Anti-Cd2 single domain antibodies, pharmaceutical compositions and kits for use in diagnosis and / or therapy of cancer

Abstract

The present invention relates to a single domain antibody binding to human CD2 for use in the diagnosis and / or therapy of cancer, preferably comprising at least one of a detectable label, a chelating agent, a prosthetic group, a therapeutically active agent and / or a pharmacologically active agent; the invention also discloses a pharmaceutical composition and a kit containing the single-domain antibody.

Description

[0001] The present invention relates to a single-domain antibody that binds to human CD2 for use in the diagnosis and / or therapy of cancer in vivo, preferably comprising at least one of a detectable label, a chelating agent, a prosthetic group, a therapeutically active agent and / or a pharmacologically active agent, and a pharmaceutical composition and kit comprising the single-domain antibody.

[0002] Immunotherapeutic approaches have been shown to be very effective in the treatment of various tumor diseases. In particular, immune checkpoint inhibitors, such as anti-PD-1 and anti-CTLA-4, have been approved for the treatment of various tumor entities. However, the possibility of adoptive cell transfer of genetically modified T cells, i.e., the use of chimeric antigen receptor T cells (CAR-T cells) or T cell receptor (TCR)-transduced T cells, has also shown promising clinical results for the first time, as evidenced by the European Medicines Agency (EMA)'s approval of two CD19-specific CAR-T cells across Europe (Druker et al., 2006).

[0003] As these new therapeutic options increasingly enter trials and clinical practice, adequate monitoring of individual therapy progress becomes increasingly important.

[0004] Established imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI), as well as functional imaging modalities such as positron emission tomography (PET), have high spatial and temporal resolution but are limited in their use for monitoring the progress of immunotherapy (Lang et al., 2020). This makes it difficult to distinguish between tumor progression and pseudoprogression, i.e., an increase in lesion size due to tumor growth or migration of immune cells (Dromain et al., 2020).

[0005] The 2009 revision of the RECIST (Response Evaluation Criteria in Solid Tumors) guidelines specifically for immunotherapy (irRECIST) represented the first attempt to improve the assessment of treatment progress, but a suitable, sensitive, and clinically usable test is still lacking. The ability to assess treatment response early to avoid premature discontinuation and unnecessary continued therapy, as well as the timely detection of T cell infiltration in healthy tissues as a manifestation of autoimmune events, is extremely important. In addition, adequate monitoring will significantly deepen the current understanding of the pharmacokinetics and pharmacodynamics of immunotherapies.

[0006] One promising imaging approach is the direct visualization of T cells, which play a key role in immunotherapy. Weist et al. described a method in which CAR-T cells and TCR-transduced T cells are radiolabeled or loaded with a reporter gene ex vivo prior to adoptive cell transfer (Weist et al., 2018). However, a disadvantage of this approach is that, in the case of radiolabeling, imaging can only be performed for a short period of time, and, for example, the effects of immune checkpoint inhibitors on endogenous T cells cannot be assessed. Furthermore, functional impairment of immune cells is often observed (Auletta et al., 2018).

[0007] Additionally, the feasibility of immuno-PET for T cell detection is currently under investigation. In this method, antibodies or their derivatives directed against specific surface antigens on T cells are radiolabeled, allowing T cells to be imaged at any time point in vivo. The key lies in selecting an appropriate target antigen with high specificity for T cells and an antibody derivative that does not affect T cell function upon antigen binding.

[0008] WO2021 / 113450A2 describes a non-invasive imaging method for the diagnosis, prediction, prognosis and treatment of a disease (particularly selected from solid tumors, non-solid tumors, autoimmune diseases, infectious diseases (including but not limited to viral, bacterial or fungal infections)), the method comprising administering to a subject a first antigen-binding construct comprising a first radionuclide tracer, wherein the antigen-binding construct selectively binds to a first target selected from CD3, CD4 and CD8, and measuring the level of the first radionuclide tracer of the subject using positron emission tomography (PET) or single photon emission computed tomography (SPECT) to estimate the distribution and / or abundance of cells expressing the first target in one or more tissues of the subject. Preferably, the antigen-binding construct is Fab', F(ab')2, Fv, rIgG (reduced IgG), scFv fragment, miniantibody, diabody, cysteine-diabody or nanobody.

[0009] Mayer et al. described the use of CD7 as a target for monitoring T cells in the context of immunotherapy (Mayer et al., 2018). T cells incubated with anti-CD2 and anti-CD7 F(ab')2 did not show significant modulation of function in vitro and provided clear and strong signals at the tumor site when imaged with the corresponding zirconium-89 labeled radiotracers. However, while T cells labeled with anti-CD7 F(ab')2 had no long-term effects on T cell function in vivo, anti-CD2 F(ab')2 led to severe T cell exhaustion and failure of tumor rejection. Unfortunately, CD7 was only slightly upregulated on specifically activated T cells and was not prominent on thymocytes.

[0010] US 10 301 389 B2 describes antigen-binding constructs for CD3 (particularly cysteine-diabodies or minibodies), and their use for modulating biological activities associated with CD3 expression on immune cells, for targeting therapeutic agents to cells expressing CD3 protein, and for detecting the presence or absence of CD3.

[0011] US 2021 / 0371527 A1 describes antigen-binding constructs (particularly cysteine-diabodies or minibodies) that bind to CD4, and their use in detection, diagnosis, surgery, staging, treatment, treatment monitoring, disease progression monitoring, and therapy monitoring.

[0012] US 10 414 820 B2 describes cysteine-diabodies or minibodies that bind to CD8 and their use for detecting human CD8, in particular for diagnostic imaging of the immune system, preferably for in vivo detection of T cell localization.

[0013] Therefore, an object of the present invention is to provide a surrogate marker associated with T cell binding for diagnosis and / or therapy of cancer.

[0014] According to the present invention, the objects of the present invention are achieved by a single domain antibody binding to human CD2, a nucleic acid, a pharmaceutical composition and a kit for use in the diagnosis and / or therapy of cancer according to the independent claims. Advantageous embodiments of the invention are indicated in the dependent claims.

[0015] A first aspect of the present invention provides a single domain antibody that binds to human CD2 for use in the diagnosis and / or therapy of cancer in vivo.

[0016] As used herein, the term "single domain antibody" (sdAb), also known as nanoantibodies, refers to an antibody fragment consisting of a single monomeric variable antibody domain and selectively binding to a specific antigen (particularly CD2). The present invention also includes single domain antibodies in multivalent form. In an embodiment, the multivalent form of the single domain antibody is formed by combining at least two single domain antibodies of the present invention by chemical methods or recombinant DNA technology. In an embodiment, the single domain antibody is in a divalent form (two combined single domain antibodies), a trivalent form (three combined single domain antibodies) or a tetravalent form (four combined single domain antibodies). The single domain antibodies included in the multivalent construct can be the same or different. Advantageously, the single domain antibodies according to the present invention are small in size and have good binding properties. The single domain antibodies can circulate rapidly through the bloodstream, penetrate target tissues and bind to immune cells, while also being rapidly excreted without being considered as harmful substances to the patient, and will not affect any physiological parameters of the patient, in particular sdAb does not impair the cytotoxicity of T cells and has no effect on the secretion of cytokines.

[0017] As used herein, the term "CD2" (Cluster of Differentiation 2) refers to a cell adhesion molecule found on the surface of T cells and Natural Killer (NK) cells, preferably human CD2 according to SEQ ID No. 1.

[0018] In an embodiment, the single domain antibody that binds to human CD2 has an affinity for CD2 with a dissociation constant K d In the range of 1 nM to 100 nM, preferably in the range of 1 nM to 15 nM. Suitably, said affinity is determined using flow cytometry.

[0019] As used herein, the term "cancer" refers to a disease involving abnormal cell growth that has the potential to invade or spread to other parts of the body.

[0020] In an embodiment, the cancer is selected from the group comprising atypical meningioma, brain tumors of different phenotypes, basal cell carcinoma, breast cancer, cervical cancer, urinary tract cancer, colon cancer / colorectal cancer, endometrial cancer, laryngeal epithelial cancer, gastric cancer, glioblastoma, gynecological cancer, hepatocellular carcinoma, Helicobacter pylori-induced MALT lymphoma, Kaposi's sarcoma, leukemia (particularly acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL)), liver cancer, lung cancer (particularly non small cell lung cancer (NSCLC), lymphoma (particularly Hodgkin lymphoma), Merkel cell carcinoma, multiple myeloma (particularly cutaneous, mucosal, or uveal myeloma), esophageal cancer, oral cancer, oral squamous cell carcinoma (OSCC), ovarian cancer, pancreatic cancer, pancreatic adenocarcinoma, peritoneal cancer, prostate cancer, renal cell carcinoma, salivary duct carcinoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma (SCC) (including SCC of the head, neck, and skin), thyroid cancer, and yolk sac tumor.

[0021] In an embodiment, a single domain antibody that binds to human CD2 (anti-CD2-sdAb) according to the present invention is used for cancer diagnosis in immunotherapy by directly imaging immune cells (particularly T cells, NK cells, thymocytes or dendritic cells, preferably T cells).

[0022] Advantageously, single-domain antibodies that bind to human CD2 can be used to assess response patterns to cancer immunotherapies. Advantageously, CD2 is present on every subset of immune cells, allowing for the evaluation of each immunotherapy. Furthermore, CD2 is present at higher densities on activated immune cells, allowing for clearer imaging of immune cells targeting tumors.

[0023] Furthermore, the binding of the single-domain antibody to the target molecule CD2 does not affect the function of immune cells (particularly T cells and / or NK cells).

[0024] In a further embodiment, the single domain antibodies according to the invention that bind to human CD2 are used for theranostic use (i.e., diagnosis combined with treatment) of immune cell-related cancers, preferably lymphomas (particularly T-cell or NK-cell lymphomas) or leukemias.

[0025] As used herein, the term "lymphoma" refers to a "malignant lymphoma" that is a cancer of the lymphatic system, which includes the lymph nodes, tonsils, and spleen, as well as lymphatic tissue in the stomach, intestines, or skin. In lymphoma, white blood cells belonging to the lymphatic system (so-called lymphocytes) grow uncontrollably.

[0026] As used herein, the term "leukemia" refers to a group of blood cancers that have large numbers of abnormal white blood cells.

[0027] In an embodiment, the diagnosis is performed before, during and / or after cancer immunotherapy.In an embodiment, the single domain antibody linked to at least one detectable label, chelator, prosthetic group, therapeutically active agent and / or pharmacologically active agent is used as a radiotracer and / or contrast agent in non-invasive medical imaging of T cells in vivo, preferably by PET or SPECT imaging; or for the synthesis of radiotracers and / or contrast agents.

[0028] Advantageously, the use of nanobodies that bind to human CD2 for cancer diagnosis during therapy enables precise observation of the presence of immune cells on and inside the tumor, indicating whether the tumor is being attacked and whether immunotherapy is effective. While CT or MRI can detect tumor growth or regression after up to two months, the infiltration of tumor cells by T cells can be revealed after only 1 to 2 weeks. In addition, if there are no immune cells on the tumor, this can also be determined quickly, and the patient can be switched to a different therapy. As a result, patients gain extremely valuable time, which can extend and / or save lives, without having to administer extremely costly and unhelpful immunotherapy for weeks. As a result, important treatment decisions can be made significantly earlier.

[0029] Furthermore, different tumor types are not limited because single-domain antibodies that bind to human CD2 do not visualize the tumor itself, but rather the immune cells used in immunotherapy. Purposefully, activated T cells (i.e., those actively fighting tumors) are bound by the single-domain antibodies. The increased density of the target molecule (CD2) also allows more tracer to bind to T cells, which in turn allows only tumor-killing cells to be more clearly visualized.

[0030] In an embodiment, the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20, or an amino acid sequence having at least 90% sequence similarity to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20.

[0031] As used herein, the term "similarity" refers to conservative substitutions of amino acid residues having similar physicochemical properties over the full length of the amino acid sequence. In an embodiment, the term "similarity" further comprises an amino acid sequence having amino acids comprising modifications selected from the group comprising: D-amino acids, pseudopeptide bonds, amino alcohols, amino acids with modified side chains (particularly cysteine ​​and selenocysteine) and / or cyclic proteins. Advantageously, the amino acids have been shown to increase the stability of the single domain antibody.

[0032] The percentage (%) of sequence similarity is determined by any reasonable similarity scoring matrix known to those skilled in the art, preferably a similarity scoring matrix selected from BLOSUM50, BLOSUM62, PMBEC, or VTML10 to VTML80.

[0033] In an embodiment, the single-domain antibody comprises an amino acid sequence having at least 80%, preferably at least 85%, and more preferably at least 90% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20. As used herein, the term "percentage (%) of sequence identity" refers to the number of identical amino acid residues relative to the length of the amino acid sequence.

[0034] Where appropriate, the percentage identity between amino acid sequences refers to the full-length alignment of the two sequences, obtained by aligning the amino acid sequences using the EM-BOSS Water PairwiseSEQuence Alignments (Protein) program (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_water). These tools for local sequence alignment, provided by the European Bioinformatics Institute (EBI) of the European Molecular Biology Laboratory (EMBL), use a modified Smith-Waterman algorithm (see https: / / www.ebi.ac.uk / jdispatcher / psa and Smith and Waterman, 1981). The default parameters defined by EMBL-EBI were used for the alignment. These parameters for amino acid sequences were: matrix = BLOSUM62, gap opening penalty = 10, gap extension penalty = 0.5.

[0035] In an embodiment, the single-domain antibody comprises complementarity determining regions (CDRs): a CDR1 having an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 29, a CDR2 having an amino acid sequence selected from the group comprising SEQ ID No. 30 to SEQ ID No. 37, and a CDR3 having an amino acid sequence selected from the group comprising SEQ ID No. 38 to SEQ ID No. 46.

[0036] In an embodiment, the single-domain antibody comprises: a CDR1 having an amino acid sequence selected from SEQ ID No. 22 or SEQ ID No. 29, a CDR2 having an amino acid sequence selected from SEQ ID No. 30, SEQ ID No. 35 or SEQ ID No. 36, and a CDR3 having an amino acid sequence selected from SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 44 or SEQ ID No. 45.

[0037] In an embodiment, the single domain antibody comprises:

[0038] having a CDR1 of SEQ ID No. 21, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 38,

[0039] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 38,

[0040] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 31 and a CDR3 of SEQ ID No. 39,

[0041] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 32, and a CDR3 of SEQ ID No. 39,

[0042] having a CDR1 of SEQ ID No. 23, a CDR2 of SEQ ID No. 31 and a CDR3 of SEQ ID No. 40,

[0043] having a CDR1 of SEQ ID No. 24, a CDR2 of SEQ ID No. 33, and a CDR3 of SEQ ID No. 41,

[0044] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 34, and a CDR3 of SEQ ID No. 42,

[0045] having a CDR1 of SEQ ID No. 25, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 43,

[0046] having a CDR1 of SEQ ID No. 26, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44,

[0047] having a CDR1 of SEQ ID No. 27, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44,

[0048] having a CDR1 of SEQ ID No. 28, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44,

[0049] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44,

[0050] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 36, and a CDR3 of SEQ ID No. 45, or

[0051] CDR1 having SEQ ID No. 29, CDR2 having SEQ ID No. 37 and CDR3 having SEQ ID No. 46.

[0052] In a preferred embodiment, the single-domain antibody comprises an amino acid sequence according to SEQ ID No. 18, an amino acid sequence having at least 90% sequence similarity to SEQ ID No. 18, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90% sequence identity to SEQ ID No. 18.

[0053] In an embodiment, the single domain antibody comprises a CDR1 having SEQ ID No. 29, a CDR2 having SEQ ID No. 36, and a CDR3 having SEQ ID No. 45.

[0054] In an embodiment, the size of the single domain antibody is in the range of 11 kDa to 16 kDa, preferably in the range of 13 kDa to 15 kDa, more preferably in the range of 14.0 kDa to 14.5 kDa. Advantageously, the single domain antibody is smaller than an antibody. Surprisingly, despite its smaller size, the single domain antibody according to the present invention can be shown to have good binding to CD2.

[0055] In an embodiment, the sequence length of the single domain antibody is in the range of 105 amino acids to 140 amino acids, preferably in the range of 110 amino acids to 130 amino acids.

[0056] In an embodiment, the single domain antibody is a multivalent form. In an embodiment, the single domain antibody in the multivalent form is formed by combining at least two single domain antibodies according to the present invention by chemical methods or recombinant DNA technology. In an embodiment, the single domain antibody is a divalent form (two combined single domain antibodies), a trivalent form (three combined single domain antibodies) or a tetravalent form (four combined single domain antibodies). The single domain antibodies included in the multivalent construct may be the same or different. In an embodiment, the single domain antibody is a divalent form, wherein one single domain antibody binds to CD2, and another single domain antibody binds to another cell adhesion molecule found on the T cell surface, particularly CD7.

[0057] In an embodiment, the single domain antibody is linked to at least one of a detectable label, a chelator, a prosthetic group, a therapeutically active agent, and / or a pharmacologically active agent.

[0058] As used herein, the term "linked" refers to the direct or indirect binding (coupling) of a single domain antibody to a detectable label, a chelator, a prosthetic group, a therapeutically active agent and / or a pharmacologically active agent, in particular through covalent or non-covalent interactions (hydrogen bonds, electrostatic interactions, hydrophobic interactions and van der Waals forces).

[0059] In an embodiment, the single domain antibody is covalently bound to a linker that is bound to a detectable label, a chelator, a prosthetic group, a therapeutically active agent, and / or a pharmacologically active agent.

[0060] In an embodiment, the single domain antibody for use in cancer treatment (particularly theranostic) is linked to at least one therapeutically active agent and / or pharmacologically active agent. Advantageously, the single domain antibody for use in cancer treatment (particularly theranostic) targets the therapeutically active agent and / or pharmacologically active agent to cancer cells.

[0061] As used herein, the term "detectable label" (also referred to as a detectable marker) refers to a detectable compound or composition that is conjugated (linked) directly or indirectly to a single domain antibody, wherein it can be detected by itself (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, it can catalyze a chemical change in a detectable substrate compound or composition. Detectable labels include detectable moieties and tracers for immunohistochemistry, optical imaging, near-infrared imaging (NIR), positron emission tomography (PET), single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI).

[0062] In an embodiment, the detectable label is selected from a tag, a dye, a fluorescent compound, a bioluminescent compound, a radioisotope, a contrast agent, an enzyme, a nanoparticle and / or a magnetic particle.

[0063] As used herein, the term "chelating agent" refers to an organic compound having two or more lone pairs of electrons and capable of forming more than one coordinate bond with a central (metal) ion (particularly with a radioisotope). Advantageously, a chelating agent is capable of fixing divalent or multivalent cations in a stable cyclic complex (chelate).

[0064] In an embodiment, the chelating agent is selected from the group comprising 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazabicyclo[5.5.2]tetradecane-4,10-diacetic acid (CB-DO2A), 1,4,8,11-tetraazabicyclo[6.6 .2] hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1-pentanedioic acid-4,7-diacetic acid (NODAGA), 1,4,7,10-tetrakis[2-(methylthio)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylthio)ethyl]-1 ,4,7,10-tetraazacyclododecane (DO3S), 1,4,7-tris[2-(methylthio)ethyl]-10-acetamido-1,4,7,10-tetraazacyclododecane (DO3SAm), 1,7-bis[2-(methylthio)ethyl]-4,10-diacetate-1,4,7,10-tetraazacyclododecane (DO2A2S), 1,4,7,10-tetra[2-(methylthio)ethyl]-1,4,7,1 0-tetraazacyclotridecane (TRI4S), 1,4,8,11-tetrakis[2-(methylthio)ethyl]-1,4,8,11-tetraazacyclotetradecane (TE4S), 6-amino-6-methylperhydro-1,4-diazacycloheptane tetraacetic acid (AAZTA), 6-amino-1,4-diazacycloheptane triacetic acid (DATA), tridentate bidentate catecholamide (TREN-CAM), preferably DOTA or NOTA.

[0065] In an embodiment, the chelating agent is selected from modified chelating agents of the above-mentioned chelating agents, wherein at least one functional group and / or side chain is modified.

[0066] As used herein, the term "prosthetic group" refers to a non-polypeptide molecule that is essential for the biological function of a protein. In embodiments, the prosthetic group is as disclosed in Wester and Schottelius (Wester and Schottelius, 2007). In embodiments, the prosthetic group is selected from SiFA (silicon-fluorine acceptor), SFB (N-succinimidyl-4-[ 18 F] fluorobenzoate), FBEM ( 18 [N-[2]-(4-fluorobenzamido)ethyl]-maleimide), halides and sulfonates, in particular tosylate, triflate, nitrobenzenesulfonate and methanesulfonate.

[0067] As used herein, the term "therapeutically active agent and / or pharmacologically active agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction of cancer cells, including radioisotopes, chemotherapeutic agents, enzymes and fragments thereof (such as nucleases); antibiotics, toxins, growth inhibitory agents, and drug moieties.

[0068] As used herein, the term "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer.

[0069] In an embodiment, the chemotherapeutic agent is selected from an immunotoxin, a chimeric antigen receptor T cell (CAR-T cell), and a switchable CAR-T cell.

[0070] As used herein, the term "immunotoxin" refers to a protein comprising at least one targeting domain linked to a toxin. When the protein binds to a cell via the targeting domain, it is taken up by endocytosis, and the toxin kills the cell.

[0071] As used herein, the term "CAR" refers to an artificial receptor consisting of a binding portion and one or more signal transduction chains derived from an immune receptor, wherein the binding portion provides antigen specificity to the target cell. Immune cells genetically modified to express CAR can be used to bind to cells or tissue structures of appropriate targets expressing the CAR binding portion. Cross-linking leads to the induction of signal pathways through the CAR signal transduction chain, which will change the biological properties of the CAR-transplanted immune cells. Advantageously, CAR-T immunotherapy is used to modify T cells to recognize cancer cells, thereby effectively targeting and destroying them.

[0072] As used herein, the term "switchable CAR-T cell" refers to a T cell engineered to express a CAR that has a binding domain that recognizes a tag or is itself a tag. Antigen specificity is provided by a soluble targeting molecule that consists of an antigen binding domain fused to a tag or a tag binding domain recognized by a switchable CAR.

[0073] In an embodiment, the chemotherapeutic agent is selected from the group comprising: A-dmDT390-bisFv (UCHT1), a bispecific CAR targeting CD19 and CD22; BL22, BM7PE, Cintredekin besudotox, Denileukin diftitox, DT2219, Hum-195 / rGel, IgG-RFB4-SMPT-dgA, IL-4 (38–37)-PE38KDEL, LMB-2, LMB-7, LMB-9, LMB-100, MOC31PE, Moxetumomab Pasudotox, MR1–1, RFT5pdgA, MT-3724, SS1 (dsFv)-PE38, T-Guard, and transferrin-CRM107.

[0074] In a further embodiment, the chemotherapeutic agent is selected from the group consisting of alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylamelamines, acetogenins, delta-9-tetrahydrocannabinol, beta-lapachone, lapachol, colchicine, betulinic acid, camptothecin, bryostatin, callystatin, CC-1065, podophyllotoxin, podophyllic acid, teniposide, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sucralose ... sarcodictyin, spongistatin, nitrogen mustards, nitrosoureas, antibiotics, dynemicin, esperamicin, aclacinomycins, actinomycins, authramycin, azaserine, bleomycins, actinomycin C, carabicin, carminomycin, carmophorin, chromomycins, actinomycin D, daunorubicin, detoximum iodide, 6-diazo-S-oxo-L-norleucine, ADRIAMYCIN TMDoxorubicin, epirubicin, esorubicin, idarubicin, marseromycin, mitomycins, mycophenolic acid, nogamycin, olivomycins, peplomycin, porphyromycin, puromycin, quelamycin, rhodorubicin, streptomycin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, androgens, antiadrenal agents, folic acid supplements, aceglucuronolactone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, besitabucil ( bestrabucil), bisantrene, edatrexate, defofamine, colcemid, diazocine, elfomithine, elliptinium acetate, epothilone, ethoxydimidine, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine compounds, mitoguanidine, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazine, procarbazine, PSK TM (Polysaccharide-K) polysaccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triiminoquinone, 2,2',2"-trichlorotriethylamine, trichothecenes, urethane, vindesine (ELDISINETM, FILDESIN TM ), dacarbazine, mannitol nitrogen mustard, dibromomannitol, dibromodulcitol, pipobroman, gacytosine, cytarabine ("Ara-C"), thiotepa, taxanes, chlorambucil, gemcitabine (GEMZAR TM ), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs, vinblastine (VELBAN TM ), platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN TM ), oxaliplatin, leucovorin, vinorelbine (NAVELBINE TM ), novantrone, edatrexate, daunomycin, aminoptern, ibandronate, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids, capecitabine (XELODA TM ), pharmaceutically acceptable salts, acids or derivatives thereof, and combinations thereof.

[0075] In an embodiment, the chemotherapeutic agent is selected from the group comprising: alkylating agents such as thiotepa and CYTOXAN TM Cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylaminopropidines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins, particularly bratasin and bratasinone; delta-9-tetrahydrocannabinol, particularly dronabinol and MARINOL TM ; β-lapachone; Lapachol; colchicine; betulinic acid; camptothecin, including its synthetic analogue topotecan (HYCAMTIN TM ), CPT-II (irinotecan, CAMPTOSAR TM ), acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; callystatin; CC-1065, including its synthetic analogs adozelesin, carzelesin, and bizelesin; podophyllotoxin; podophyllic acid; teniposide; cryptophycins, particularly cryptophycin 1 and cryptophycin 8; dolystine; duocarmycins, including its synthetic analogs KW-2189 and CB1-TM1; eleutherobin; pancratistatin; chrysogenum sclerotin, spongestatin, nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, hydrochloride), melphalan, nembice, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II); lidamycins, including lidamycin A; ramycins; and neocarcinogens and related chromogenic protein enediyne antibiotic chromophores, aclarubics, actinomycins, authramycin, azaserine, bleomycins, actinomycin C, carabicin, carminomycin, carmosin, chromomycins, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN TMDoxorubicin, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and desoxydoxorubicin; epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolic acid, nogamycin, olivomycins, peplomycin, porfiromycin, puromycin, quinamycin, rhodorubicin, streptomycin, streptomycin Urexacil, tuberculin, ubenimex, zoloft, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine glycosides, doceflurane, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, cyclothiocarbamate, melastane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bispyribacil Stabucil; bisantrene; edatrexate; deferoxamine; colcemid; diazocine; eflornithine; elliptinium acetate; epothilone; ethoxydimidine; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine compounds, such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidol; nitrosamine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; PSK TM (Polysaccharide-K) polysaccharide complex; razoxane; rhizoxin; sizolan; spirogermanium; alternating spore acid; triimidoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes, especially T-2 toxin, verrucosporin A, baculosporin A, and serpentin; urethane; vindesine (ELDISINE) TM FILDESIN TM ); dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; cytarabine ("Ara-C"); thiotepa; taxanes, such as TAXOL TM , paclitaxel, ABRAXANE TM and TAXOTERE TM Docetaxel; Chlorambucil; Gemcitabine (GEMZAR TM ); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN TM ); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN TM); oxaliplatin; leucovorin; vinorelbine (NAVELBINE TM ); noantolone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine (XELODA TM ); pharmaceutically acceptable salts, acids or derivatives thereof, and combinations thereof.

[0076] In a preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: thiotepa and CYTOXAN TM Cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquinone, methandopa, uredopa, ethyleneimines, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bratasin, bratasinone, dronabinol, MARINOL TM , β-lapachone, lapachol, colchicine, betulinic acid, topotecan (HYCAMTIN TM ), CPT-11 (irinotecan, CAMPTOSAR TM ), acetylcamptothecin, scopoletin, 9-aminocamptothecin, bryostatin, callystatin, CC-1065, CC-1065 adozelain analog, CC-1065 kazelain analog, CC-1065 bitzelain analog, podophyllotoxin, podophyllic acid, teniposide, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1; eleutherobin, pancratistatin, chlorambucil, naphthalene nitrogen mustard, cholephosphamide, estramustine, ifosfamide, nitrogen mustard, nitric oxide mustard hydrochloride, melphalan, nembice, phenylephrine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, enediyne antibiotics, calicheamicin, lidamycin, ramycin, neocarzinostatin chromophore, aclarubicin, actinomycin, authramycin, azaserine, bleomycins, actinomycin C, carabicin, carminomycin, carmosin, chromomycins, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN TMDoxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marseromycin, mitomycins, mycophenolic acid, nogamycins, olivomycins, peplomycin, porfibrinocin, puromycin, quinamycin, rhodorubicin, streptozocin, streptozotocin, tuberculin, ubenimex, zoloft, doxycycline, methotrexate, 5-fluorouracil (5-FU), dimethylfolate, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiopurine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, Dideoxyuridine, doxefiridine, enocitabine, floxuridine, captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglucuronolide, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, besitabucil, bisantrene, edatrexate, deferoxamine, colcemid, diazocine, eflornithine, elliptonium acetate, epothilone, ethoxydimidine, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocins, mitoguanidine, mitoxantrone, mopidol, nitrosamine, pentostatin, benamine, pirarubicin, losoxantrone, 2-ethylhydrazine, procarbazine, PSK TM (Polysaccharide-K) polysaccharide complex, razoxane, rhizoxin, sizolan, spirogermanium, alternaria acid, triiminoquinone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verrucosporin A, baculozolin A, serpentine, urethane, vindesine (ELDISINE TM FILDESIN TM ), dacarbazine, mannitol mustard, dibromomannitol, dibromodulcitol, pipobroman, cytarabine ("Ara-C"), thiotepa, TAXOL TM , paclitaxel, ABRAXANE TM 、TAXOTERE TM Docetaxel, chlorambucil, gemcitabine (GEMZAR TM ), 6-thioguanine, mercaptopurine, methotrexate, cisplatin, carboplatin, vinblastine (VELBAN TM ), platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN TM ), oxaliplatin, leucovorin, vinorelbine (NAVELBINE TM ), Noantolone, edatrexate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoic acid, capecitabine (XELODA TM ), its pharmaceutically acceptable salts or acids, and combinations thereof.

[0077] As used herein, the term "toxin" refers to a naturally occurring organic substance that is produced by the metabolic activity of living cells or organisms (particularly bacteria, fungi, plants or animals) and has an adverse effect on the growth or proliferation of cells. In embodiments, the toxin is a small molecule, a peptide or a protein. In embodiments, the toxin is selected from an exotoxin or an endotoxin.

[0078] In an embodiment, the toxin is selected from the group comprising abrin, auristatins (such as auristatin E, auristatin F, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)); calicheamicins, cholera toxin, chlortetracycline, diphtheria toxin, daunorubicin, duocarmycins, dolastatins, liposome-encapsulated doxorubicin, maytansine compounds (such as maytansine compounds DM1 and DM4); maytansine, mitomycin, paclitaxel, Pseudomonas exotoxin, ricin, ricin A and vinblastine.

[0079] In an embodiment, the detectable label, therapeutically active agent or pharmacologically active agent is a radioisotope selected from the group comprising: 68 Ga, 18 F. 89 Zr, 99m Tc, 61 Cu, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 47 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 123 I. 124 I. 177 Lu, 225 Ac, 213 Bi, 211 At 212 Pb and 223 Ra, preferably 68 Ga.

[0080] In an embodiment, the single domain antibody is conjugated to at least one chelator and / or prosthetic group and at least one radionuclide (particularly 68 Ga, 18 F. 89 Zr, 99m Tc, 61 Cu, 64 Cu, 67 Cu, 43 Sc, 44 Sc,47 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 123 I. 124 I. 177 Lu, 225 Ac, 213 Bi, 211 At 212 Pb and 223 Ra, preferably 68 Ga) are connected.

[0081] In an embodiment, the single domain antibody is conjugated to at least one chelator (preferably DOTA or NOTA) and at least one radionuclide (particularly 68 Ga, 18 F. 89 Zr, 99m Tc, 61 Cu, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 47 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 123 I. 124 I. 177 Lu, 225 Ac, 213 Bi, 211 At 212 Pb and 223 Ra, preferably 68 Ga) are connected.

[0082] In an embodiment, the single domain antibody comprises an amino acid sequence according to SEQ ID No. 18, an amino acid sequence having at least 90% sequence similarity to SEQ ID No. 18, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90% sequence identity to SEQ ID No. 18; and is conjugated to a chelator (preferably DOTA or NOTA) and a radionuclide (particularly 68 Ga, 18 F. 89 Zr, 99m Tc, 61 Cu, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 47Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 123 I. 124 I. 177 Lu, 225 Ac, 213 Bi, 211 At 212 Pb and 223 Ra, preferably 68 Ga), preferably covalently linked.

[0083] Yet another aspect of the present invention provides a pharmaceutical composition for use in the diagnosis and / or therapy of cancer in vivo, comprising at least one single domain antibody according to the present invention.

[0084] The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously. In an embodiment, the pharmaceutical composition is in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion or suspension.

[0085] In an embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable diluent (diluent) or carrier. In an embodiment, the carrier is selected from water, an aqueous buffer solution, a 0.9% saline solution, 5% glucose, 5% xylitol, a 0.3% glycine solution, Ringer's solution or an amino acid solution. In another embodiment, the aqueous buffer solution is selected from an aqueous histidine, sodium succinate, sodium acetate, sodium citrate, sodium phosphate or potassium phosphate buffer solution, and the pH value is in the range of 5.0 to 7.0. In an embodiment, the buffer concentration of the aqueous buffer solution is in the range of 1 mmol / l (mM) to 500 mM, preferably in the range of 5 mM to 20 mM, and particularly preferably in the range of 5 mM to 10 mM.

[0086] In an embodiment, the carrier comprises sodium chloride, preferably at a concentration in the range of 1 mM to 300 mM, particularly preferably 150 mM.

[0087] In an embodiment, the pharmaceutical composition further comprises a stabilizer, preferably at a concentration in the range of 1 mM to 900 mM, particularly preferably in the range of 50 mM to 600 mM. In an embodiment, the stabilizer is sucrose, trehalose and / or L-methionine.

[0088] In an embodiment, the pharmaceutical composition further comprises a free radical scavenger, in particular an antioxidant, preferably at a concentration in the range of 1 mM to 900 mM, particularly preferably in the range of 20 mM to 200 mM. In an embodiment, the free radical scavenger is ascorbic acid ((5R)-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one, vitamin C) or gentisic acid (2,5-dihydroxybenzoic acid). Advantageously, free radical scavengers, in particular antioxidants, can react with and neutralize free radicals formed by radioactivity, which are highly reactive molecules that can trigger oxidation reactions. Thus, free radical scavengers prevent or delay oxidative degradation of sensitive substances (in particular single domain antibodies according to the present invention).

[0089] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a compound that provides approximate physiological conditions and / or increases stability, such as agents and buffers for adjusting pH, agents for adjusting toxicity, etc. In an embodiment, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and polysorbate-80, preferably in the range of 0.0001% (w / v) to 1% (w / v), particularly preferably in the range of 0.001% (w / v) to 0.1% (w / v) of polysorbate-80.

[0090] In a preferred embodiment, the pharmaceutical composition comprises a single domain antibody in a dosage amount ranging from 0.01 mg / day to 1.0 mg / day, preferably in a dosage amount ranging from 0.01 mg / day to 0.1 mg / day.

[0091] In another embodiment, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized by conventional known techniques, including but not limited to sterile filtration.

[0092] In an embodiment, the pharmaceutical composition is for administration to a subject.

[0093] In an embodiment, the pharmaceutical composition is lyophilized prior to storage, or stored in solution at ambient temperature or lower, including but not limited to frozen storage.

[0094] In an embodiment, the pharmaceutical composition is reconstituted and / or diluted in an infusion and stabilizer solution prior to administration to a subject.The solution for reconstitution or infusion / stabilization may contain any of the components mentioned in the pharmaceutical composition or similar components.

[0095] Another aspect of the present invention is a kit for use in the diagnosis and / or therapy of cancer in vivo, comprising:

[0096] i. at least one single domain antibody according to the present invention; and ii. at least one pharmaceutically acceptable excipient or a solution thereof.

[0097] In an embodiment of the kit, the at least one single domain antibody is linked to at least one chelator and / or prosthetic group.

[0098] The at least one pharmaceutically acceptable excipient or a solution thereof is used to reconstitute and / or dilute the at least one single domain antibody prior to administration to a subject.

[0099] In an embodiment, said at least one single domain antibody and / or said at least one pharmaceutically acceptable excipient or solution thereof is sterile.

[0100] In an embodiment, the at least one pharmaceutically acceptable excipient or a solution thereof is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and polysorbate-80, preferably sodium acetate.

[0101] In an embodiment, the solution concentration of the at least one pharmaceutically acceptable excipient is in the range of 0.0001% (w / v) to 1% (w / v), particularly preferably in the range of 0.001% (w / v) to 0.1% (w / v).

[0102] In an embodiment, the kit further comprises an inorganic acid. Advantageously, the inorganic acid is used for preparing radioisotopes, in particular for eluting from a radioisotope generator.

[0103] In embodiments, the inorganic acid is hydrochloric acid.

[0104] In embodiments, the radioisotope is 68 Ga, and the generator is a gallium / germanium generator.

[0105] In an embodiment, the kit is used to prepare a single domain antibody linked to a radioisotope in vitro or ex vivo for use in the diagnosis and / or therapy of cancer in vivo. Advantageously, the kit can be used to prepare a diagnostic and / or therapeutic agent immediately before use.

[0106] Yet another aspect of the present invention is a single domain antibody comprising an amino acid sequence according to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20, or an amino acid sequence having at least 90% sequence similarity to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20.

[0107] In an embodiment, the single domain antibody comprises an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90% sequence identity to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20.

[0108] In an embodiment, the single-domain antibody comprises complementarity determining regions (CDRs): a CDR1 having an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 29, a CDR2 having an amino acid sequence selected from the group comprising SEQ ID No. 30 to SEQ ID No. 37, and a CDR3 having an amino acid sequence selected from the group comprising SEQ ID No. 38 to SEQ ID No. 46.

[0109] In an embodiment, the single-domain antibody comprises: a CDR1 having an amino acid sequence selected from SEQ ID No. 22 or SEQ ID No. 29, a CDR2 having an amino acid sequence selected from SEQ ID No. 30, SEQ ID No. 35 or SEQ ID No. 36, and a CDR3 having an amino acid sequence selected from SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 44 or SEQ ID No. 45.

[0110] In an embodiment, the single domain antibody comprises:

[0111] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 38,

[0112] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 39,

[0113] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 44,

[0114] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 45,

[0115] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 35, a CDR3 of SEQ ID No. 38,

[0116] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 35, a CDR3 of SEQ ID No. 39,

[0117] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44,

[0118] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 35, a CDR3 of SEQ ID No. 45,

[0119] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 38,

[0120] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 39,

[0121] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 44,

[0122] having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 45,

[0123] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 38,

[0124] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 39,

[0125] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 44,

[0126] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 45,

[0127] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 38,

[0128] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 35, a CDR3 of SEQ ID No. 39,

[0129] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44,

[0130] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 35, a CDR3 of SEQ ID No. 45,

[0131] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 38,

[0132] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 39,

[0133] having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 36, a CDR3 of SEQ ID No. 44,

[0134] The CDR1 has SEQ ID No. 29, the CDR2 has SEQ ID No. 36, and the CDR3 has SEQ ID No. 45.

[0135] In a preferred embodiment, the single-domain antibody comprises an amino acid sequence according to SEQ ID No. 18, an amino acid sequence having at least 90% sequence similarity to SEQ ID No. 18, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90% sequence identity to SEQ ID No. 18.

[0136] In an embodiment, the single domain antibody comprises a CDR1 having SEQ ID No. 29, a CDR2 having SEQ ID No. 36, and a CDR3 having SEQ ID No. 45.

[0137] In an embodiment, the size of the single domain antibody is in the range of 11 kDa to 16 kDa, preferably in the range of 13 kDa to 15 kDa, more preferably in the range of 14.0 kDa to 14.5 kDa.

[0138] In an embodiment, the sequence length of the single domain antibody is in the range of 105 amino acids to 140 amino acids, preferably in the range of 110 amino acids to 130 amino acids.

[0139] In an embodiment, the single domain antibody is in a multivalent form. In an embodiment, the multivalent form of the single domain antibody is formed by combining at least two single domain antibodies according to the present invention by chemical methods or by recombinant DNA technology. In an embodiment, the single domain antibody is in a divalent form (two combined single domain antibodies), a trivalent form (three combined single domain antibodies) or a tetravalent form (four combined single domain antibodies). The single domain antibodies included in the multivalent construct can be the same or different.

[0140] In an embodiment, the single domain antibody is linked to at least one of a detectable label, a chelator, a prosthetic group, a therapeutically active agent, and / or a pharmacologically active agent.

[0141] In an embodiment, the single domain antibodies according to the invention are used for the ex vivo or in vitro detection of CD2 in a sample.

[0142] In an embodiment, the single domain antibodies according to the present invention are used to detect immune cells, in particular T cells and NK cells. Advantageously, the distribution of immune cells (in particular T cells) in the human body can be detected to assess the activity of inflammatory processes in cancer, infection and chronic inflammatory diseases.

[0143] In an embodiment, the single domain antibodies according to the invention are used in the diagnosis of cancer. In an embodiment, the single domain antibodies according to the invention are used to diagnose cancer by directly imaging immune cells (particularly T cells or NK cells) in immunotherapy.

[0144] In embodiments, the diagnosis is performed before, during and / or after cancer immunotherapy.In embodiments, the single domain antibody linked to at least one detectable label, chelator, prosthetic group, therapeutically active agent and / or pharmacologically active agent is used as a radiotracer and / or contrast agent in non-invasive medical imaging of T cells in vivo, preferably by PET or SPECT imaging; or for synthesizing radiotracers and / or contrast agents in vitro or ex vivo.

[0145] In an embodiment, the single domain antibodies according to the invention are used to treat cancer or autoimmune diseases.

[0146] In yet another embodiment, the single domain antibody according to the invention is used for theranostic use (i.e., combined diagnosis and treatment) of immune cell-related cancers, preferably lymphomas (particularly T-cell or NK-cell lymphomas) or leukemias.

[0147] In an embodiment, the single domain antibody according to the invention is used for the preparation of a medicament for the treatment of cancer or autoimmune diseases.

[0148] Another aspect of the present invention provides a nucleic acid comprising a nucleic acid sequence encoding the single domain antibody according to the present invention.

[0149] Yet another aspect of the present invention provides a pharmaceutical composition comprising at least one single domain antibody according to the present invention.

[0150] Yet another aspect of the present invention is a method for diagnosing a subject in need thereof, comprising administering to said subject in need thereof (preferably a human suffering from cancer) a diagnostically effective amount of a single domain antibody according to the present invention.

[0151] Yet another aspect of the present invention is a method for treating a subject in need thereof, comprising administering to a subject in need thereof (preferably a human suffering from cancer or autoimmune disease) a therapeutically effective dose of a single domain antibody according to the present invention.

[0152] The terms "subject," "patient," and "individual" are used interchangeably to refer to an entity that is being diagnosed and / or treated. This can include, for example, a mammal, particularly a human or non-human primate. The mammal can also be a laboratory mammal, such as a mouse, rat, rabbit, or hamster. In some embodiments, the mammal can be an agricultural mammal (e.g., a horse, sheep, cattle, pig, or camelid) or a domestic mammal (e.g., a dog or cat).

[0153] The term "diagnostically effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result. In embodiments, a diagnostically effective amount or a therapeutically effective amount does not induce or cause undesirable side effects. A diagnostically effective amount or a therapeutically effective amount can be determined by initially administering a low dose and then gradually increasing the dose until the desired effect is achieved.

[0154] For diagnostic or therapeutic use, a sterile pharmaceutical composition according to the invention or a sterile kit according to the invention (comprising a single domain antibody according to the invention) is administered to a subject in order to diagnose or treat a disease.

[0155] In an embodiment, the pharmaceutical composition is administered parenterally, preferably intravenously.

[0156] In an embodiment of the diagnostic method, said diagnosis is performed after administration of the single domain antibody according to the present invention, preferably in the range of 10 min to 240 min after administration of the single domain antibody according to the present invention.

[0157] In an embodiment of the diagnostic method, the diagnosis is performed by non-invasive medical imaging of T cells in vivo, preferably by NIR, PET, PET / CT, PET / MR, SPECT or MR imaging, more preferably by PET, PET / CT, PET / MR or SPECT imaging.

[0158] In an embodiment of the method of treatment, the single domain antibody according to the invention is administered in at least one dose, preferably in at least two doses.

[0159] In an embodiment of the method of treatment, the single domain antibody according to the invention is administered in at least two doses, wherein the time interval between said two doses is in the range of 6 to 12 weeks.

[0160] In an embodiment of the method of treatment, the single domain antibody according to the invention is administered in combination with at least one other therapeutically active agent and / or pharmacologically active agent and / or at least one other therapy (in particular surgical resection of the tumor, radiotherapy, chemotherapy, stem cell transplantation, hyperthermia treatment and / or immunotherapy).

[0161] The present invention is not limited to the embodiments shown and described, but includes all equivalent embodiments within the meaning of the invention. Furthermore, the present invention is not limited to the specific feature combinations described, but may be defined by any other specific feature combination from all the disclosed individual features, as long as the individual features are not mutually exclusive or a specific combination of individual features is not explicitly excluded. Example

[0162] The present invention will be described in more detail below by way of an embodiment example. The embodiment is intended to illustrate the present invention but is not intended to limit the present invention. By way of example only, embodiments of the present invention will be described with reference to the accompanying drawings, in which: Figure 1 Figure 2 shows the ability of anti-CD2 single-domain antibodies (CD2-sdAbs) to bind to human CD8+ T cells. The binding specificity of CD2-sdAbs to human CD8+ T cells was determined based on flow cytometry. A non-targeted, unrelated sdAb (R3b23-sdAb) was used as a control to exclude nonspecific binding of the sdAb basic framework structure (first figure). A monoclonal antibody against CD2 was used as a positive control to verify the possible binding of the corresponding target antigen.

[0163] Figure 2Analysis showing binding of anti-CD2 single-domain antibodies to retrovirally transduced U698M cells. Flow cytometry-based determination of binding specificity to the B-cell lymphoma tumor cell line U698M, retrovirally transduced with the CD2 target antigen. (A) Exemplary binding of R3b23 to CD2-transduced U698M cells, with wild-type U698M used as a negative control. (B) Binding of CD2-sdAbs to CD2-transduced U698M cells, with wild-type U698M used as a negative control.

[0164] Figure 3 The binding affinity of anti-CD2 single domain antibodies to human CD8+ T cells is shown. Affinity binding curves of CD2-sdAb (B) at different concentrations on human CD8+ T cells were determined. R3b23-sdAb was used as a negative control for nonspecific binding (A). The binding of CD2-sdAb (B) was determined with maximum specific binding (B max ) fraction, and the binding strength of R3b23-sdAb (A) is represented by the mean fluorescence intensity (MFI) at different concentrations. The measurement was repeated three times, and the data are plotted on a semi-logarithmic scale. The dissociation constant (K) of CD2 D ) was calculated based on single-site specific binding and was 2.3 × 10 -9 [M].

[0165] Figure 4 Analysis of the thermal stability of the ability of anti-CD2 single domain antibodies to bind to human CD8+ T cells is shown by flow cytometry analysis. CD2-sdAbs were kept at 4°C, 37°C, 60°C or 90°C for 0.5h, 1h, 2h, 4h or 6h, after which their binding ability to human CD8+ T cells was analyzed by flow cytometry. The temperature of the sdAb was kept stable at 37°C, 60°C and 90°C as well as at 4°C. The measurements were repeated three times, and the flow cytometry data were depicted as MFI.

[0166] Figure 5 Figure 2 shows the analysis of cytokine secretion by ELISA of T cells (CD8+ T cells) activated after co-incubation with an anti-CD2 single domain antibody according to the present invention. IFNγ levels were determined by ELISA after 4 hours of co-culture of human CD8+ T cells, corresponding target cell lines, CD2-sdAb, and R3b23-sdAb as a control. The measurements were performed in triplicate.

[0167] Figure 6Figure 2 shows cytokine secretion analysis of IL-2, GM-CSF, and TNF-α by ELISA after co-incubation with CD2-sdAb. After 4 hours of co-incubation of human CD8+ T cells and the corresponding target cell lines with CD2-sdAb and R3b23-sdAb as a control, cytokine secretion levels of IL-2, GM-CSF, and TNF-α were determined by ELISA. The target cell lines used were ML2-B7 (A), NB4-B7 (B), and HL60-B7 (C). Measurements were performed in triplicate.

[0168] Figure 7 Figure 3 shows in vivo analysis of T cell function of T cells after injection of a single domain antibody according to the present invention. Schematic diagram of the experimental setup of the CD8+ T cell in vivo tumor rejection model. NSG mice were injected subcutaneously (sc) with ML2-B7 cells in the right flank and ML2-B15 cells in the left flank. Eight days later, TCR transgenic human CD8+ T cells were injected intravenously (iv) via the tail vein, and three days later, R3-b23-sdAb, OKT11, RPA, or CD2-sdAb were injected iv. Tumor growth was monitored from the time tumors appeared until the end of the experiment on day 12.

[0169] Figure 8 Figure 3 shows tumor growth kinetics monitoring of ML2-B15 (A) and ML2-B7 tumors (B) in NSG mice. On day 0, 8 days after subcutaneous tumor injection, mice were injected intravenously with TCR 2.5D6 transgenic CD8+ T cells and three days later with PBS, R3b23-sdAb, CD2-F(ab')2(OKT11), CD2-F(ab')2(RPA-2.10), or anti-CD2 single domain antibody (CD2-sdAb). Tumor growth kinetics were monitored daily for 12 days after T cell injection. The experiment was terminated on day 12, at which time all relevant non-control tumors had been completely rejected. Tumor size is expressed in mm 2 The mean and SD of each group of mice are shown. PBS n=4, R3b23-sdAb n=3, CD2-F(ab')2(OKT11) n=6, CD2-F(ab')2(RPA-2.10) n=4, CD2-sdAb n=5. Significance was calculated using the Mann-Whitney test (*p≤0.05, **p≤0.01, ***p≤0.001). Application of CD2-sdAb did not impair T cell cytotoxicity in vivo.

[0170] Figure 9 shows the use of 68In vivo imaging of Ga-NOTA labeled anti-CD2 single domain antibody (CD2-sdAb) in NSG mice. A) Schematic diagram of the experimental setup for in vivo imaging. Experimental design for in vivo imaging studies using intravenously injected CD8+ T cells after ML2-B7 and -B15 tumor injection. Four mice per group, two groups injected with each condition, were scanned using PET / MRI 1 hour after injection. B) Using [ 68 Ga]Ga-NOTA-CD2-sdAb tracks intravenously injected TCR transgenic CD8+ T cells in vivo in ML2-B7 tumors. 68 Ga]Ga-NOTA-CD2-sdAb (left) or [ 68 PET / MRI images of mice injected with Ga]Ga-NOTA-R3b23-sdAb (right) at 1 hour post-injection. Example mice are shown in coronal, sagittal, and axial directions (A) or coronal only (B). 7 TCR transgenic CD8+ T cells were injected at a dose of 13 ± 1 MBq of tracer per mouse. The scale bar represents the percentage of injected dose per gram (%ID / g), 0.4-1.5% ID / g. B = bladder, K = kidney. Striped arrow = ML2-B7 tumor, white arrow = ML2-B15 tumor. (Bottom) 1.5 hours after injection and after previous PET / MR image acquisition, [ 68 Ga]Ga-NOTA-CD2-sdAb and [ 68 Biodistribution data of Ga]Ga-NOTA-R3b23-sdAb in mice bearing ML2-B7 and -B15 tumors. Mean %ID / g ± SD for each group of mice are depicted. 68 Ga]Ga-NOTA-R3b23-sdAb n=4,[ 68 Ga]Ga-NOTA-CD2-sdAb n = 4. Significance was calculated using the Mann-Whitney test (*p≤0.05).

[0171] Figure 10 showed that [ 68 Quality control of Ga]Ga-NOTA-anti-CD2-sdAb, showing radiochemical purity >99%. The chromatogram shows an overlay of three chromatograms acquired 1 hour, 2 hours, and 5 hours after production.

[0172] Figure 11 shows the PET / MR axial measurements of different organs and tumors in the mouse model: A) Biodistribution data (n=6 per group), where [ 68The corresponding tumor uptake of Ga]Ga-NOTA-anti-CD2-sdAb was 1.80±0.67, 1.44±0.22, and 1.58±0.21% injected dose / gram (%ID / g) in Group 1 (3.74±0.97 MBq (1.63±0.42 μg)), Group 2 (5.88±0.35 MBq (3.67±0.22 μg)), and Group 3 (9.52±0.39 MBq (6.35±0.26 μg)), respectively. B) [ 68 Accumulation of Ga]Ga-NOTA-anti-CD2-sdAb in CD2-positive tumors (ML2-B7) for different injected doses (G1 - Group 1, G2 - Group 2, G3 - Group 3).

[0173] Figure 12 The [ 68 Biodistribution data of Ga]Ga-NOTA-anti-CD2-sdAb in different organs and tumors in mouse models (n=5 per group).

[0174] Figure 13 show[ 68 Ga]Ga-NOTA-anti-CD2-sdAb and [ 68 The results of the binding study of Ga]Ga-NOTA-control-sdAb showed that [ 68 Ga]Ga-NOTA-anti-CD2-sdAb has higher activity in Jurkat cells and human peripheral blood mononuclear cells (PBMCs).

[0175] The functionality and specificity of the single domain antibody according to SEQ ID No. 18 were investigated by examining its binding to T cells and to the target marker CD2 (see Figures 1 to 3 For this purpose, the human tumor cell line U698M (B-cell lymphoma) was transduced with a specific gene encoding CD2. Flow cytometry showed specific binding to the transduced cells compared to untransduced controls. In addition, the strength of the binding affinity of the single domain antibody was determined by binding to human CD8+ T cells and was 2.30×10 - 9 M, is within the expected range. This not only proves the functionality of the single domain antibody, but also proves its specificity for the target.

[0176] In addition, the thermal stability was investigated (see Figure 4 ), cytokine secretion (see Figure 5 and 6 ), and aspects such as damage to the cytotoxic function of T cells after co-incubation with the single domain antibody.

[0177] The results showed that co-incubation had no negative effect on the cytotoxic potential of T cells, that is, the binding of the single domain antibody to the CD2 surface marker on T cells did not impair their function of recognizing and destroying tumor cells in vitro.

[0178] Regarding the binding of anti-CD2 single-domain antibodies, no changes in IFNγ secretion were observed in all tumor cell lines examined (ML-2, NB-4, and HL-60) compared to the control group using an irrelevant binding single-domain antibody (R3b23). Therefore, it can be assumed that the binding of single-domain antibodies has no effect on the secretion of cytokines that can trigger cytokine storm syndrome in patients.

[0179] In addition, the in vivo effects of CD2-binding F(ab')2 fragments and single domain antibodies were compared (see Figure 7 and 8 The group receiving the single-domain antibody showed a similar pattern to the PBS control group. Tumors were detected and rejected. Thus, it was demonstrated that the single-domain antibody had no effect on T cell function in vitro and in vivo in a mouse model. In vitro testing using CD2-binding F(ab')2 fragments revealed a significant increase in tumor volume four days after T cell injection, indicating impaired T cell function.

[0180] With NOTA and 68 Coupling of Ga

[0181] 1 mg of preGMP NOTA-anti-CD2-sdAb was produced and analyzed for concentration (3.157 mg / ml) using a NanoDrop spectrophotometer and purity (99.6%) using UPLC-MS or SDS-PAGE. 68 Ga]Ga-NOTA-anti-CD2-sdAbs and reported a specific activity of 2.98 MBq / μg. The radiochemical yield was >98%, which was determined using radioactive thin-layer chromatography.

[0182] Purification was performed using a PD-10 column and a buffer containing NaCl (0.9%) and ascorbic acid (5 mg / ml). 68 Ga]Ga-NOTA-sdAbs to achieve >99% radiochemical yield.

[0183] Size exclusion chromatography (SEC) was performed on a Superdex 30 plus 3.2 / 300 column (GE Healthcare) to assess radiochemical purity (RCP, R t =14.1 min) and the absence of aggregates. The isocratic method was performed using PBS (pH 7.4) as eluent.

[0184] To assess the presence of product fragments formed due to radiolysis (which otherwise could not be assessed using the above methods), an alternative HPLC method was used. A reversed-phase HPLC column (Agilent PLRP-S 4.6×150 mm, 5 μm), and the following elution method was used: solvent A: H 2 O; solvent B: MeCN+0.1% formic acid (FA); flow rate: 1 ml / min (see Table 1).

[0185] Table 1 Solvent gradient used in reverse phase HPLC

[0186] Time (min) Solvent B (MeCN + 0.1% formic acid, %) 0 5 4 5 16 95 19 95 20 5 22 5

[0187] Up to 5 hours after production, [ 68 The stability of the incorporated [Ga]Ga-NOTA-anti-CD2-sdAb was evaluated. 68 Ga] gallium (R t =10.8min) was maintained at ≥99% ( Figure 10 ). Figure 10 Shown is an overlay of three chromatograms acquired 1 hour, 2 hours, and 5 hours after production.

[0188] Based on the in vitro and in vivo data on purity, binding strength and specificity, as well as the lack of functional restriction of T cells after single domain antibody binding, the potential of CD2 single domain antibodies in immunoPET imaging was subsequently investigated (see Figure 9). In this mouse experiment, four mice received [ 68 Ga]Ga-NOTA-R3b23-sdAb and used for single-domain antibody-based PET imaging, and another 4 mice were injected with an irrelevant single-domain antibody [ 68 Ga]Ga-NOTA-R3b23-sdAb was used as a control. For this purpose, ML-2 tumor cells were transduced with genes encoding HLA-B07:02 (B7) or HLA-B15:01 (B15) and injected into the left (B15) and right (B7) shoulders of mice. Tumors carrying HLA-B15 were used as negative controls. After successful growth of tumor cells 8 days after injection, human CD8+ T cells were injected intravenously through the tail vein. 5 days later, radiolabeled nanobodies were injected intravenously.

[0189] To enable imaging by PET imaging, the single domain antibodies were previously conjugated to the p-SCN-Bn-NOTA chelator and stained with the radioisotope gallium-68 ( 68The short half-life of 67 minutes, which is highly advantageous for potential clinical applications, suggests that the CD2-targeting single-domain antibody can bind to T cells in vivo and be imaged using PET.

[0190] In addition, the radiolabeling of the single-domain antibody was developed and optimized, which is crucial for PET imaging. The tracer was labeled with the radioisotope gallium-68, and its imaging properties were then evaluated. PET imaging results showed that one hour after tracer injection, clear accumulation occurred in the treated experimental tumor ML2-B7 that had been infiltrated by T cells. This emphasizes the rapid and specific accumulation of the tracer. In addition, except for the expected accumulation in the kidneys (due to excretion), no visible tracer accumulation was observed in other organs or control tumors (ML2-B15), thus indicating that the radiolabeled single-domain antibody can bind to T cells in a mouse model without loss of function and can be specifically and clearly visualized by PET imaging.

[0191] Different doses of anti-CD2-sdAb were tested in the same experimental setting as above. Mice were injected with different doses of [ 68 The anti-CD2-sdAb of Ga]Ga-NOTA- was injected into the rats and was scanned by PET / MRI for 15 minutes with Mediso small animal scanner 1 hour after injection. The relevant organs and tumors were collected subsequently for biodistribution study. The samples were then measured with a γ counter (PerkinElmer), and the biodistribution data were presented in Figure 11 with %ID / g form. The injected dose (ID) was: Group 1: 3.74 ± 0.97 MBq (1.63 ± 0.42 μg), Group 2: 5.88 ± 0.35 MBq (3.67 ± 0.22 μg), Group 3: 9.52 ± 0.39 MBq (6.35 ± 0.26 μg). Figure 11A The results show that different injection doses [ 68 Biodistribution data of [Ga]Ga-NOTA-anti-CD2-sdAb (n=6 per group) and corresponding tumor uptake. Figure 11B Tumor comparisons are shown. All groups (G1-Group 1, G2-Group 2, G3-Group 3) showed strong tracer accumulation in positive tumors (ML2-B7, whose HLA type allows intravenously injected CD2-positive human CD8+ T cells to bind and attack B7 tumor cells) compared to negative cell lines (ML2-M15, which have different HLA types and therefore human T cells cannot attack and infiltrate tumor cells). The Mann-Whitney test showed a P value of 0.0022 for all groups. All groups showed similar kidney / tumor ratios, indicating that there were no major quality-related differences in biodistribution within the scope of this study.

[0192] In the same animal model described above, PET / MR imaging was performed [ 68 Pharmacokinetics of Ga]Ga-NOTA-anti-CD2-sdAb. Mice were injected with 3.88±0.46μg of [ 68 Ga]Ga-NOTA-anti-CD2-sdAb, and then collected relevant organs and tumors at selected time points for biodistribution examination (n=5 for each time point). The samples were then measured using a γ counter (PerkinElmer), and the biodistribution data were displayed in the form of % ID / g. Figure 12 In addition, in the injection [ 68 One hour after the administration of [Ga]Ga-NOTA-anti-CD2-sdAb, dynamic PET / MR scanning was performed using a Mediso small animal scanner (n=3).

[0193] Compare[ 68 Ga]Ga-NOTA-anti-CD2-sdAb and [ 68 Binding properties of Ga]Ga-NOTA-control-sdAb to different types of human blood mononuclear cells (PBMC). The number of cells used for each type was 1 Mio ML2 cells (CD2 negative cell line), 0.3 Mio Jurkat cells (CD2 positive cell line), 1 Mio human PBMC, 1 Mio non-human primate (NHP) PBMC, 0.5 Mio rabbit PBMC, 0.4 Mio pig PBMC and 1 Mio dog PBMC. As a control, a single domain antibody that does not target the CD2 receptor was used. After an incubation time of 30 minutes, the precipitate was separated from the supernatant, washed with PBS, and the sample was measured using a gamma counter (PerkinElmer). The results are displayed in counts per minute (cmp) Figure 13 In the middle, it is shown that 68 Compared with [Ga]Ga-NOTA-control-sdAb, 68 Ga]Ga-NOTA-anti-CD2-sdAb has significantly higher binding capacity in CD2+ Jurkat cells and human PBMCs.

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Claims

1. A single domain antibody that binds to human CD2 for use in the diagnosis and / or therapy of cancer in vivo.

2. The single-domain antibody for use in the diagnosis and / or treatment of cancer according to claim 1, comprising: a. One of the following CDR combinations: having a CDR1 of SEQ ID No. 21, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 38, having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 38, having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 31 and a CDR3 of SEQ ID No. 39, having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 32, and a CDR3 of SEQ ID No. 39, having a CDR1 of SEQ ID No. 23, a CDR2 of SEQ ID No. 31 and a CDR3 of SEQ ID No. 40, having a CDR1 of SEQ ID No. 24, a CDR2 of SEQ ID No. 33, and a CDR3 of SEQ ID No. 41, having a CDR1 of SEQ ID No. 22, a CDR2 of SEQ ID No. 34, and a CDR3 of SEQ ID No. 42, having a CDR1 of SEQ ID No. 25, a CDR2 of SEQ ID No. 30, and a CDR3 of SEQ ID No. 43, having a CDR1 of SEQ ID No. 26, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44, having a CDR1 of SEQ ID No. 27, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44, having a CDR1 of SEQ ID No. 28, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44, having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 35, and a CDR3 of SEQ ID No. 44, having a CDR1 of SEQ ID No. 29, a CDR2 of SEQ ID No. 36, and a CDR3 of SEQ ID No. 45, or a CDR1 having SEQ ID No. 29, a CDR2 having SEQ ID No. 37, and a CDR3 having SEQ ID No. 46; and / or b. an amino acid sequence selected from one of the sequences of SEQ ID No. 2 to SEQ ID No. 20, or an amino acid sequence having at least 90% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No.

20. 3 . The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 1 , wherein the size of the single domain antibody is in the range of 11 kDa to 16 kDa.

4. The single domain antibody for use in the diagnosis and / or therapy of cancer according to any one of claims 1 to 3, wherein the single domain antibody is linked to at least one of a detectable label, a chelating agent, a prosthetic group, a therapeutically active agent and / or a pharmacologically active agent.

5. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 4, wherein the detectable label, therapeutically active agent and pharmacologically active agent is a radioactive isotope selected from the group comprising: 68 Ga, 18 F. 89 Zr, 99m Tc, 61 Cu, 64 Cu, 67 Cu, 43 Sc, 44 Sc, 47 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 123 I. 124 I. 177 Lu, 225 Ac, 213 Bi, 211 At 212 Pb and 223 Ra.

6. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 4, wherein the detectable label is selected from the group consisting of a tag, a fluorophore or a magnetic particle.

7. A single domain antibody for use in the diagnosis and / or therapy of cancer according to any one of claims 4 to 6, wherein the single domain antibody linked to at least one of a detectable label, a chelator, a prosthetic group, a therapeutically active agent and / or a pharmacologically active agent is used as a radiotracer and / or contrast agent in in vivo non-invasive medical imaging, preferably by PET or SPECT imaging.

8. A pharmaceutical composition for use in the diagnosis and / or therapy of cancer in vivo, comprising at least one single domain antibody as defined in any one of claims 1 to 6.

9. A kit for use in the diagnosis and / or treatment of cancer in vivo, comprising: i. at least one single domain antibody as defined in any one of claims 1 to 6; and ii. at least one pharmaceutically acceptable excipient or a solution thereof.

10. The kit according to claim 9, wherein the at least one single domain antibody is linked to at least one chelator and / or prosthetic group.

11. A single domain antibody that binds to human CD2, comprising an amino acid sequence according to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 20, or an amino acid sequence having at least 90% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No.

20. 12 . The single domain antibody according to claim 11 , wherein the size of the single domain antibody is in the range of 11 kDa to 16 kDa.

13. The single domain antibody according to claim 11 or 12, wherein the single domain antibody is linked to at least one of a detectable label, a chelating agent, a prosthetic group, a therapeutically active agent and / or a pharmacologically active agent. 14 . A nucleic acid comprising a nucleic acid sequence encoding the single domain antibody according to claim 11 .

15. A pharmaceutical composition comprising at least one single domain antibody according to any one of claims 11 to 13.

16. Use of a single domain antibody as defined in any one of claims 1 to 6, a pharmaceutical composition as defined in claim 8 and / or a kit as defined in claim 9 or 10 in the diagnosis and / or therapy of cancer in vitro or ex vivo or in the synthesis of radiotracers and / or contrast agents.

Images