure or derived therefrom. FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and these are as further defined herein.

[0333] SEQ ID NOs: 21 and 22 (see Table 5) provide the amino acid sequences of the heavy chain variable domains raised against human FOLR1.

[0334]

Table 5

[0335] The present invention is not limited with respect to the origin of the VHH or its fragment (or the nucleotide sequence for expressing them), nor is it limited with respect to the method by which the VHH or its fragment, preferably the VHH or its fragment or the nucleotide sequence disclosed herein, is generated or obtained (or has been obtained). Therefore, the VHH or its fragment disclosed herein can be a naturally occurring amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence. Methods for isolating the VHH or its fragment and methods for generating the VHH or its fragment, as well as nucleic acid molecules encoding the VHH or its fragment, constructs containing these nucleic acid molecules, and cells containing these constructs are disclosed in detail in the definition section at the end of the description.

[0336] In a specific but non-limiting embodiment of the present invention, the amino acid sequence of the VHH or its fragment is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, and as such, but not limited to, "humanized" immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized VHH sequences), "camelized" immunoglobulin sequences, and (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences) affinity maturation, CDR grafting, veneering, combinations of fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those skilled in the art, and immunoglobulin sequences obtained by such techniques or any suitable combination of any of the foregoing. The VHH or its fragment according to the present invention can also be suitably humanized as described in more detail herein to provide one or more further (partial or complete) humanized amino acid sequences of the present invention.

[0337] In one embodiment, the VHH of the present invention or a fragment thereof is obtained from the aforementioned VHH or a fragment thereof using CDR grafting. Preferred VHHs or fragments thereof include: a CDR1 region having SEQ ID NO: 23, a CDR2 region having SEQ ID NO: 31, and a CDR3 region having SEQ ID NO: 25, a CDR1 region having SEQ ID NO: 23, a CDR2 region having SEQ ID NO: 31, and a CDR3 region having SEQ ID NO: 32, a CDR1 region having SEQ ID NO: 23, a CDR2 region having SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 32, a CDR1 region having SEQ ID NO: 30, a CDR2 region having SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 32, a CDR1 region having SEQ ID NO: 30, a CDR2 region having SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 25, or a CDR1 region having SEQ ID NO: 30, a CDR2 region having SEQ ID NO: 31, and a CDR3 region having SEQ ID NO: 25.

[0338] Each of these VHHs or fragments thereof may have the FRs of VHH2 or VHH3, as identified in Table 5. However, other FRs may exist.

[0339] In another embodiment, the VHH of the present invention or a fragment thereof is obtained from the above VHH or a fragment thereof using CDR grafting. Preferred VHHs or fragments thereof are a CDR1 region having SEQ ID NO: 23 and a CDR3 region having SEQ ID NO: 25, a CDR1 region having SEQ ID NO: 23 and a CDR3 region having SEQ ID NO: 32, a CDR1 region having SEQ ID NO: 30 and a CDR3 region having SEQ ID NO: 32, a CDR1 region having SEQ ID NO: 30 and a CDR3 region having SEQ ID NO: 25 and include.

[0340] Each of these VHHs or fragments thereof may contain a CDR2 region from another VHH and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist.

[0341] In another embodiment, the VHHs or fragments thereof of the present invention are obtained from the above VHHs or fragments thereof using CDR grafting. Preferred VHHs or fragments thereof are a CDR1 region having SEQ ID NO: 23 and a CDR2 region having SEQ ID NO: 24, a CDR1 region having SEQ ID NO: 23 and a CDR2 region having SEQ ID NO: 31, a CDR1 region having SEQ ID NO: 30 and a CDR2 region having SEQ ID NO: 31, a CDR1 region having SEQ ID NO: 30 and a CDR2 region having SEQ ID NO: 24 and contain.

[0342] Each of these VHHs or fragments thereof may contain a CDR3 region from another VHH and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist.

[0343] In another embodiment, the VHHs or fragments thereof of the present invention are obtained from the above VHHs or fragments thereof using CDR grafting. Preferred VHHs or fragments thereof are a CDR2 region having SEQ ID NO: 24 and a CDR3 region having SEQ ID NO: 25, a CDR2 region having SEQ ID NO: 24 and a CDR3 region having SEQ ID NO: 32, a CDR2 region having SEQ ID NO: 31 and a CDR3 region having SEQ ID NO: 32, a CDR2 region having SEQ ID NO: 31 and a CDR3 region having SEQ ID NO: 25 and contain.

[0344] Each of these VHHs or fragments thereof may contain a CDR1 region from another VHH and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist.

[0345] In another embodiment, the VHH or fragment thereof of the present invention is obtained from the above VHH or fragment thereof using CDR grafting. Preferred VHH or fragment thereof may include a CDR1 region having SEQ ID NO: 23 or 30, may include CDR2 and CDR3 regions from another VHH, and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist. In another embodiment, the VHH or fragment thereof of the present invention is obtained from the above VHH or fragment thereof using CDR grafting. Preferred VHH or fragment thereof may include a CDR2 region having SEQ ID NO: 24 or 31, may include CDR1 and CDR3 regions from another VHH, and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist.

[0346] In another embodiment, the VHH or fragment thereof of the present invention is obtained from the above VHH or fragment thereof using CDR grafting. Preferred VHH or fragment thereof may include a CDR3 region having SEQ ID NO: 25 or 19, may include CDR1 and CDR2 regions from another VHH, and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist. In another embodiment, the VHH or fragment thereof of the present invention is obtained from the above VHH or fragment thereof using CDR grafting. Preferred VHH or fragment thereof may include a CDR3 region having SEQ ID NO: 25 or 19, may include CDR1 and CDR2 regions from another VHH, and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist.

[0347] In another embodiment, the VHH or fragment thereof of the present invention is obtained from the above VHH or fragment thereof using CDR grafting. Preferred VHH or fragment thereof may include a CDR3 region having SEQ ID NO: 25 or 19, may include CDR1 and CDR2 regions from another VHH, and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist. In another embodiment, the VHH or fragment thereof of the present invention is obtained from the above VHH or fragment thereof using CDR grafting. Preferred VHH or fragment thereof may include a CDR3 region having SEQ ID NO: 25 or 19, may include CDR1 and CDR2 regions from another VHH, and may have the FRs of VHH2 or VHH3 as identified in Table 5. However, different FRs may exist.

[0348] Similarly, when the amino acid sequence includes a synthetic or semi-synthetic sequence (such as a partially humanized sequence), the sequence may also be optionally further preferably humanized as described herein so as to provide one or more additional (partially or fully) humanized amino acid sequences disclosed herein. Finally, more detailed definitions of "agonist", "antagonist", "variant", and "post-translational structure property evaluation" are provided.

[0349] In particular, the humanized VHH can be represented by an amino acid sequence that is a humanized substitution and / or has at least one amino acid residue (especially in at least one framework residue) corresponding to the humanized substitution. Additionally or alternatively, the sequence of the framework region of a naturally occurring VHH sequence is compared with the corresponding framework sequences of one or more closely related human V H sequences to identify other potentially useful humanized substitutions, and then one or more (or combinations thereof) of the potentially useful humanized substitutions thus determined can be introduced into the VHH sequence in any manner known per se (as further described herein), and the resulting humanized VHH sequence or its functional fragment can be tested for affinity for the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited amount of trial and error, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art.

[0350] In one embodiment, the VHH or fragment thereof according to the invention is represented by the first, second, third, and / or fourth structural features identified herein. Instead of or in combination with the structural features, the VHH or fragment thereof is characterized by the functional feature of specifically binding to human folate receptor α (FOLR1) but not (preferably) to mouse FOLR1, and not binding to human folate receptor β (FOLR2) and human folate receptor γ (FOLR3). In one embodiment, the VHH or fragment thereof is characterized by the functional feature of specifically binding to human folate receptor α (FOLR1) but not (preferably) to mouse FOLR1, and not binding to human folate receptor β (FOLR2) and human folate receptor γ (FOLR3).

[0351] In a preferred embodiment, the combination according to the invention comprises: - A VHH or fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3) (or a VHH or fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150, and / or 176-184, and a labeled compound comprising iodine-131 and GMIB, a VHH or fragment thereof according to the present invention, - A VHH or fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3) (or a VHH or fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150, and / or 176-184, and a labeled compound comprising DOTA or DTPA and lutetium-177, preferably, the labeled compound is 177 Lu-DOTA-PEG 7 -Tz or 177 Lu-DTPA-PEG 7 -Tz, a VHH or fragment thereof according to the present invention, comprising a labeled compound that can be represented by - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150 and / or 176-184, and a labeled compound comprising DOTA and actinium-225, preferably, the labeled compound is 225 Ac-DOTA-PEG 7 -Tz, and a VHH or a fragment thereof according to the present invention comprising the labeled compound, or - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or regions 89-94, 140-150 and / or 176-184, and a labeled compound comprising technetium-99m, and a VHH or a fragment thereof according to the present invention.

[0352] In a preferred embodiment, the combination according to the present invention comprises: - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or a combination of regions 89-94, 140-150, and 176-184, and a labeled compound comprising iodine-131 and GMIB, a VHH or a fragment thereof according to the present invention, - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1, human folate receptor β (FOLR2), or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or a combination of regions 89-94, 140-150, and 176-184, and a labeled compound comprising DOTA or DTPA and lutetium-177, preferably, the labeled compound is 177 Lu-DOTA-PEG 7 -Tz or 177 Lu-DTPA-PEG 7 -Tz, and a VHH or a fragment thereof according to the present invention comprising a labeled compound that can be represented by - A VHH or a fragment thereof according to the invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or a combination of regions 89 - 94, 140 - 150 and 176 - 184, and a labeled compound comprising DOTA and actinium-225, preferably, the labeled compound is 225 Ac-DOTA-PEG 7 -Tz, and a VHH or a fragment thereof according to the invention comprising the labeled compound, or - A VHH or a fragment thereof according to the invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the epitope is a linear amino acid stretch of SEQ ID NO: 14 or a combination of regions 89 - 94, 140 - 150 and 176 - 184, and a labeled compound comprising technetium-99m, and a VHH or a fragment thereof according to the invention.

[0353] In a preferred embodiment, the combination according to the invention comprises: - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the antibody fragment is represented by an amino acid sequence comprising at least one of SEQ ID NOs: 21, 22, 16, 17, 18, 19 or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence, a VHH or a fragment thereof according to the present invention, and a labeled compound comprising iodine-131 and GMIB, - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the antibody fragment is represented by an amino acid sequence comprising at least one of SEQ ID NOs: 21, 22, 16, 17, 18, 19 or 20 and having at least 63% sequence identity over the full length of the sequence or at least 50% of the length of the sequence, a VHH or a fragment thereof according to the present invention, and a labeled compound comprising DOTA or DTPA and lutetium-177, preferably, the labeled compound is 177 Lu-DOTA-PEG 7 -Tz or 177 Lu-DTPA-PEG 7 -Tz, and can be represented by a labeled compound, - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the antibody fragment is represented by an amino acid sequence that includes at least one of SEQ ID NOs: 21, 22, 16, 17, 18, 19, or 20 and has at least 63% sequence identity over the full length of the sequence or over at least 50% of the length of the sequence, a VHH or a fragment thereof according to the present invention, and a labeled compound comprising DOTA and actinium-225, preferably, the labeled compound can be represented by 225 Ac-DOTA-PEG 7 -Tz, a labeled compound, or - A VHH or a fragment thereof according to the present invention that specifically binds to an epitope of human folate receptor α (FOLR1) but does not bind to any of mouse FOLR1 or human folate receptor β (FOLR2) or human folate receptor γ (FOLR3) (or a VHH or a fragment thereof according to the present invention that specifically binds to human FOLR1 but does not bind to either human FOLR2 or human FOLR3), wherein the antibody fragment is represented by an amino acid sequence that includes at least one of SEQ ID NOs: 21, 22, 16, 17, 18, 19, or 20 and has at least 63% sequence identity over the full length of the sequence or over at least 50% of the length of the sequence, a VHH or a fragment thereof according to the present invention, and a labeled compound comprising technetium-99m.

[0354] Human epidermal growth factor 2 (HER2) In multiple embodiments, the VHH or fragment thereof according to the present invention is particularly suitable for binding to human epidermal growth factor receptor 2. Example 8 shows that the TCO conjugate HER2-binding VHH can be used for pretargeting applications. Preferably, the VHH or fragment thereof does not compete with the monoclonal antibody trastuzumab (Herceptin®) or the monoclonal antibody pertuzumab (Perjeta®) for binding to HER2, as determined using an appropriate competition assay.

[0355] In multiple embodiments, the VHH or fragment thereof according to the present invention - a CDR1 region having SEQ ID NO: 40, a CDR2 region having SEQ ID NO: 41, and a CDR3 region having SEQ ID NO: 42, and / or - a CDR1 region having SEQ ID NO: 43, a CDR2 region having SEQ ID NO: 44, and a CDR3 region having SEQ ID NO: 45 comprises one of the CDR combinations selected from the group consisting of.

[0356] In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with at least one of the amino acid sequences of SEQ ID NO: 46 or 47.

[0357] In multiple embodiments, the VHH or fragment thereof according to the present invention comprises the amino acid sequence of SEQ ID NO: 46 or 47.

[0358] In multiple embodiments, the VHH or fragment thereof according to the present invention comprises or consists of any of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 60% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 70% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 90% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 95% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88.

[0359] In multiple embodiments, the VHH or fragment thereof according to the present invention comprises or consists of any of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 60% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 70% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 90% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88. In multiple embodiments, the VHH or fragment thereof according to the present invention has at least 95% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48 to 88.

[0360] In multiple embodiments, the VHH or fragment thereof according to the present invention exists in a monovalent form.

[0361] In multiple embodiments, the VHH or fragment thereof according to the present invention lacks a cysteine-containing tag, preferably a GGC tag.

[0362] In multiple embodiments, the VHH or fragment thereof according to the present invention is non-life-extending.

[0363] In multiple embodiments, the VHH or fragment thereof according to the present invention has no carboxy-terminal polypeptide tag, and preferably, the VHH or fragment thereof is untagged.

[0364] A combination comprising an antibody and antibody fragment that binds to FAP or FOLR1 In one aspect of the present invention, - an antibody or fragment thereof comprising a click group, and - a labeled compound comprising a label and a click group A combination for use in the treatment and / or diagnosis of cancer is provided, wherein the antibody or fragment thereof can specifically bind to human FAP and / or mouse FAP, or can specifically bind to an epitope of human folate receptor α (FOLR1), but does not bind to either human folate receptor β (FOLR2) or human folate receptor γ (FOLR3), the use comprising an initial administration of the antibody or fragment thereof to a subject in need thereof and a subsequent administration of the labeled compound, and the click group contained in the antibody or fragment thereof can undergo a click reaction with the click group contained in the labeled compound in the subject.

[0365] All preferred embodiments and implementations related to the combinations according to the present invention can be applied to the combinations according to this embodiment with necessary modifications. For example, the preferred click groups included in the VHH or its fragments, which are part of the combinations according to the present invention, are also the preferred click groups included in the aforementioned antibodies or their fragments. Furthermore, the preferred sequences of the VHH or its fragments that can be part of the combinations according to the present invention, as well as other structural and functional modifications, can all be applied to antibodies or their fragments. Clearly, other preferred features of the combinations according to the present invention, which are related to but not limited to elements such as cancer, labeled compounds, first administration, second administration, and click reactions, can be applied to the aforementioned combinations with necessary modifications.

[0366] Composition In yet another aspect, there is provided a composition comprising, consisting of, or essentially consisting of a VHH or its fragment according to the present invention. Such a composition is referred to as a composition according to the present invention in the context of this application. All preferred embodiments related to the VHH or its fragment according to the present invention, which are optionally included in the combinations according to the present invention, can be applied to such a composition with necessary modifications. In the combinations according to the present invention, the VHH or its fragment can be included in the composition according to the present invention.

[0367] In another aspect, there is provided a composition comprising, consisting of, or essentially consisting of a labeled compound as used in the combinations according to the present invention. In the combinations according to the present invention, the labeled compound can be included in such a composition.

[0368] The above composition may contain excipients. The excipients should be acceptable for diagnostic and / or therapeutic purposes. In one embodiment, the composition is a pharmaceutical composition. In another embodiment, the composition is a diagnostic composition. It is also encompassed by the present invention that the composition is both a pharmaceutical composition and a diagnostic composition. Preferred formulations of the present invention are disclosed in the definition section at the end of the description.

[0369] The composition may contain one or more acceptable carriers.

[0370] The composition may contain at least one other compound.

[0371] The pharmaceutical compositions contemplated herein can be used for the prevention and / or treatment of cancer.

[0372] Diagnostic use of the combination In yet another aspect, there is provided a method for assessing the expression of an antigen, preferably human FAP or human FOLR1, in a subject or an isolated sample of said subject using the combination according to the invention. This method comprises a) providing the combination according to the invention; b) administering a first dose of said VHH or a fragment thereof comprised in said combination and a subsequent dose of said labeled compound comprised in said combination to a subject in need thereof or an isolated sample of the subject; c) assessing the expression of the antigen in the subject or the isolated sample of the subject. may comprise.

[0373] This method may also be referred to as a diagnostic method. This method can be an in vitro or in vivo method. This method can enable the localization of the expression of an antigen, preferably human FAP or human FOLR1, in a subject or an isolated sample of said subject, and can enable the prediction and / or prognostic diagnosis of a specific disease and / or disorder and / or condition in said subject. In one embodiment, the method can be a stratification method for identifying patients who are likely to respond to a specific treatment such as cancer treatment or wound healing treatment or fibrosis treatment. Thus, in a further aspect, there is provided a method for stratifying a subject and assessing whether the subject is likely to respond to a specific treatment such as cancer treatment or wound healing treatment or fibrosis treatment using the combination according to the invention. This method comprises a) providing the combination according to the invention; and b) administering the VHH or fragment thereof included in the combination to a subject in need thereof or to an isolated sample of the subject, followed by administering the labeled compound included in the combination; c) evaluating the expression of an antigen in the subject or the isolated sample of the subject; d) determining whether the subject is likely to be responsive to medical treatment may be included.

[0374] Preferably, the label included in the combination is a radionuclide. The subject is preferably human. Detailed information is disclosed in the definition section at the end of this specification for the purpose of manufacturing / providing the combination according to the present invention and for the purpose of administration. The same applies to the administration of the combination for diagnostic or therapeutic purposes. The method according to this aspect may be an in vitro or ex vivo method.

[0375] In one embodiment, a screening dose or biomarker dose is administered to a subject or an isolated sample of the subject. Detailed definitions will be provided later, especially in comparison with the definition of therapeutic dose.

[0376] In one embodiment of the diagnostic method, the labeled compound included in the combination according to the present invention comprises [I-131]GMIB, or comprises a DTPA or DOTA linker and a lutetium-177 nuclide, or comprises a technetium-99m nuclide.

[0377] The evaluation of the expression of an antigen in a subject, preferably human FAP or human FOLR1, is preferably carried out using imaging as disclosed in the part for the purpose of the definition at the end of the description. Alternatively, the evaluation of the expression of an antigen in a subject, preferably human FAP or human FOLR1, is preferably carried out using an isolated sample of the subject. In the context of the present invention, an isolated sample of the subject may be a tissue or liquid sample from the subject. The liquid may be serum. A sample isolated from a patient may also be called a biopsy or tumor biopsy.

[0378] Therapeutic use of the combination In one embodiment, the combination according to the invention and the corresponding VHH or fragment thereof according to the invention for use in the treatment and / or diagnosis of solid cancer, preferably solid cancer derived from epithelial cells or tissues.

[0379] In one embodiment, the combination according to the invention comprises a label which is a molecule to be delivered to cells, tissues, organs that express or overexpress the corresponding antigen, preferably human FAP or human FOLR1. The molecule can be a peptide or a small molecule, a nucleic acid. The peptide can be a cytokine. The small molecule can be a chemotherapeutic agent. The entity can be a cell such as a CAR-T cell, a CAR-NK cell, a BITE or a LITE. The present compound or a composition comprising the same can be a pharmaceutical for treating a disease or condition associated with the expression or overexpression of an antigen, preferably FAP or FOLR1.

[0380] Any moiety, molecule or pharmaceutical known to act on cells, tissues, organs that express an antigen, preferably human FAP or human FOLR1, can be encompassed by the present invention and can be the label comprised in the combination according to the invention.

[0381] In multiple embodiments, the combination according to the present invention is for use in the treatment of cancer. In another embodiment, said cancer is related to the expression of human FAP in cancer or tumor cells or metastasized lesions. The cancer to be treated can be metastatic, and preferably, the metastatic cells are present in the brain, bone, liver, and lung. Cancers related to the expression of FAP include leukemia, bone, uterus, pancreas, GEP-NET (gastroenteropancreatic neuroendocrine tumor), skin, muscle, brain, breast, colorectal, esophagus, stomach, liver, lung, NSCLC (non-small cell lung cancer), ovary, parathyroid, renal cancer cells, CUP (cancer of unknown primary origin), prostate, small intestine, CCC (cholangiocarcinoma), sarcoma (Pure et al 2018, Oncogene Aug;37(32):4343-4357 and Frederik Giesel et al J Nucl Med May 1, 2019 vol.60 no.supplement 1 Abstract 289). However, the present invention is not limited to these types of cancers. As soon as a subject is suspected of having cancer cells that express or overexpress human FAP, the combination according to the present invention can be used.

[0382] In an alternative embodiment, said cancer is related to the expression of human FOLR1. In one embodiment, the label included in the combination is a molecule to be delivered to the central nervous system (CNS). This molecule preferably does not pass through the blood-brain barrier (BBB) alone. The above molecule can be a peptide or a small molecule, nucleic acid. The peptide can be a cytokine. The small molecule can be a chemotherapeutic agent. The entity can be a cell such as a CAR-T cell, CAR-NK cell, BITE or LITE. The present compound can be a pharmaceutical for treating a disease or condition related to the brain, or a pharmaceutical for treating a disease or condition in which a change in brain activity affects another organ of the subject.

[0383] More preferably, the molecule is a pharmaceutical that acts on the CNS, preferably the brain. Even more preferably, the molecule passes through the blood-brain barrier (BBB) and / or the blood-cerebrospinal fluid barrier (BCSFB) by transport via human FOLR1 (a mechanism called receptor-mediated transcytosis (RMT)). Any pharmaceutical known to act on the CNS or the brain may be encompassed by the present invention.

[0384] A disease or condition or disorder related to the CNS or the brain can be any disease, condition or disorder known to those skilled in the art as being related to the CNS or the brain. Such a disease, condition or disorder can result in a change in CNS activity or a change in brain activity. Examples of such diseases, conditions or disorders include epilepsy, autism spectrum disorder, autism, changes in food intake, changes in thermoregulation, changes in pain perception, chronic pain, depression, migraine, hearing loss, bipolar disorder, Alzheimer's disease, schizophrenia, brain injury, blindness, stroke, Parkinson's disease, multiple sclerosis, spinal cord injury, amyotrophic lateral sclerosis.

[0385] In one embodiment, the cancer is related to the expression of human FOLR1 on cancer or tumor cells or metastases. The cancer to be treated can be metastatic, and preferably, in this case, the metastatic cells are present in the brain. Since the labeled compound can pass through the BBB, this is an attractive embodiment.

[0386] The cancer related to the expression of FOLR1 can be any of ovarian cancer, endometrial cancer, brain tumor, lung cancer, adrenal cancer, head and neck cancer, breast cancer, gastric cancer, colorectal cancer. However, the present invention is not limited to these types of cancers. As soon as a subject is suspected of having cancer cells that express or overexpress human FOLR1, the combination according to the present invention can be used.

[0387] In one embodiment, the subject is treated with the same or a different label, preferably a radionuclide, after being first diagnosed using the combination of the present invention. The identity of the radionuclide need not be the same for diagnostic and therapeutic uses.

[0388] In one embodiment of this treatment method or use, the labeled compound included in the combination according to the present invention comprises [I-131]GMIB, or comprises a DTPA or DOTA linker and a lutetium-177 nuclide, or comprises a technetium-99m nuclide.

[0389] In connection with the present invention, a disease or condition or disorder is prevented or treated when the combination according to the present invention is administered, and - improvement of at least one symptom associated with said disease or condition or disorder, and / or - improvement of at least one parameter associated with said disease or condition or disorder is obtained.

[0390] Improvement can be observed at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week after administration of the combination. Alternatively, improvement can be observed at least 1 month, 6 months after administration of the compound, each labeled compound. The dosage and mode of administration envisaged are further disclosed in the definition section at the end of this specification.

[0391] The (therapeutic) use of the combination according to the present invention results in or exhibits anti-cancer activity when at least one of the following is satisfied: (compared to an untreated or a treated control or the subject at the start of treatment) - can kill tumor cells, cancer cells and / or CAFs expressing an antigen, preferably human FAP or human FOLR1, - can reduce or delay the growth and / or proliferation of such tumor cells or cancer cells, - can reduce the size of the primary tumor or metastases, - can delay the occurrence of metastasis and / or tumor cell migration, - can delay the increase in tumor weight or growth, - can extend the survival period of the patient for at least 1 month, several months or more.

[0392] When the number of viable cancer cells and / or viable tumor cells after administration of the combination according to the present invention is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the number of initial viable cancer cells and / or initial viable tumor cells, anti-cancer activity can be identified or determined. When the size of the primary tumor and / or the size of the metastatic foci after administration of the combination according to the present invention is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the size of the primary tumor and / or the size of the metastatic foci, there is a possibility that anti-cancer activity has been identified or determined.

[0393] Tumor cell death can be evaluated by measurement of radiolabeled annexin A5, a molecular imaging agent for measuring cell death in vitro, and non-invasive measurements in cancer patients such as ICH (Schutters K. et al., "Apoptosis" (2010); de Saint-Hubert M. et al., "Methods" Vol. 48: p. 178, 2009). ICH is defined in the Definitions section at the end of the description.

[0394] Tumor growth can be inhibited by at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75% or more. Tumor growth can be evaluated using techniques known to those skilled in the art. Tumor growth can be evaluated using MRI (magnetic resonance imaging) or CT (computed tomography imaging).

[0395] In certain embodiments, tumor weight gain or tumor growth can be inhibited by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Tumor weight or tumor growth can be evaluated using techniques known to those of ordinary skill in the art. Detection of tumor growth or detection of tumor cell proliferation can be evaluated in vivo by measuring changes in glucose utilization by positron emission tomography using the glucose analog 2-[18F]-fluoro-2-deoxy-D-glucose (FDG-PET) or [18F]-’3-fluoro-’3-deoxy-L-thymidine (FLT-PET). An ex vivo alternative can be staining of tumor biopsies with Ki67.

[0396] The occurrence of metastasis and / or the delay in tumor cell migration can be a delay of at least 1 week, 1 month, several months, 1 year or more. The presence of metastasis can be evaluated using MRI, CT or echography or techniques that enable the detection of circulating tumor cells (CTC). An example of the latter test is the CellSearch CTC test (Veridex), which is an EpCam-based magnetic selection of CTC from peripheral blood.

[0397] In certain embodiments, tumor growth can be delayed or inhibited for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week, 2 weeks, 3 weeks, 1 month, 2 months or more. In certain embodiments, the occurrence of metastasis is delayed for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more.

[0398] The combination according to the invention exerts its anti-cancer activity by the mechanism of radiation toxicity when it binds to antigens, preferably cancers expressing human FAP or human FOLR1, or tumor cells, or metastases, or CAF.

[0399] The doses of tumor cells, cancer cells, lesions, metastases and the above combination are defined in the section entitled Definition.

[0400] In a further aspect, there is provided a method for the prevention and / or treatment of a disease, and / or disorder, and / or condition, the method comprising administering to a subject in need thereof a combination according to the present invention. All features of this method are defined hereinbefore.

[0401] Definitions The following terms or definitions are provided solely to facilitate understanding of the present invention. Unless specifically defined herein, all terms used herein have the same meaning to those skilled in the art of the present invention. Those skilled in the art are referred to the definitions and terms of the art, in particular Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al. "Current Protocols in Molecular Biology" (Supplement 47), John Wiley & Sons, New York (1999). The definitions provided herein should not be construed as having a scope less than that understood by those skilled in the art.

[0402] Unless otherwise indicated, all methods, procedures, techniques and operations not specifically described in detail can be carried out in a manner known per se, as will be apparent to those skilled in the art, and are carried out. For example, standard handbooks, the general background art referred to above and further references cited therein are referred to again.

[0403] As used herein, the singular forms "a", "an" and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0404] As used herein, the terms "comprising", "including" and "consisting of" are synonymous with "containing", "including" or "having", "containing", and are inclusive or non-limiting and do not exclude additional unrecited members, elements or method steps. The expression "consisting essentially of" as used in connection with a product or composition ("product consisting essentially of" or "composition consisting essentially of") means that additional molecules may be present, but such molecules do not change / alter the characteristics / activities / functionalities of the said product or composition. For example, if the composition itself exhibits the same characteristics / activities / functionalities as the VHH according to the present invention, the said VHH may consist essentially of an antibody fragment.

[0405] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within each range, as well as the recited endpoints.

[0406] As used herein, the term "about" when referring to a measurable value such as a parameter, amount, duration, etc. means including variations of + / - 10% or less, preferably + / - 5% or less, more preferably + / - 1% or less, still more preferably + / - 0.1% or less of a particular value, and such variations are only included if they are suitable for carrying out the disclosed invention. It is to be understood that the value itself to which the modifier "about" refers is also specifically and preferably disclosed.

[0407] In this application, the terms "for use in medicine" and "for use as a medicament" are used interchangeably.

[0408] Polypeptide, nucleic acid molecule, identity, similarity As used herein, amino acid residues are designated either by their full names or according to the standard three-letter or one-letter amino acid codes.

[0409] As used herein, the terms "polypeptide" or "protein" are used interchangeably and refer to a polymeric form of amino acids of any length that can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. A "peptide" is also a polymer of amino acids, typically having a length of up to 50 amino acids. A polypeptide or peptide is represented by an amino acid sequence.

[0410] As used herein, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length of either deoxyribonucleotides or ribonucleotides or analogs thereof. A nucleic acid molecule is represented by a nucleic acid sequence that primarily characterizes its base sequence. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule can be linear or circular.

[0411] As used herein, the term "homology" means at least secondary structural identity or similarity between two macromolecules derived from the same or different taxa, particularly between two polypeptides or polynucleotides, said similarity being due to a common ancestor. Thus, the term "homolog" refers to such related macromolecules having said secondary structure similarity and optionally tertiary structure similarity. To compare two or more nucleotide sequences, the "percent (of) sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using methods known to those of skill in the art, for example, by dividing the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions of the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100%, or by using known computer algorithms for sequence alignment such as NCBI Blast. In determining the degree of sequence similarity between two amino acid sequences, one of skill in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure and which have little or essentially no effect on the function, activity or other biological properties of the polypeptide. Possible conservative amino acid substitutions will be apparent to those of skill in the art. An amino acid sequence and a nucleic acid sequence are said to be "identical" if they have 100% sequence identity over their entire length.

[0412] Throughout this application, whenever a sequence number for a particular amino acid sequence is referred to (by way of example, taking sequence number Y), this can be replaced by a polypeptide comprising an amino acid sequence having at least 80% sequence identity or similarity to the amino acid sequence of sequence number Y.

[0413] Each amino acid sequence described herein has, by its percentage identity (at least 80%) with a given amino acid sequence, and in further preferred embodiments, at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity with a given amino acid sequence, respectively. In preferred embodiments, sequence identity is determined by comparing the full length of the sequences identified herein. Each amino acid sequence described herein has, by its percentage similarity (at least 81%) with a given amino acid sequence, and in further preferred embodiments, at least 81%, 85%, 90%, 95%, 97%, 98%, 99% or more similarity with a given amino acid sequence, respectively. In preferred embodiments, sequence similarity is determined by comparing the full length of the sequences identified herein. Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO. means identity or similarity based on the full length of said sequence (i.e., over its entire length or as a whole).

[0414] "Sequence identity" is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences determined by comparing the sequences. Identity between two amino acid sequences is preferably defined by assessing their identity within the entire SEQ ID NO. identified herein or a portion thereof. The portion can mean at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length of the SEQ ID NO.

[0415] In the art, "identity" also means the degree of sequence relatedness between amino acid sequences, such that it can be determined by a match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods including, but not limited to, "Computational Molecular Biology", Lesk, A.M. ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects", Smith, D.W. ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data", Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology", von Heine, G., Academic Press, 1987; and "Sequence Analysis Primer", Gribskov, M. and Devereux, J. eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., "SIAM J. Applied Math.", Vol. 48: p. 1073 (1988).

[0416] Preferred methods for determining identity are designed to give the maximum match between the sequences tested. Methods for determining identity and similarity are embodied in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J. et al., Nucleic Acids Research, Vol. 12, No. 1, p. 387 (1984)), BestFit, FASTA, BLASTN and BLASTP (Altschul, S. F. et al., J. Mol. Biol., Vol. 215, pp. 403-410 (1990)), EMBOSS Needle (Madeira, F. et al., Nucleic Acids Research, Vol. 47, No. W1, pp. W636-W641 (2019)). The BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol., Vol. 215, pp. 403-410 (1990)). The EMBOSS programs are publicly available from EMBL-EBI. Identity can also be determined using the well-known Smith Waterman algorithm. The preferred program used is the EMBOSS Needle program.

[0417] Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, "J. Mol. Biol." Vol. 48 (No. 3): pp. 443-453 (1970); Comparison matrix: BLOSUM62 from Henikoff and Henikoff, "Proc. Natl. Acad. Sci. USA." Vol. 89: pp. 10915-10919 (1992); Gap Open Penalty: 10; and Gap Extend Penalty: 0.5. A program useful with these parameters is publicly available from EMBL-EBI as the EMBOSS Needle program. The aforementioned parameters are the default parameters for Global Pairwise Sequence alignment of proteins (no penalty for terminal gaps).

[0418] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, "J. Mol. Biol." Vol. 48: pp. 443-453 (1970); Comparison matrix: DNAfull; Gap Open Penalty: 10; Gap Extend Penalty: 0.5. A program useful with these parameters is publicly available from EMBL-EBI as the EMBOSS Needle program. The aforementioned parameters are the default parameters for Global Pairwise Sequence alignment of nucleotide sequences (no penalty for terminal gaps).

[0419] Optionally, when determining the degree of amino acid similarity, as will be apparent to those skilled in the art, those skilled in the art may also consider so-called "conservative" amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains are glycine, alanine, valine, leucine and isoleucine. The group of amino acids having aliphatic-hydroxyl side chains are serine and threonine. The group of amino acids having amide-containing side chains are asparagine and glutamine. The group of amino acids having aromatic side chains are phenylalanine, tyrosine and tryptophan. The group of amino acids having basic side chains are lysine, arginine and histidine. The group of amino acids having acidic side chains are aspartic acid and glutamic acid. The group of amino acids having sulfur-containing side chains are cysteine and methionine. The preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala~Ser; Arg~Lys or Gln; Asn~Asp, His or Ser; Asp~Glu or Asn; Gln~Glu, Lys or Arg; Glu~Lys, Asp, Gln; His~Tyr or Asn; Ile~Leu, Val or Met; Leu~Ile, Met or Val; Lys~Arg, Gln or Glu; Met~Val, Leu or Ile; Phe~Trp or Tyr; Ser~Thr, Ala or Asn; Thr~Ser; Trp~Tyr or Phe; Tyr~His, Trp or Phe; and Val~Ile, Leu or Met. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence is removed and a different residue is inserted in its place. Preferably, the amino acid changes are conservative.

[0420] antibody As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies and fragments thereof, such as Fab F(ab')[ 2, scFv, VHH, and other fragments that retain the antigen-binding function of the parental antibody, etc. Thus, an antibody can refer to a construct containing an antigen-binding portion within an immunoglobulin or glycoprotein or a fragment or portion thereof or a modified immunoglobulin-like framework, or an antigen-binding portion contained within a construct containing a non-immunoglobulin-like framework or scaffold.

[0421] As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous population of antibodies. This term is not limited with respect to the species or source of the antibody and is not intended to be limited by the manner in which it is made. This term encompasses whole immunoglobulins as well as fragments and others that retain the antigen-binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in the present invention. However, in practice, due to the availability of rat or mouse cell lines for use in generating the hybrid cell lines or hybridomas required to produce monoclonal antibodies, antibodies are typically of rat or mouse origin.

[0422] As used herein, the term "polyclonal antibody" refers to an antibody composition having a heterogeneous population of antibodies. Polyclonal antibodies are often derived from immunized animals or pooled sera from selected humans.

[0423] As used herein, "the variable domain of the heavy chain of an antibody or a fragment thereof" (i) includes, but is not limited to, the variable domain of the heavy chain of a heavy-chain antibody of a camelid or shark, the variable domain of the heavy chain of a heavy-chain antibody that is naturally lacking a light chain (hereinafter also referred to as VHH), or (ii) the camelized (further defined herein) variable domain of the heavy chain of a conventional four-chain antibody (hereinafter also referred to as camelized V H also referred to as), and includes, but is not limited to, the variable domain of the heavy chain of a conventional four-chain antibody (hereinafter also referred to as V HAlso shown (or but not limited to) is particularly suitable for binding to a tumor antigen or an antigen present on cancer cells, and is present in and / or can be incorporated into a VHH disclosed herein (or based on and / or derived from the CDR sequences of the VHHs disclosed herein), and means any fragment thereof, such as one or more stretches of amino acid residues (i.e., small peptides). In one embodiment, the fragment of the VHH is a functional fragment.

[0424] As further described hereinbelow, the amino acid sequence and structure of the variable domain of the heavy chain of an antibody can be considered to be composed of four framework regions or "FRs", which are respectively referred to as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3" and "framework region 4" or "FR4" in the art and herein, although not limited thereto, and these framework regions are interrupted by three complementarity determining regions or "CDRs", which are respectively referred to as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2" and "complementarity determining region 3" or "CDR3" in the art.

[0425] As used herein, in the context of an antibody, the term "complementary determining region" or "CDR" refers to the variable region of either the H (heavy) or L (light) chain (also abbreviated VH and VL, respectively), and includes an amino acid sequence capable of specifically binding to an antigen target. These CDR regions account for the fundamental specificity of the antibody for a particular antigen determinant structure. Such regions are also referred to as "hypervariable regions". CDRs represent discontinuous stretches of amino acids within the variable region, and regardless of species, the placement of these important amino acid sequences within the variable heavy and variable light chain regions has been found to have a similar arrangement within the amino acid sequence of the variable chain. All canonical antibodies have three CDR regions each in their variable heavy and variable light chains, and for each light (L) and heavy (H) chain, each is discontinuous from the other CDR regions (designated L1, L2, L3, H1, H2, H3).

[0426] As further described herein below, the total number of amino acid residues in the heavy chain variable domain (VHH or V H including) of an antibody can also be in the region of 110 - 130. However, it should be noted that a portion, fragment or analog of the heavy chain variable domain of an antibody is not particularly limited with respect to its length and / or size, provided that such portion, fragment or analog retains (at least in part) the functional activity of the original heavy chain variable domain of the antibody from which these portions, fragments or analogs are derived, and / or retains (at least in part) the binding specificity. A portion, fragment or analog that retains (at least in part) the functional activity of the original heavy chain variable domain of the antibody from which these portions, fragments or analogs are derived, and / or retains (at least in part) the binding specificity is further referred to herein as a "functional fragment" of the heavy chain variable domain.

[0427] The variable domain of an antibody (VHH or V HThe amino acid residues (including those) are preferably numbered according to the IMGT unique numbering of the V domain (immunoglobulin and T cell receptor) given by the described IMGT nomenclature (Lefranc M.P. et al., 1997, Immunology today, Vol. 18: p. 509, PMID: 9386342; Lefranc, M.-P., 1999, The Immunologist, Vol. 7: pp. 132 - 136 and Lefranc M.P. et al., 2003, Dev. Comp. Immunol., Vol. 27: pp. 55 - 77, PMID: 12477501). According to this numbering (see, for example, Table 1 of Lefranc (2003)), conserved amino acids, such as cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP), always have the same positions. The unique numbering of IMGT provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 - 26, FR2-IMGT: positions 39 - 55, FR3-IMGT: positions 66 - 104 and FR4-IMGT: positions 118 - 128) and the complementarity-determining regions: CDR1-IMGT: 27 - 38, CDR2-IMGT: 56 - 65 and CDR3-IMGT: 105 - 117. Gaps represent non-occupied positions. The gaps in CDR1-IMGT and CDR2-IMGT (less than 12 and 10 amino acids in length respectively) are placed at the top of the CDR-IMGT loop. The base length of the rearranged CDR3-IMGT is 13 amino acids (positions 105 - 117), which corresponds to the 15-amino acid JUNCTION (2nd-CYS104 relative to J-TRP or J-PHE118). When the length of CDR3-IMGT is less than 13 amino acids, gaps are created in the following order 111, 112, 110, 113, 109, 114, etc. from the top of the loop. When the length of CDR3-IMGT exceeds 13 amino acids, additional positions are created in the following order 112.1, 111.1, 112.2, 111.2, 112.3, 111.3, etc. at positions 111 - 112 at the top of the CDR3-IMGT loop.

[0428] In this regard, as is well known in the art with respect to VHH domains, the total number of amino acid residues in each of the CDRs can vary and need not correspond to the total number of amino acid residues indicated by IMGT numbering (i.e., one or more positions according to IMGT numbering may not be occupied by the actual sequence, or the actual sequence may contain more amino acid residues than the number permitted by IMGT numbering). It should be noted that this generally means that the numbering by IMGT may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0429] Alternatively, the amino acid residues of the variable domain of an antibody (including VHH or VH) can be numbered according to Kabat numbering (Kabat et al., 1987, National Institutes of Health, USA; 1987, page 804, publication number 165 - 462). The correspondence between IMGT and Kabat numbering of immunoglobulin V regions can be found, for example, in Table 2 of Lefranc et al. (2003).

[0430] For a general description of heavy chain antibodies and their variable domains, see, inter alia, as general background art, Muyldermans S. et al. (2013) "Annual Review of Biochemistry" Vol. 82: pp. 775 - 797.

[0431] In general, it should be noted that the term "heavy chain variable domain" as used herein is, in its broadest sense, not limited to a particular biological source or a particular method of preparation. For example, as will be discussed in more detail below, the heavy chain variable domain derived from a heavy chain antibody (i.e., VHH) according to the present invention can be obtained by (1) isolating the VHH domain of a naturally occurring heavy chain antibody, (2) expressing the nucleotide sequence encoding a naturally occurring VHH domain, (3) "camelization" of a naturally occurring V domain from any animal species, particularly mammalian species such as humans (described below) or such camelized V H domain HBy expressing a nucleic acid encoding the domain, the "camelization" of the "domain antibody" or "dAb" described in (4) Weizao C. et al., "Methods Mol Biol" (2009), Vol. 525: pp. 81-99), or such camelized V H By expressing a nucleic acid encoding the domain, (5) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences, (6) preparing a nucleic acid encoding VHH using techniques for nucleic acid synthesis and subsequently expressing the nucleic acid thus obtained, and / or (7) can be obtained by any of the above combinations. Suitable methods and techniques for performing the above are apparent to those skilled in the art based on the disclosure herein and include, for example, the methods and techniques described in more detail below herein.

[0432] A VHH or a fragment thereof according to the invention is considered to be in the "(essentially) isolated (form)" as used herein when it is extracted or purified from the host cell and / or medium in which it was produced.

[0433] Method for isolating a suitable VHH against an antigen In certain embodiments, the VHH or fragment thereof according to the invention is present on and / or specific to solid tumors and / or cancer cells, and is preferably obtained by affinity selection for an antigen, preferably a human and / or mouse FAP or human FOLR1, present on and / or specific to solid tumors and / or cancer cells. Acquisition of a suitable polypeptide by affinity selection for a specific solid tumor antigen or cancer cell can be carried out by screening a set, aggregate or library of cells that express the VHH on their surface (e.g., bacteriophage) for binding to a tumor-specific antigen and / or a cancer cell-specific antigen, all of which can be carried out in a manner known per se, essentially comprising the following non-limiting steps: a) obtaining an isolated solution or suspension of a tumor-specific or cancer cell-specific protein target molecule, the molecule of which is known to be a potential target for a cancer drug; b) biopanning phage or other cells from a VHH library against the protein target molecule; c) isolating phage or other cells that bind to the tumor-specific or cancer cell-specific protein target molecule; d) determining the nucleotide sequence encoding the VHH insert from the individual binding phage or other cells; e) generating a predetermined amount of VHH according to this sequence using recombinant protein expression; f) determining the affinity of the VHH domain for the tumor-specific or cancer cell-specific protein target molecule, and optionally g) testing the tumoricidal or anti-cancer activity of the VHH domain in a bioassay. To determine the affinity between the VHH domain and the tumor-specific or cancer cell-specific protein target molecule, various methods can be used, including, for example, enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR) assay, which are common practices in the art, as described in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The equilibrium dissociation constant is generally used to describe the affinity between a polypeptide and its target molecule. Typically, the equilibrium dissociation constant is 10 -7Less than M. Preferably, the equilibrium dissociation constant is 10 -8 Less than M or 10 -9 Less than M or more preferably 10 -9 In the range of M to 10 -12 M.

[0434] Variant of VHH It should be noted that the VHH or fragment thereof according to the present invention is not limited with respect to the origin of the VHH or fragment thereof (or the nucleotide sequence of the present invention used to express them). Furthermore, the present invention is also not limited with respect to the method by which the VHH sequence or nucleotide sequence was produced or obtained. Accordingly, the amino acid sequences disclosed herein can be synthetic or semi-synthetic amino acid sequences, polypeptides or proteins.

[0435] The present invention also encompasses polypeptides comprising or consisting essentially of one or more of a portion, fragment, analog, mutant, variant and / or derivative of a VHH or fragment thereof and / or such portion, fragment, analog, mutant, variant and / or derivative, provided that such portion, fragment, analog, mutant, variant and / or derivative is suitable for the purposes contemplated herein (preferably, delivery to an antigen, preferably FAP or a CNS molecule expressing FOLR1, cell, tissue or organ which, when linked thereto and to a radionuclide, is suitable for use in diagnostic and therapeutic applications). Such portion, fragment, analog, mutant, variant and / or derivative according to the present invention preferably can specifically bind to said antigen, preferably - Specifically binds to human and / or mouse FAP, preferably both FAPs, and / or - Preferably is not a modulator of FAP, preferably not an inhibitor of FAP, and / or - Specifically binds to human FOLR1 and cannot specifically bind to mouse FOLR1, human FOLR2 and human FOLR3.

[0436] For example, the present specification provides sequences having at least 80% identity with SEQ ID NO: 4, which represents the sequence of a VHH or fragment thereof that is also referred to as a stretch of amino acid residues (i.e., small peptides), identified as SEQ ID NOs: 1, 2, 3, and is particularly suitable for binding to human and / or mouse FAP, etc.

[0437] For example, the present specification provides sequences having at least 60% identity with at least one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, which represents the sequence of a VHH or fragment thereof that is also referred to as a stretch of amino acid residues (i.e., small peptides) and is particularly suitable for binding to human FOLR1.

[0438] These stretches can be regarded as antibody fragments of VHH and can be present in and / or incorporated into any suitable scaffold (protein), particularly such that they form the antigen-binding site (or part thereof) of their suitable scaffold or VHH. However, in its broadest sense, the present invention is not limited to the specific structural role or function that these stretches of amino acid residues can have in the scaffolds or VHHs disclosed herein, as long as the scaffolds or VHHs disclosed herein are enabled to specifically bind to the antigen by these stretches of amino acid residues.

[0439] Further evaluation of the post-translational structural properties of VHH or its fragments In certain embodiments, the VHH or fragment thereof according to the invention may optionally be linked via one or more linkers to one or more additional groups, substructures or residues. These one or more additional groups, substructures or residues may serve to bind to other targets of interest. It should be apparent that such additional groups, residues, substructures and / or binding sites may or may not provide additional functionality to the VHH and may or may not modify its properties as disclosed herein. Such groups, residues, substructures or binding units may be, for example, chemical groups that may be biologically active.

[0440] In certain embodiments, these groups, substructures or residues are linked to the heavy chain variable domain at the N-terminus or C-terminus, particularly at the C-terminus.

[0441] In certain embodiments, the VHH domain or fragment thereof according to the invention may also be chemically modified. For example, such modifications may include the introduction or linkage of one or more functional groups, residues or substructures to or onto the VHH. These groups, residues or substructures may confer one or more desired properties or functionalities to the VHH. Examples of such functional groups will be apparent to those skilled in the art.

[0442] For example, the introduction or linkage of such functional groups to the VHH may result in an increase in their solubility and / or stability, a reduction in their toxicity or the elimination or attenuation of any undesirable side effects and / or other advantageous properties.

[0443] In certain embodiments, one or more groups, residues or substructures are linked to the VHH or fragment thereof of the invention via one or more suitable linkers or spacers.

[0444] When all of the two or more binding sites of the VHH or a fragment thereof of the present invention are directed to or specifically bind to the same site, determinant, moiety, epitope, domain or stretch of amino acid residues of the antigen disclosed herein, it is said to be "bivalent" (in the case of two binding sites in a VHH or a fragment thereof) or multivalent (in the case of more than two binding sites in a VHH or a fragment thereof), for example trivalent, etc.

[0445] In one embodiment, the VHH or a fragment thereof according to the present invention exists in a monovalent form.

[0446] As used herein, the term "monovalent" when referring to a VHH or a fragment thereof refers to a VHH or a fragment thereof in monomeric form. A monovalent VHH or a fragment thereof contains only one binding site. In this regard, the binding site of a VHH or a fragment thereof includes one or more "complementary determining regions" or "CDRs" that are directed to or specifically bind to a particular site, determinant, substructure, epitope, domain or stretch of an antigen.

[0447] In a plurality of embodiments, the binding site of the VHH or a fragment thereof according to the present invention is directed to or specifically binds to a particular site, determinant, substructure, epitope, domain or stretch of amino acid residues of human and / or mouse FAP, and includes one or more "complementary determining regions" or "CDRs" represented by SEQ ID NO: 1, 2 and / or 3 and / or one or more regions identified herein as having at least 80% identity with SEQ ID NO: 4 of the VHH.

[0448] In several embodiments, the binding site of the VHH or fragment thereof according to the invention is directed to or specifically binds to a specific site, determinant, substructure, epitope, domain or stretch of amino acid residues of human FOLR1, and includes one or more "complementary determining regions" or "CDRs" and / or one or more regions identified herein as having at least 60% identity with at least one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31.

[0449] The VHH or fragment thereof according to the invention is attractive for therapeutic / diagnostic use. In certain applications, such a small size can also be attractive when high tissue penetration is required to achieve an optimal therapeutic effect.

[0450] Some particularly suitable linkers or spacers include, for example, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine- and serine-rich linkers, etc., or linkers composed mostly of polar polypeptide fragments or homo- or heterobifunctional chemical crosslinking compounds such as glutaraldehyde or optionally maleimide or NHS ester with a PEG spacer, but are not limited thereto.

[0451] For example, the polypeptide linker or spacer can be a suitable amino acid sequence having a length of 1 to 50 amino acids, such as 1 to 30, particularly 1 to 10 amino acid residues. It should be apparent that the length, degree of flexibility and / or other properties of the linker can have some effect on the properties of the VHH or fragment thereof according to the invention (including but not limited to affinity, specificity or binding activity for a tumor target or target on cancer cells) or the pharmacological behavior. It is clear that when two or more linkers are used, these linkers can be the same or different.

[0452] As used herein, the term "untagged", when referring to a VHH or fragment thereof according to the invention, refers to a VHH or fragment thereof that does not contain a foreign polypeptide sequence (e.g., contains only the VHH or fragment thereof). Exemplary foreign polypeptide sequences include carboxy-terminal polypeptide tags such as His tags, cysteine-containing tags (e.g., GGC tags as described in Pruszynski et al 2013 Nucl Med Biol 40:52-59) and / or Myc tags. The His tag may contain 4, 5, 6, 7, 8, 9, 10 histidines. In one embodiment, six histidines are present.

[0453] In one embodiment, one or more groups, residues or substructures that may be present do not also induce multimerization, such as dimerization, of a VHH or fragment thereof according to the invention. Thus, in one embodiment, a VHH or fragment thereof according to the invention lacks a tag that induces multimerization such as dimerization, preferably lacks a cysteine-containing tag, preferably a GGC tag.

[0454] Thus, in one embodiment, a VHH or fragment thereof according to the invention lacks a carboxy-terminal polypeptide tag and is preferably untagged.

[0455] Advantageously, it has been demonstrated that renal retention is significantly reduced when using antibody fragments without a carboxy-terminal polypeptide tag as compared to polypeptide tagging such as His-tagging and Myc-His-tagged antibody fragments (D’Huyvetter et al. (2014), Theranostics. 4(7):708-20).

[0456] When referring to a VHH or a fragment thereof according to the present invention, the term "bispecific" means that a) when two or more binding sites of the VHH or a fragment thereof according to the present invention are directed to or specifically bind to an antigen, but are not directed to or do not specifically bind to the same site, determinant, moiety, epitope, domain or stretch of amino acid residues of that antigen (i.e., are different), the VHH or a fragment thereof according to the present invention is said to be "bispecific" (in the case of two binding sites on the VHH or fragment) or multispecific (in the case of three or more binding sites on the VHH or fragment), or b) two or more binding sites of the VHH or fragment indicate that they are directed to or specifically bind to different target molecules of interest. The term "multispecific" is used when there are three or more binding sites on the VHH or fragment.

[0457] The "half-life" of a VHH or a fragment thereof according to the present invention can generally be defined as the time required for the in vivo serum concentration of the VHH or fragment to decrease by 50%. The in vivo half-life of a VHH or fragment according to the present invention can be determined in any manner known to those skilled in the art, such as pharmacokinetic analysis. As will be apparent to those skilled in the art, the half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta and area under the curve (AUC). An increase in the half-life in vivo is generally characterized by an increase in one or more, preferably all three, of the parameters t1 / 2-alpha, t1 / 2-beta and area under the curve (AUC).

[0458] When referring to "half-life extension" in the context of a VHH of the present invention or a fragment thereof, the term is used to indicate that the VHH or fragment has been modified to extend its half-life. Strategies for extending the half-life of a VHH are well known in the art and include, but are not limited to, the (chemical or other) conjugation of one or more groups or substructures that extend half-life, such as polyethylene glycol (PEG) or bovine serum albumin (BSA) or human serum albumin (HSA), or the targeting of serum proteins such as antibody Fc fragments or serum albumin to antigen-binding antibody fragments.

[0459] Accordingly, in one embodiment, the VHH or fragment thereof according to the present invention is non-half-life extended.

[0460] Binding Specific binding of a VHH or fragment thereof according to the present invention can be determined in any suitable manner known per se, including, for example, biopanning, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) (also called enzyme-linked immunosorbent assay, ELISA), sandwich competitive assay, surface plasmon resonance (SPR) or biolayer interferometry and various variants thereof known in the art. Each of these assays can be performed in vitro using an antigen that can be immobilized on a support or in solution, preferably a human and / or mouse FAP recombinant protein or a human FOLR1 recombinant protein or a human HER2 recombinant protein. Alternatively, in some specific situations, some of these assays can be performed in vitro using cells that express an antigen, preferably human and / or mouse FAP, or human FOLR1, or human HER2. Such cells can endogenously express or overexpress an antigen, preferably human and / or mouse FAP or mouse FAP, or human FOLR1, or human HER2. The evaluation is usually carried out in vitro in culture medium or PBS or in a suitable medium or buffer.

[0461] Preferred cells are fibroblasts expressing human and / or mouse FAP. Preferred cell lines can be GM05389 or U-87 MG. Alternatively, preferred cells are transfected cells expressing human and / or mouse FAP. A preferred transfected cell line can be HEK293.

[0462] Preferred cells are SKOV3 or OVCAR3 expressing human FOLR1.

[0463] Alternatively, binding of the VHH or a fragment thereof according to the invention can preferably be evaluated in vivo in an animal expressing the corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, using imaging techniques. Preferred imaging techniques are SPECT / CT, PET / CT, SPECT / MRI or PET / MRI. Cells overexpressing the antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, can be xenografted into the animal. It is also possible to use the human FAP or FOLR1 or HER2 knock-in mice of the invention expressing human FAP or FOLR1 or HER2.

[0464] Thus, the expression "in vitro" is used herein in relation to cell-free assays or to cells in culture when the human and / or mouse FAP or human FOLR or human HER2 recombinant protein is immobilized on a support or in a solution. In contrast, the terms "in vivo" or "ex vivo" are used herein in relation to non-human animals or to tissues or organs of this non-human animal. Usually, "ex vivo" is used when quantifying non-human or human animal tissues or organs, and "in vivo" is used when performing imaging-based quantification on non-human or human animals. Usually, "binding" is evaluated using in vitro conditions and further confirmed using in vivo conditions.

[0465] In the in vitro, in vivo and ex vivo assays disclosed herein, it is preferred to use a negative control. Specific binding to an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, can also be evaluated in the presence of other antigens as described later herein.

[0466] As used herein, the terms "affinity", "specific binding", "binding", "binding activity" or "specific binding activity" mean that the VHH or fragment thereof of the present invention binds to an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, and shifts the equilibrium between the VHH or fragment and the antigen towards the presence of the complex formed by their binding. Binding can be evaluated using SPR or biolayer interferometry. Thus, for example, when combining human and / or mouse FAP with a VHH or fragment at relatively equal concentrations, a high-affinity VHH or fragment will bind to the available human and / or mouse FAP so as to shift the equilibrium towards a high concentration of the resulting complex. The equilibrium dissociation constant (K D ) is generally used to describe the affinity between a protein binding domain (VHH or fragment) and an antigen.

[0467] Typically, the equilibrium dissociation constant is less than 10 -7 M. Preferably, the equilibrium dissociation constant is less than 10 -8 M or less than 10 -9 M or more preferably in the range of 10 -9 M to 10 -12 M.

[0468] Any VHH or fragment as disclosed herein preferably has an equilibrium dissociation constant (K -9 ~10 -12 mol / liter or 10 -10 ~10 -12 mol / liter for human and / or mouse FAP or human FOLR1 or human HER2. D) that specifically binds thereto, and is preferably evaluated using the biolayer interferometry method.

[0469] The "specificity" of the VHH or fragment thereof according to the present invention can be determined based on affinity and / or binding activity. The "affinity" of the VHH or fragment thereof according to the present invention is represented by the equilibrium constant for the dissociation of the VHH or fragment and the antigen to which it binds. K D The lower the value, the stronger the binding strength between the VHH or fragment and the target protein (antigen) of interest to which it binds. Alternatively, the affinity can be expressed as the equilibrium association constant (K D ) corresponding to 1 / K A . The binding affinity of the VHH or fragment thereof according to the present invention can be determined in a manner known to those skilled in the art according to the specific target protein of interest. The "binding activity" of the VHH or fragment thereof according to the present invention is a measure of the binding strength between the VHH or fragment and the relevant target protein of interest. The binding activity is related to both the affinity between the binding site on the target protein of interest and the binding site on the VHH or fragment, and the number of relevant binding sites present on the VHH or fragment. The VHH has only a single domain and thus has only one binding site. The affinity exhibited by the VHH is in the sub-nanomolar concentration range and is thus very exceptional considering the presence of a single binding site. A K D value exceeding about 1 millimolar is generally considered to indicate non-binding or non-specific binding. K D is the dissociation rate constant of the complex shown as k off or k d (expressed in seconds -1 or s -1 ) and the association rate constant of the complex shown as k on or k a (expressed in moles -1 seconds -1 or M -1 seconds -1 ), and is also generally known in the art to be represented as the ratio thereof. In particular, the VHH or fragment thereof according to the present invention has a dissociation rate constant of 0.1 to 0.00001 seconds-1 in the range of, or 10 -2 ~10 -5 seconds -1 in the range of, or 10 -3 ~10 -5 seconds -1 in the range of, or 10 -4 ~10 -5 seconds -1 over the range of k on and / or 1,000 to 10,000,000 M -1 seconds -1 in the range of, or 10 4 ~10 7 M -1 seconds -1 in the range of, or 10 5 ~10 7 M -1 seconds -1 over the range of k off can bind to the target protein (i.e., antigen) of interest. The binding affinity, k off and k on rates can be determined by methods known to those skilled in the art, such as ELISA, isothermal titration calorimetry, SPR, biolayer interferometry, fluorescence-activated cell sorting analysis, etc.

[0470] In a preferred embodiment, an antibody fragment as disclosed herein specifically binds to human and / or mouse FAP in the range of 0.1 to 0.00001 seconds -1 in the range of, or 10 -2 ~10 -5 seconds -1 or 10 -3 ~10 -5 seconds -1 in the range of, or 10 -4 ~10 -5 seconds -1 over the range of k off and is preferably evaluated using biolayer interferometry.

[0471] In a preferred embodiment, the VHH of the present invention or a fragment thereof as disclosed herein is preferably evaluated using biolayer interferometry and is 10 -9 ~10 -12K in the range of moles per liter D and / or 10 -2 ~10 -5 seconds -1 k in the range of off more preferably 10 -9 ~10 -12 K in the range of moles per liter D and 10 -2 ~10 -5 seconds -1 k in the range of off which specifically binds to human and / or mouse FAP (as defined herein).

[0472] Thus, a VHH or a fragment thereof according to the present invention is said to "specifically bind" to an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, when the VHH or the fragment thereof has an affinity and specificity for the antigen (or at least one part or fragment thereof) and / or is specifically directed against the antigen.

[0473] Regarding the VHH or its fragment according to the present invention, the terms "binding region", "binding site" or "interaction site" as present in the antibody fragment as disclosed herein have the meaning of a specific site, portion, locus, domain or stretch of amino acid residues present in the VHH or fragment that is responsible for binding or specific binding to the corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2. This binding region present on the VHH or fragment is called a paratope. Such a binding region comprises, consists of or consists essentially of specific amino acid residues derived from the amino acid sequence as disclosed herein of the VHH or fragment that is in contact with the corresponding antigen, preferably the extracellular domain region or part or discontinuous amino acids of human and / or mouse FAP or human FOLR1 or human HER2, which may be called an epitope and is defined later herein.

[0474] As used herein, the terms "specifically binds" and "specific binding" generally refer to the ability of a polypeptide, particularly an immunoglobulin such as an antibody or a VHH or its fragment of the present invention, for example, the VHH or its fragment of the present invention, to preferentially bind to a specific antigen such as human and / or mouse FAP or human FOLR1 or human HER2. Such a VHH or its fragment according to the present invention can also be identified as the VHH or its fragment of the present invention that is elicited against a specific antigen.

[0475] Method for purifying and producing VHH The VHH or its fragment according to the present invention can be generated and produced by any of the methods described below.

[0476] As will be apparent to those skilled in the art, one particularly useful method for preparing the VHH or its fragment according to the present invention generally (a) A step of expressing a nucleotide sequence encoding a VHH or a fragment; (b) Optionally, a step of isolating and / or purifying the above VHH or fragment; comprises.

[0477] The nucleic acid encoding the VHH or fragment may be contained in a vector or a gene construct.

[0478] In certain embodiments, the VHH or a fragment thereof can be obtained by a method involving generating a random library of VHH sequences and screening this library for VHH sequences that can specifically bind to a corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2.

[0479] Accordingly, a specific method for preparing a VHH or a fragment thereof according to the present invention is (a) providing a set, collection or library of amino acid sequences of the VHH domain; (b) screening the set, collection or library of amino acid sequences for amino acid sequences that can bind to a corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, and / or can have an affinity therefor; (c) isolating an amino acid sequence that can bind to the above antigen and / or can have an affinity therefor comprises.

[0480] In such a method, the set, collection or library of VHH sequences can be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences can be a set, collection or library of immunoglobulin fragment sequences (described herein), such as a naive set, collection or library of immunoglobulin fragment sequences (a synthetic or semi-synthetic set, collection or library of immunoglobulin fragment sequences and / or a set, collection or library of immunoglobulin fragment sequences that have been subjected to affinity maturation).

[0481] In certain embodiments of this method, the set, collection or library of VHH sequences can be an immune set, collection or library of immunoglobulin fragment sequences, for example, the corresponding antigen, preferably human FAP and / or mouse FAP or human FOLR1 or human HER2 or a suitable antigenic determinant based on or derived from them (e.g., its antigenic portion, fragment, region, domain, loop or other epitope, etc.), and can be an immune set, collection or library of immunoglobulin fragment sequences derived from a mammalian animal suitably immunized with it. In a particular aspect, the antigenic determinant can be an extracellular portion, region, domain, loop or other extracellular epitope.

[0482] In the above method, the set, collection or library of VHH sequences can be presented, for example, on phage, phagemid, ribosome or a suitable microorganism (such as yeast) to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (the set, collection or library) amino acid sequences will be apparent to those skilled in the art, for example, based on further disclosure herein. See also the review by Hoogenboom in Nature Biotechnology, Vol. 23, No. 9, pp. 1105-1116 (2005).

[0483] The method for producing a VHH or a fragment thereof according to the present invention comprises at least (a) Providing a collection or sample of cells that express a VHH domain amino acid sequence; (b) Screening the collection or sample of cells for cells that express an amino acid sequence capable of binding to a corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, and / or having an affinity therefor; (c) (i) Isolating the amino acid sequence, or (ii) isolating a nucleic acid sequence encoding the amino acid sequence from the cells and subsequently expressing the amino acid sequence; comprising.

[0484] The collection or sample of cells can be, for example, a collection or sample of B cells. In this method, the sample of cells can also be derived from a mammal suitably immunized with a corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2 or a suitable antigenic determinant based on or derived from them (e.g., its antigenic portion, fragment, region, domain, loop or other epitope, etc.). In a particular embodiment, the antigenic determinant can be an extracellular portion, region, domain, loop or other extracellular epitope.

[0485] Specific methods for producing a VHH or a fragment thereof according to the present invention include at least (a) Providing a set, collection or library of nucleic acid sequences encoding a VHH domain amino acid sequence; (b) Screening the set, collection or library of nucleic acid sequences for a nucleic acid sequence encoding an amino acid sequence capable of binding to a corresponding antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, and / or having an affinity therefor; (c) Isolating the nucleic acid sequence and subsequently expressing the amino acid sequence; may be included.

[0486] In the above method, the set, collection or library of nucleic acid sequences encoding an amino acid sequence can be, for example, a set, collection or library of nucleic acid sequences encoding a naive set, collection or library of immunoglobulin fragment sequences, a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin fragment sequences, and / or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin fragment sequences that have been subjected to affinity maturation.

[0487] In particular, in such a method, the set, collection or library of nucleic acid sequences encodes a set, collection or library of the VHHs of the present invention or fragments thereof against an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2.

[0488] In the above method, the set, collection or library of nucleotide sequences can be displayed on, for example, phage, phagemid, ribosome or a suitable microorganism (such as yeast) to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding an amino acid sequence will be apparent to those skilled in the art, for example, based on further disclosure herein. See also the review by Hoogenboom in Nature Biotechnology, Vol. 23, No. 9, pp. 1105-1116 (2005).

[0489] The present invention relates to whether the VHH or fragment thereof of the present invention can be obtained by a method comprising the above method or alternatively one of the above methods, and in addition, a step of determining at least the nucleotide sequence or amino acid sequence of the VHH or fragment, and a step of expressing or synthesizing the VHH or fragment thereof in a manner known per se, for example, by expression in a suitable host cell or host organism or by chemical synthesis.

[0490] In some cases, the method for generating the VHH or fragment thereof of the present invention that specifically binds to an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, may further include isolating at least one VHH or fragment thereof having a detectable binding affinity for the antigen or a detectable in vitro effect on these from an amino acid sequence library.

[0491] These methods may further include amplifying a sequence encoding at least one VHH or fragment thereof having a detectable binding affinity for the antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, or a detectable in vitro effect on its activity. For example, a phage clone showing a specific amino acid sequence obtained from the selection step of the method described herein can be amplified by reinfection of host bacteria and incubation in a growth medium.

[0492] A particular method may include determining the sequence of one or more amino acid sequences that can bind to an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2.

[0493] When the VHH or fragment thereof of the present invention contained in a set, aggregate or library of amino acid sequences is presented on a suitable cell or phage or particle, it is possible to isolate the nucleotide sequence encoding its amino acid sequence from said cell or phage or particle. In this way, the nucleotide sequence of a selected amino acid sequence library member can be determined by conventional sequencing methods.

[0494] A specific method for generating the VHH or fragment thereof according to the present invention includes expressing the nucleotide sequence in a host organism under suitable conditions to obtain the actual desired amino acid sequence. This step can be carried out by methods known to those skilled in the art.

[0495] In addition, the obtained VHH or its fragment (which has a detectable binding affinity for an antigen, preferably human and / or mouse FAP or human FOLR1 or human HER2, and / or has no detectable in vitro effect on its activity) can optionally be synthesized as a soluble protein construct after identifying their sequences.

[0496] As an example, the VHH or its fragment of the p...

Claims

1. A heavy chain variable domain derived from a heavy chain antibody (VHH) containing a click group or a fragment thereof for use as a medicament, preferably for the treatment and / or diagnosis of cancer, wherein the click group does not cause a click reaction when the medicament is administered to a patient.

2. A heavy chain variable domain derived from a heavy chain antibody (VHH) or a fragment thereof, comprising a Click group, preferably for use as a pharmaceutical, said VHH or a fragment thereof comprising 10 －２ s －１ Less than 10, more preferably －３ s －１ Below, most preferably 10 －４ s －１ The dissociation constant (k ｏｆｆ ) capable of specifically binding to an antigen, said click group not forming a click pair when said medicament is administered to a patient, more preferably said VHH or fragment thereof is for use in the treatment and / or diagnosis of a cancer associated with said antigen.

3. The click group is an alkene, alkyne, azide, nitrone or tetrazine, preferably the alkene is trans-cyclooctene, preferably the alkyne is BCN, BARAC or DIBAC, the VHH or fragment thereof according to claim 2 or the VHH or fragment thereof for use according to claim 1 or 2.

4. The antigen is present on the surface of tumor cells or within the tumor microenvironment, the VHH or fragment thereof according to claim 2 or 3 or the VHH or fragment thereof for use according to claim 2 or 3.

5. The antigen is human fibroblast activation protein (FAP), human folate receptor alpha (FOLR1), human epidermal growth factor receptor 2 (HER2), the VHH or fragment thereof according to any one of claims 2 to 4 or the VHH or fragment thereof for use according to any one of claims 2 to 4.

6. A combination for use in the treatment and / or diagnosis of cancer, - a heavy chain variable domain derived from a heavy chain antibody (VHH) or a fragment thereof according to any one of claims 1 to 5, - a labeled compound containing a label and a click group comprising The VHH or a fragment thereof is －２ s －１ Less than 10, more preferably －３ s －１ Below, most preferably 10 －４ s －１ The dissociation constant (k ｏｆｆ ) capable of specifically binding to the cancer-associated antigen; the use includes an initial administration of the VHH or fragment thereof to a subject in need thereof and a subsequent administration of the labeled compound, the click group contained in the VHH or fragment thereof forms a click pair with the click group contained in the labeled compound.

7. The click group contained in the labeled compound is an alkene, alkyne, azide, nitrone or tetrazine, preferably the alkene is trans-cyclooctene or the alkyne is BCN, BARAC or DIBAC, more preferably the click group contained in the labeled compound is tetrazine, the combination for use according to claim 6.

8. The labeling compound is CL-L １ -L ２ -R ＊ and can be represented by CL-L-L-R, where CL is the click group, L １ is a first linker or bond, L ２ is a second linker, and R ＊ is the label, a combination for use according to claim 6 or 7.

9. L １ is PEG ｎ wherein n is an integer from 1 to 10, preferably n is an integer from 5 to 9, and more preferably n is 7, the combination for use according to claim 8.

10. L ２ is a chelating agent or a benzoic acid linker, preferably, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (Macropa), 2,2',2'',2'''-(1,10-dioxa-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid (Crown) or 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), preferably, the benzoic acid linker is guanidinomethyl benzoate (GMIB), a combination for use according to claim 8 or 9.

11. The combination for use according to any one of claims 6 to 10, wherein the label is a radionuclide.

12. The radionuclide is an α-emitting radionuclide or a β-emitting radionuclide. Preferably, the radionuclide is actinium-225, astatine-211, bismuth-212, bismuth-213, cesium-137, chromium-51, cobalt-60, copper-67, dysprosium-165, erbium-169, fermium-255, gold-198, holmium-166, iodine-125, iodine-131, iridium-192, iron-59, lead-212, lutetium-177, molybdenum-99, palladium-103, phosphorus-32, potassium-42, rhenium-186, rhenium-188, samarium-153, radium-223, radium-224, ruthenium-106, sodium-24, strontium-89, scandium-47, terbium-149, terbium-161, terbium-149, thorium-227, xenon-133, ytterbium-169, ytterbium-177 or yttrium-90. More preferably, the radionuclide is lutetium-177, terbium-161 or iodine-131. Most preferably, the radionuclide is lutetium-177 or iodine-131. The combination for use according to claim 11.

13. The radionuclide is a positron-emitting radioisotope (PET) or a γ-emitting radioisotope (SPECT). Preferably, the radionuclide is iodine-131, yttrium-90, iodine-125, lutetium-177, rhenium-186, rhenium-188, terbium-161, terbium-149, technetium-99m, indium-111, xenon-133, thallium-201, fluorine-18, gallium-68, gallium-67, copper-67, scandium-44, scandium-43, iodine-123, iodine-124, zirconium-89 or copper-64. More preferably, the radionuclide is lutetium-177, terbium-161 or iodine-131. Most preferably, the radionuclide is lutetium-177 or iodine-131. The combination for use according to claim 11.

14. The labeling compound is １７７ Lu-DOTA-PEG ７ -Tz, where Tz is tetrazine, for use in the combination according to any one of claims 6 to 13.

15. The blood purification agent is a combination for use according to any one of claims 6 to 14, which is not administered to the subject between the first administration and the subsequent administration.

16. The time between the first administration and the subsequent administration is at least 2 hours, preferably at least 4 hours, more preferably at least 8 hours, for a combination for use according to any one of claims 6 to 15.

17. The cancer is a solid cancer, for a VHH or a fragment thereof for use according to any one of claims 1 to 5 or a combination for use according to any one of claims 6 to 16.

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