Anti-CD7 single domain antibody, a pharmaceutical composition and a kit for use in the diagnosis and / or therapy of cancer

Abstract

The present disclosure relates a single domain antibody which binds to human CD7 for use in the diagnosis and / or therapy of cancer, optionally comprising at least one of a detectable label, a chelator, a prosthetic group, a therapeutically and / or pharmacologically active agent, a pharmaceutical composition comprising the single domain antibody and a kit.

Description

[0001] Anti-CD7 single domain antibody, a pharmaceutical composition and a kit for use in the diagnosis and / or therapy of cancer

[0002] The present disclosure relates to a single domain antibody (sdAb) which binds to human CD7 for use in the diagnosis and / or therapy of cancer, preferably comprising at least one of a detectable label, a chelator, a prosthetic group, a therapeutically and / or pharmacologically active agent. In another aspect, this disclosure relates to a pharmaceutical composition comprising the single domain antibody, and a kit.

[0003] Immunotherapeutic approaches have proven 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 already been authorised for the therapy of many tumor entities. 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, also showed first promising clinical results, which is reflected in the European-wide approval of two CD19-specific CAR T cells by the EMA (European Medicines Agency) (Druker et al. 2006).

[0004] With increasing integration of these new therapeutic options into trials and clinical practice, adequate monitoring of individual therapy progress is becoming increasingly important.

[0005] Established imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), and functional imaging modalities such as positron emission tomography (PET) have high spatial and temporal resolution but are limited in their application for monitoring the progress of immunotherapies (Chalian et al. 2016, McCarthy et al. 2020). This results in poor differentiation between progression and pseudoprogression of tumors, i.e., increase in size of lesions due to tumor growth or immigration of immune cells (Dromain et al. 2020).

[0006] The 2009 revision of the RECIST (Response Evaluation Criteria in Solid Tumors) guidelines specifically for immunotherapies (irRECIST) represents a first attempt to improve the assessment of therapy progression, but there is still a lack of an appropriate, sensitive, clinically applicable imaging modality. It would be extremely important to be able to evaluate the response to therapy at an early stage in order to avoid premature discontinuation and unnecessary continuation of therapy, as well as to promptly detect T-cell infiltration in healthy tissue as an expression of an autoimmune event. Furthermore, adequate monitoring would significantly deepen the current understanding of pharmacokinetics and dynamics of immunotherapies.

[0007] A promising imaging approach is the direct visualization of T cells, which play the key role in immunotherapies. Weist et al. describes an approach, wherein CAR T cells and TCR- transduced T cells are radiolabeled ex vivo or equipped with a reporter gene prior to adoptive cell transfer (Weist et al. 2018). However, this has the disadvantage that in the case of radioactive labeling, these can only be imaged over a short period of time, while, for example, the effects of immune checkpoint inhibitors on endogenous T cells cannot be assessed. Furthermore disadvantageously, functional impairment of immune cells was often shown (Auletta et al. 2018).

[0008] Alternatively, immuno-PET is currently examined for the feasibility of detection of T cells. Here, antibodies or their derivatives directed against specific surface antigens of T cells are radioactively labeled, allowing T cells to be imaged at any time point in vivo. The selection of a suitable target antigen, which is highly specific for T cells, as well as an antibody derivative, which does not affect the functionality of the T cells when the antigen is bound, is decisive.

[0009] WO 2021 / 113450 A2 describes a non-invasive imaging method for diagnosis, prediction, prognosis, and treatment of a disease, in particular selected from a solid tumor, a non-solid tumor, an autoimmune disease, an infectious disease (including without limitation, viral, bacterial, or fungal infections), comprising administering to a subject a first antigen-binding construct comprising a first radionuclide tracer, wherein the antigen-binding construct selectively binds a first target selected from CD3, CD4, and CD8, and estimating a distribution and / or abundance of cells expressing the first target in one or more tissues of the subject using positron emission tomography (PET) or single photon emission computed tomography (SPECT) to measure a level of the first radionuclide tracer in the subject. Preferably, the antigen-binding construct is a Fab’, F(ab’)2, Fv, rlgG (reduced IgG), a scFv fragment, a minibody, a diabody, a cys-diabody, or a single domain antibody.

[0010] Mayer et al. describe CD7 as target for monitoring T cells in the context of immunotherapies (Mayer et al. 2018). T cells incubated with anti-CD2 and anti-CD7 F(ab')2 showed no major modulation of functionality in vitro, and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. T-cell tracking by anti-CD7 F(ab')2 had no long-term impact on T-cell functionality in vivo and anti-CD7 F(ab')2 imaging did not alter tumor rejection. Additionally, Mayer et al. have shown that CD7 is slightly upregulated on specifically activated T cells and present on both CD4+ and CD8+ T cells. US 10,301 ,389 B2 describes antigen-binding constructs, in particular cys-diabodies or minibodies, to CD3 and the use for modulating the biologic activity associated with CD3 expression on immune cells, for targeting therapeutic agents to cells that express the CD3 protein, and for detecting the presence or absence of CD3.

[0011] US 2021 / 0371527 A1 describes antigen binding constructs, in particular cys-diabodies or minibodies, binding to CD4 and the use for detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression, and monitoring therapy.

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

[0013] US 2017 / 0226204 A1 discloses nanometer antibodies for human CD7 molecule and an encoding DNA sequence thereof. The nanometer antibody is expressed in Escherichia coli, used for preparing an agent for the detection of CD7 molecule and targeted therapy, and for flow cytometry and cellular immunofluorescence assays.

[0014] Furthermore, Kasbauer et al. and Tang et al. describe anti-CD7 single domain antibodies for flow cytometry and cellular immunofluorescence assays (Kasbauer et al. 2019, Tang et al. 2016)

[0015] It would be useful to provide an alternative marker for the diagnosis and / or therapy of cancer related to T cell binding. For example, it would be useful to have improved methods and molecules for cancer diagnosis and therapy. Improvement is needed as regards influencing T cell function, exposure of patients to radiation, stability and pharmacokinetics of the molecule.

[0016] Summary of the disclosure

[0017] According to this disclosure, the object is solved by a single domain antibody which binds to human CD7, including single domain antibodies for use in the diagnosis and / or therapy of cancer, nucleic acids, pharmaceutical compositions, and kits as described herein.

[0018] It was found that the single domain antibodies, their uses, nucleic acids, pharmaceutical compositions, kits and methods provide for improved options of cancer diagnosis and therapy, particularly in the sense that therapy, therapy monitoring and diagnosis of disease progression can be performed very effectively. The single domain antibodies of this disclosure have superior pharmacokinetic properties in that they distribute within a patient’s system very quickly allowing for almost immediate imaging of immune cells in and around a tumor, or anti-tumor activity. Quick distribution and quick elimination both contribute to short hospitalization because patients can be released shortly after administration, when residual radioactivity is below a predetermined threshold. Moreover, because of their strong affinities to human CD7 the single domain antibodies quickly and effectively accumulate at sites of increased T cell activity, thereby limiting the dosage necessary to perform the methods disclosed herein. Furthermore, because of the strong affinities and quick distribution of the single domain antibodies, detectable labels of short half-lives can be employed. Particularly with radioisotopes, short-lived radioactive substances have the additional advantage that radioactivity declines quickly so that patients can be released from the hospital shortly after administration of the single domain antibodies. Thus, in an embodiment, the methods disclosed herein are performed without patient hospitalization. Importantly, the single domain antibodies of this disclosure, if not intentionally equipped with a radioisotope, a chemotherapeutic agent or toxin, do not relevantly affect immune cell response, neither positively (e.g. inducing T cell induced immune response), nor negatively (e.g. impairing T cell induced immune response against tumors). Because of the unique properties of the single domain antibodies disclosed herein, tumor infiltration can be determined using the methods of this disclosure, providing superior means of monitoring treatment efficacy in immunotherapy patients.

[0019] A first aspect of this disclosure provides a single domain antibody which binds to human CD7 for use in the diagnosis and / or therapy of cancer in vivo, the single domain antibody comprising complementarity determining regions (CDR):

[0020] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14 to SEQ ID No. 17,

[0021] CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 to SEQ ID No. 20 and

[0022] CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 25.

[0023] In a second aspect, this disclosure relates to a pharmaceutical composition comprising at least one of the single domain antibodies according to the first aspect for use in the diagnosis and / or therapy of cancer in vivo.

[0024] In a third aspect, this disclosure relates to a kit for use in the diagnosis and / or therapy of cancer in vivo comprising i. at least one single domain antibody according to the first aspect and ii. at least one pharmaceutically acceptable excipient or a solution thereof.

[0025] In a fourth aspect, this disclosure relates to a single domain antibody which binds to human CD7 comprising an amino acid sequence according to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13 or at least 90% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13.

[0026] In a fifth aspect, this disclosure relates to a nucleic acid comprising a nucleic acid sequence encoding for the single domain antibody according to the fourth aspect.

[0027] In a sixth aspect, this disclosure relates to a pharmaceutical composition comprising at least one single domain antibody according to the fourth aspect.

[0028] In a seventh aspect, this disclosure relates to the use of the single domain antibody according to the first aspect, the pharmaceutical composition according to the second aspect, and / or the kit according to the third aspect in the diagnosis and / or therapy of cancer in vitro or ex vivo or for the synthesis of radioactive tracers and / or contrast agents.

[0029] In an eighth aspect, this disclosure relates to a single domain antibody for use in therapy of cancer, the single domain antibody binding to human CD7 with an affinity of less than 15 nM, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with a detectable label, which detectable label is a radioactive isotope with a half-life at most 750 minutes, at most 500 minutes, or at most 250 minutes, wherein the therapy includes administering immunotherapy to a patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0030] In a ninth aspect, this disclosure relates to a single domain antibody, that binds to human CD7 for use in cancer therapy, wherein the single domain antibody is associated with a detectable label, the detectable label being a radioactive isotope, wherein the cancer therapy comprises administering cancer therapy, such as immunotherapy, to a patient in need thereof, administering an effective amount of the single domain antibody to the patient, monitoring a presence and / or amount of immune cells expressing human CD7 at the tumor site, such as in the vicinity of the tumor and / or in the tumor, optionally, deciding whether the cancer therapy is effective based on the presence and / or amount of the immune cells at the tumor site, further wherein the single domain antibody binds human CD7 with an affinity, the single domain antibody having a molecular weight and the detectable label having a radioactive half-life, wherein a figure of merit defined as - - - - - —r- - - , affinity [nmol / Ljx molecular weight [g / mol\xradioactive half-life [s] is at least 0.05 or at least 0.50 [L / g*s],

[0031] In a tenth aspect, this disclosure relates to a single domain antibody that binds to human CD7 for use in cancer therapy, wherein the single domain antibody is associated with a radioactive isotope, a chemotherapeutic agent and / or a toxin.

[0032] The single domain antibodies of this disclosure can be used in a variety of diagnostic, therapeutic and theranostic methods that can be summarized in the categories of imaging, treatment and combinations of both. Imaging can be performed using radioisotopes or other detectable markers, wherein if radioisotopes are used those with short half-lives provide relevant benefits. Treatment with single domain antibodies of this disclosure may include delivery of a toxin, a chemotherapeutic agent and / or radioisotope to a target cell, such as a tumor cell. In that case, the target cell will typically express CD7 on its surface so that the single domain antibody may deliver its radioactive payload to the target and, in particular, be internalized into the target cell upon binding.

[0033] An eleventh aspect of this disclosure provides a single domain antibody which binds to human CD7 for use in the diagnosis and / or therapy of cancer in vivo, the single domain antibody comprising complementarity determining regions (CDR):

[0034] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14 to SEQ ID No. 17,

[0035] CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 to SEQ ID No. 20 and CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 25; wherein the single domain antibody comprises a chelator.

[0036] The single domain antibody according to the eleventh aspect may find utility as an intermediate product, i.e. this single domain antibody may be free of a radioactive isotope. The chelator may be suitable for chelating a radioactive isotope so that the single domain antibody according to the eleventh aspect may be used for preparing a diagnostic and / or therapeutic final antibody, i.e. carrying a radioactive isotope, e.g. at the hospital or point of care. This allows for the use of particularly short-lived isotopes.

[0037] Brief of the fi

[0038] Fig. 1 shows the analysis of the capability of the anti-CD7 single-domain antibody (CD7-single domain antibody) of this disclosure to bind human CD8+ T cells.

[0039] Fig. 2 shows the analysis of binding of an anti-CD7 single domain antibody of this disclosure to retrovirally transduced LI698M cells.

[0040] Fig. 3 shows binding affinity of an anti-CD7 single domain antibody of this disclosure to human CD8+ T cells.

[0041] Fig. 4 shows the analysis of the thermostability of the ability of the anti-CD7 single domain antibody of this disclosure to bind to human CD8+ T cells via flow cytometry analysis.

[0042] Fig. 5 shows the analysis of the cytokine secretion of Interferon-y of activated T cells (CD8+ T cells) after co-incubation with the anti-CD7 single domain antibody according to this disclosure by ELISA.

[0043] Fig. 6 shows cytokine secretion analysis of IL2, GM-CSF and TNF-a by ELISA after coincubation with an CD7-single domain antibody of this disclosure.

[0044] Fig. 7 shows an in vivo analysis of T cell functionality of T cells after injection of a single domain antibody according to this disclosure.

[0045] Fig. 8 shows monitoring of tumor growth kinetics of ML2-B15 (A) and ML2-B7 tumors (B) in NSG mice. Fig. 9 shows in vivo imaging using [68Ga]Ga -NOTA-labeled anti-CD7 single domain antibody (CD7-single domain antibody) of this disclosure in NSG mice.

[0046] Detailed description

[0047] As used herein, the term “single domain antibody” (abbreviation: “sdAb”) refers to an antibody fragment consisting of a single monomeric variable antibody domain that selectively binds to a specific antigen, in particular CD7. Advantageously, the single domain antibodies according to the invention have a small size and good binding properties, wherein binding includes specific binding. The single domain antibody can circulate rapidly through the bloodstream, penetrate the target tissue and bind to immune cells, but is also rapidly excreted and not perceived as a harmful substance in a patient, and in particular does not affect any physiological parameters of a patient to any relevant extent.

[0048] As used herein, the term “CD7” (cluster of differentiation 7) refers to a cell adhesion molecule found on the surface of T cells, natural killer (NK) cells and thymocytes, preferably human CD7 according to SEQ ID No. 1.

[0049] As used herein, the term “cancer” refers to a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body.

[0050] As used herein, the term “lymphoma” refers to "malignant lymphomas", which are cancers of the lymphatic system, including the lymph nodes, tonsils and spleen, and lymphatic tissue in the stomach, intestines or skin. In lymphoma, white blood cells, the so-called lymphocytes, which belong to the lymphatic system, grow uncontrollably. As used herein, the term “at the tumor site” may include locations in the tumor or in the vicinity of the tumor. “In the vicinity of the tumor” means within a distance of at most 0.5 cm from any cancerous tissue.

[0051] As used herein, the term “leukaemia” refers to a group of blood cancers with a high number of abnormal white blood cells.

[0052] As used herein, the term “similarity”, or “sequence similarity” refers to the sequence homology, wherein conservative substitutions of amino acid residues having similar physicochemical properties over the length of the amino acid sequence are comprised. The % sequence similarity is determined with any reasonable similarity-scoring matrix known by the person skilled in the art, such as with a similarity-scoring matrix selected from BLOSUM50, BLOSUM62, PMBEC or VTML10 to VTML80. As used herein, the term “% sequence identity” refers to the number of identical amino acid residues in relation to the length of the amino acid sequence.

[0053] As used herein, the “framework regions” form part of the single domain antibodies of this disclosure. A single domain antibody is composed of four framework regions (FRs) and three complementarity-determining regions (CDRs). The framework regions provide structural support and stability to the nanobody, while the CDRs are responsible for antigen binding. The positions of the CDRs and FRs within the single domain antibody’s sequence may be determined using the IMGT method (https: / / www.imgt.org / IMGTindex / FR.php, https: / / www.imgt.org / IMGTindex / CDR.php, February 8, 2024).

[0054] In this disclosure, affinity (dissociation constant KD) of a single domain antibody may be determined using surface plasmon resonance (at 25°C, e.g. Biacore), or mean fluorescence intensity (MFI) methods, such as the method described in the context of Figure 3 below. As used herein, an affinity is indicated by reference to the dissociation constant, meaning that a lower value represents higher affinity. Affinity to human CD7 may be determined on human CD8+ T cells.

[0055] The terms “subject,” “patient,” and “individual” interchangeably refer to an entity that is being diagnosed and / or treated. This can include, for example, a mammal, in particular a human or a non-human primate mammal. The mammal can also be a laboratory mammal, e.g., mouse, rat, rabbit, hamster. In some embodiments, the mammal can be an agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g., canine, feline).

[0056] The term “diagnostically effective dose” or “therapeutically effective dose” refers to an amount sufficient to achieve the desired result. In embodiments, a diagnostically effective dose or therapeutically effective dose do not induce or cause undesirable side effects. A diagnostically effective dose or therapeutically effective dose can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.

[0057] In embodiments, the single domain antibody which binds to human CD7 (anti-CD7-single domain antibodies) according to this disclosure is used for the diagnosis of cancer by the imaging (such as direct imaging) of immune cells, in particular T cells, NK cells, thymocytes or dendritic cells, preferably T cells, in immunotherapies. Particularly, the radioisotope chosen for this application may be selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb,123l,124l,213Bi and combinations thereof in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof. The terms “radioisotope” and “radioactive isotope” are used interchangeably herein.

[0058] The single domain antibody that binds to human CD7 may be used in cancer therapy, wherein the single domain antibody is associated with a radioactive isotope, a chemotherapeutic agent and / or a toxin. Particularly, the radioisotope chosen for this application may be selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0059] In an embodiment of this disclosure the single domain antibody used in cancer diagnosis and / or therapy has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 of less than 12 nM or less than 10 nM.

[0060] Advantageously, response patterns of cancer immunotherapies can be evaluated using the single domain antibody which binds to human CD7. Advantageously, CD7 occurs on each subgroup of the immune cells and thus each kind of immunotherapy can be evaluated. Further advantageously, CD7 occurs on activated immune cells at a slightly higher density and thus, the tumor treating immune cells can be imaged more clearly.

[0061] Further advantageously, the binding of the single domain antibody to the target molecule CD7 does not affect the function of the immune cells, in particular T cells and / or NK cells. In an embodiment, the feature that the single domain antibody does not affect the function of the immune cells may include one or more or all of the following: the single domain antibody does not alter IFNy secretion of activated T cells (CD8+ T cells), e.g. compared to control after co-incubation with the single domain antibody with IFNy levels optionally determined by ELISA after 4 h co-culture of human CD8+ T cells, and the respective target cell lines, wherein R3b23-single domain antibody served as control (see Fig. 5); the single domain antibody does not alter IL2, GM-CSF and / or TNF-a secretion of activated T cells (CD8+ T cells), e.g. compared to control after co-incubation with the single domain antibody with IL2, GM-CSF and / or TNF-a levels optionally determined by ELISA after 4 h co-culture of human CD8+ T cells, and the respective target cell lines, wherein R3b23-single domain antibody served as control (see Fig. 6), optionally using ML2-B7, NB4-B7 or HL60-B7 as target cell lines; the single domain antibody does not have an influence on tumor growth, e.g. as compared to control in a tumor rejection model for CD8+ T cells, wherein NSG mice are subcutaneously (s.c.) injected with ML2-B7 cells in the right flank and ML2-B15 cells in the left flank, and after eight days, TCR-transgenic human CD8+ T cells are injected intravenously (i.v.) through the tail vein, followed three days later by i.v. injection of R3- b23-single domain antibody (control), OKT11 , RPA, or CD7-single domain antibody. Optionally, influence on tumor growth is judged at day 12 (Fig. 7); the single domain antibody does not have an influence on tumor growth of ML2-B15 and / or ML2-B7 tumors, e.g. as compared to control in NSG mice, wherein on day 0, eight days after subcutaneous tumor injection, mice were intravenously injected with TOR 2.5D6-transgenic CD8+ T cells and three days later with either PBS, R3b23-single domain antibody (control), CD2-F(ab')2 (OKT11), CD2-F(ab')2 (RPA-2.10) or CD7-single domain antibody; wherein kinetics of tumor growth were monitored daily for twelve days post T-cell injection. Optionally, influence on tumor growth is judged at day 12 (Fig. 8).

[0062] In further embodiments, the single domain antibody which binds to human CD7 according to this disclosure is used for theranostics, i.e. the combined diagnosis and therapy, of immune cell related cancer, preferably lymphomas, in particular ? cell or NK lymphomas; or leukemia in vivo.

[0063] In an embodiment, this disclosure relates to a method of treating cancer comprising administering to a subject having cancer an amount of the single domain antibody which binds to human CD7 according to this disclosure, wherein the single domain antibody is associated with a radioisotope, a chemotherapeutic agent or a toxin, wherein the cancer cells express human CD7. The cancer may be immune cell derived, such as T cell derived cancer. For example, the cancer may be selected from lymphomas, in particular T cell or NK lymphomas; or leukemia. Optionally, the cancer may be metastasized, in particular with solid metastases. Particularly, the radioisotope chosen for this application may be selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0064] In an embodiment, this disclosure relates to a method of theranostics, i.e. combined diagnosis and therapy of cancer, comprising administering to a subject an amount of a single domain antibody which binds to human CD7, particularly a single domain antibody according to this disclosure, wherein the single domain antibody is associated with a first radioisotope, performing imaging by detecting the presence of the radioisotope in the subject, administering to the subject an amount of a single domain antibody which binds to human CD7, particularly a single domain antibody according to this disclosure, wherein the single domain antibody is associated with a second radioisotope, wherein the cancer cells express human CD7. The cancer may be immune cell derived, such as T cell derived cancer. For example, the cancer may be selected from lymphomas, in particular T cell or NK lymphomas; or leukemia in vivo. Optionally, the cancer may be metastasized, in particular with solid metastases.

[0065] The subject may be known to have cancer when the single domain antibody with the first radioisotope is administered, or the step may be performed to learn whether or not the subject has cancer. The single domain antibody with the second radioisotope may in particular be administered to a subject who has been diagnosed with cancer.

[0066] Particularly, the second radioisotope chosen for this application may be selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof, and / or the first radioisotope may be selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb,123l,124l,213Bi and combinations thereof in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof. In an embodiment, the first radioisotope and the second radioisotope are different isotopes of the same element. In particular, the element may be Cu, Tb or Sc. In an embodiment, the first radioisotope is61Cu and / or64Cu, and the second radioisotope is67Cu. In another embodiment, the first radioisotope is43Sc and / or44Sc, and the second radioisotope is47Sc. In yet a further embodiment, the first radioisotope is149Tb,152Tb and / or155Tb, and the second radioisotope is149Tb and / or161Tb.

[0067] Optionally, diagnosis and / or theranostics of cancer includes determining the existence, localization and / or size of a tumor or metastasis of a CD7 positive cancer.

[0068] In an embodiment of this disclosure, the single domain antibody binding to human CD7 is internalized into the CD7 positive cell upon binding of the single domain antibody.

[0069] In embodiments, the diagnosis is carried out before, during and / or after immunotherapy of cancer. Diagnosis and / or therapy may be performed in vivo. In embodiments, the single domain antibody is used as radioactive tracer 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 radioactive tracers and / or contrast agents ex vivo. Advantageously, the use of a single domain antibody which binds to human CD7 for diagnosis of cancer during therapy allows precise observation of whether immune cells are present on and in the tumor, which in turn indicates whether the tumor is being fought and the immunotherapy is effective. In other words, as used herein “diagnosis” includes monitoring immune cells during immunotherapy. Additionally, if there are no or insufficient immune cells on the tumor, this can also be determined promptly and the patients can be switched to a different therapy. Thus, patients gain enormously valuable time and at the same time an extremely costly immunotherapy without benefit does not have to be administered over several weeks. The time gained for patients is essential, because while CT or MRI can only detect tumor growth or tumor regression after up to two months, infiltration of the tumor with T cells can be made visible after only 1 to 2 weeks. Accordingly, important therapy decisions can be made much earlier.

[0070] Furthermore, different tumor types are no limitation, since the single domain antibody which binds to human CD7 does not visualize the tumor itself, but the immune cells used for immunotherapy. Purposefully, the activated T cells, i.e. those that actively fight the tumor, are bound by the single domain antibody. The increased density of the target molecules (CD7) also allows more tracer to bind to the T cells, which in turn allows just the tumor-fighting cells to be visualized more clearly.

[0071] Thus, in an embodiment, diagnosis and / or therapy of cancer includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0072] Evaluating a response pattern may include: concluding based on a presence or concentration or accumulation of immune cells at a tumor site, e.g. in the vicinity of and / or in the tumor, whether immunotherapy is effective.

[0073] Imaging of immune cells may be performed from 10 minutes to 240 minutes, particularly less than 180 minutes after administration of the single domain antibody. Imaging may include detection of a detectable label attached to the single domain antibody of this disclosure while binding to the immune cell. In some embodiments, the immune cell is a CD7 positive cell, such as a T cell. As used throughout this disclosure, “imaging” may include “direct imaging”. Immunotherapy is a type of treatment that uses the body’s own immune system to fight diseases, including cancer. It can work by either stimulating the immune system to work harder or smarter to attack cancer cells, or by providing the immune system with components, such as man-made immune system proteins. There are several types of immunotherapy including: immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies binding to targets on cancer cells, cancer vaccines, and / or immune system modulators.

[0074] Thus, administering immunotherapy to a patient may include administration of T-cell transfer therapy, monoclonal antibodies binding to targets on cancer cells, cancer vaccines, and / or immune system modulators, or of one or more checkpoint inhibitors, such as checkpoint inhibitory antibodies. Exemplary checkpoint inhibitory antibodies include anti-PD-1, anti-PD-L1 anti-CTLA-4, anti-TIGIT, anti-PVRIG, anti-TIM3, anti-B7-H3, anti-CD47-SIRPa, anti-NKG2A, anti-VISTA, anti-PVRL2, anti-A2aR, anti-BTLA, anti-CD96, anti-SIGLEC-15 and anti-LAG3.

[0075] Diagnosis and / or therapy of this disclosure may include administration of the single domain antibody, e.g. within a pharmaceutical composition, in a dosage quantity in the range of 0.005 mg / day to 25.0 mg / day, such as dosage quantities in the range of 0.01 mg / day to 15.0 mg / day, or from 0.02 to 10.0 mg / day. In some embodiments, the dosage applied in imaging, e.g. in a method of diagnosis, can be very low, such as from 0.005 to 1.0 mg / day, or from 0.01 to 0.5 mg / day, or from 0.02 to 0.10 mg / day. In therapy, the dosage may be higher, such as from 1.0 to 25 mg / day, or from 1.5 to 15 mg / day, or from 2.0 to 10.0 mg / day. In an embodiment, the daily amount of single domain antibody is administered in a single dose.

[0076] In an embodiment, this disclosure relates to a single domain antibody that binds to human CD7 for use in cancer therapy, wherein the single domain antibody is associated with a detectable label, the detectable label being a radioactive isotope, wherein the cancer therapy comprises administering cancer therapy, such as immunotherapy, to a patient in need thereof, administering an effective amount of the single domain antibody to the patient, monitoring a presence and / or amount of immune cells expressing human CD7 at the tumor site, e.g. in the vicinity of the tumor and / or in the tumor, optionally, deciding whether the cancer therapy is effective based on the presence and / or amount of the immune cells at the tumor site. In embodiments, 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. 13. It was found that the single domain antibody of this disclosure has excellent thermal stability and affinity. Thermal stability is a relevant property because some radioisotopes require increased reaction temperatures for their attachment to the antibody. Thus, excellent thermal stability allows for a broad range of radioisotope options.

[0077] In embodiments, the single domain antibody comprises an amino acid sequence with at least 90% sequence similarity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13, such as an amino acid sequence with at least 95% sequence similarity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13, particularly an amino acid sequence with at least 99% sequence similarity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13.

[0078] In embodiments, the single domain antibody comprises an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99%; sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13. In an embodiment, the single domain antibody comprises an amino acid sequence selected from SEQ ID No. 2 to SEQ ID No. 13 with at least 90%, such as at least 95%, particularly at least 99% (including 100%) sequence identity within the framework regions of these sequences. Thus, this embodiment allows some deviations from the disclosed sequences in the framework regions. Desirably, there is 100% sequence identity in the CDR sequences.

[0079] In embodiments, the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, particularly an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9.

[0080] In embodiments, the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 10 and SEQ ID No. 11, such as an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 10 and SEQ ID No. 11.

[0081] In embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID No. 2, an amino acid sequence with at least 90% sequence similarity with SEQ ID No. 2 or an amino acid sequence with at least 90% (or at least 95% or at least 99%) sequence identity with SEQ ID No. 2. In an embodiment, the single domain antibody comprises an amino acid sequence of SEQ ID No. 2 with at least 90%, such as at least 95%, particularly at least 99% (including 100%) sequence identity within the framework regions of these sequences. Thus, this embodiment allows some deviations from the disclosed sequences in the framework regions. Desirably, there is 100% sequence identity in the CDR sequences.

[0082] In embodiments, the single domain antibody comprises:

[0083] - CDR1 with SEQ ID No. 14, CDR2 with SEQ ID No. 18 and CDR3 with SEQ ID No. 21,

[0084] - CDR1 with SEQ ID No. 15, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 22,

[0085] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 22,

[0086] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 23,

[0087] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 24, or

[0088] - CDR1 with SEQ ID No. 17, CDR2 with SEQ ID No. 20 and CDR3 with SEQ ID No. 25.

[0089] In embodiments, the single domain antibody comprises:

[0090] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16,

[0091] CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 and SEQ ID No. 19, and

[0092] CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 24.

[0093] In embodiments, the single domain antibody comprises:

[0094] CDR1 with an amino acid sequence according to SEQ ID No. 14, CDR2 with an amino acid sequence according to SEQ ID No. 18 and CDR3 with an amino acid sequence according to SEQ ID No. 21.

[0095] In embodiments, the single domain antibody has a size in the range of 11 kDa to 16 kDa, or in the range of 13 kDa to 15 kDa, particularly in the range of 14.0 kDa to 14.5 kDa.

[0096] Advantageously, a single domain antibody is smaller than an antibody. Surprisingly, good binding to CD7 could be demonstrated with the single domain antibody according to this disclosure despite its small size.

[0097] In embodiments, the single domain antibody has a sequence length in the range of 105 amino acids to 140 amino acids, such as in the range of 110 amino acids to 130 amino acids. In embodiments, the single domain antibody is in a multivalent form. In embodiments, the multivalent form of the single domain antibody is formed by bonding, chemically or by recombinant DNA techniques, of at least two single domain antibodies according to this disclosure. In embodiments, the single domain antibody is in a bivalent form (two bound single domain antibodies), a trivalent form (three bound single domain antibodies) or tetravalent form (four bound single domain antibodies). The single domain antibodies comprised within a multivalent construct may be identical or different. In an embodiment, the single domain antibody used in cancer diagnosis and / or therapy is monovalent.

[0098] In embodiments, the single domain antibody is associated with at least one of a detectable label, a chelator, a prosthetic group, a therapeutically and / or pharmacologically active agent. In an embodiment, “associated with” includes association by direct or indirect chemical and / or physical bonds, wherein indirect bonds are those mediated by additional chemical structures such as linkers or chelators, whereas direct bonding occurs without such additional chemical structures. Chemical bonds include covalent bonds and hydrogen bonds. Physical bonds include ionic bonds, coordination complexes and van der Waals interaction.

[0099] In a particular embodiment, the single domain antibody of this disclosure may be covalently bound to a chelator, e.g. NOTA (1,4,7-Triazacyclononane-1,4,7-triacetic acid), which forms a coordination complex with a radioactive isotope, e.g.68Ga (cf. example underlying Fig. 9). In an embodiment, the single domain antibody comprises an amino acid sequence of SEQ ID No. 2 with at least 90%, such as at least 95%, particularly at least 99% (including 100%) sequence identity within the framework regions of these sequences, the single domain antibody being covalently bound to a chelator, e.g. NOTA (1 ,4,7-Triazacyclononane-1,4,7-triacetic acid), which forms a coordination complex with a radioactive isotope, e.g.68Ga. In another embodiment, the single domain antibody of this disclosure is covalently bound to a chelator which complexes a metal ion (e.g. Al3+), the metal ion forming a bond (e.g. an ionic bond) to a radioactive isotope (e.g.18F-).

[0100] In embodiments, the single domain antibody for use in the therapy of cancer, in particular theranostics, is associated with at least one therapeutically and / or pharmacologically active agent. Advantageously, the single domain antibody for use in the therapy of cancer, in particular theranostics, targets the therapeutically and / or pharmacologically active agent to the cancer cells.

[0101] As used herein, the term “detectable label” (also detectable marker) refers to a detectable compound or composition which is conjugated directly, or indirectly associated with the single domain antibody, wherein it is detectable by itself (e. g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The detectable label includes detectable moieties and tracers for immuno-histochemistry, optical imaging, near infrared imaging (NIR), positron emission tomography (PET), single photon emission computed tomography (SPECT) or magnetic resonance imaging (MRI).

[0102] In embodiments, the detectable label is selected from a tag, a dye, a fluorescent compound, a bioluminescent compound, a radioactive isotope, a contrast agent, an enzyme, a nanoparticle and / or a magnetic particle.

[0103] As used herein, the term “prosthetic group” refers to a non-polypeptide molecule required for the biological function of proteins. In an embodiment, a “prosthetic group” is a non-polypeptide molecule that contains one or more covalently bound radioisotopes, such as18F or99mTc. In embodiments, prosthetic groups are disclosed in Wester and Schottelius (Wester and Schottelius 2007). In embodiments, prosthetic groups are selected from SiFA (silicon-fluoride- acceptor), SFB (N-Succinimidyl-4-[18F]Fluorobenzoate), FBEM (18F-N-[2](4- Fluorobenzamido)ethyl]-maleimide), halides and sulfonates, in particular tosylates, triflates, nosylates and mesylates.

[0104] As used herein, the term “therapeutically and / or pharmacologically active agent” refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction of a cancer cells, including radioactive isotopes, chemotherapeutic agents, enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics, toxins, growth inhibitory agents and drug moieties.

[0105] As used herein, the term “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. In embodiments, chemotherapeutic agents are selected from immunotoxins, chimeric antigen receptor T cells (CAR T cells) and switchable CAR T cells. As used herein, the term “immunotoxin” refers to a protein comprising at least a targeting domain linked to a toxin. When the protein binds to that cell by the targeting domain, it is taken in through endocytosis, and the toxin kills the cell.

[0106] As used herein, the term “CAR” refers to an artificial receptor consisting of a binding moiety, which provides an antigen-specificity for a target cell and one or several signaling chains derived from immune receptors. Immune cells, genetically modified to express CARs, can be used to bind cells or tissue structures expressing the appropriate target of the CAR binding moiety. Cross-linking leads to an induction of signal pathways via the CAR signaling chains, which will change the biologic properties of the CAR-engrafted immune cell. Advantageously, CAR T immunotherapy is used to modify T cells to recognize cancer cells in order to effectively target and destroy them.

[0107] As used herein, the term “switchable CAR T cells” refers to T cells engineered to express a CAR with a binding domain recognizing a tag or being a tag. Antigen-specificity is provided by soluble targeting molecules, which consist of an antigen-binding domain fused to the tag or a tag-binding domain recognized by the switchable CAR.

[0108] In embodiments, chemotherapeutic agents are selected from the group comprising A- dmDT390-bisFv(UCHT1), bispecific CARs targeting CD19 and CD22; BL22, BM7PE, Cintredekin besudotox, Denileukin diftitox, DT2219, Hum-195 / rGel, lgG-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.

[0109] In further embodiments, chemotherapeutic agents are selected from the group comprising alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, delta-9-tetrahydrocannabinol, beta-lapachone, lapachol, colchicines, betulinic acid, camptothecin, bryostatin, callystatin, CC-1065, podophyllotoxin, podophyllinic acid, teniposide, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards, nitrosoureas, antibiotics, dynemicin, an esperamicin, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo- S-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, folic acid analogues, purine analogs, pyrimidine analogs, androgens, anti-adrenals, folic acid replenisher, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfomithine, elliptinium acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSKTM(Polysaccharide- K) polysaccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2, 2’, 2"- trichlorotriethylamine, trichothecenes, urethane, Vindesine (ELDISINE™, FILDESIN™), dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), thiotepa, taxoids, chloranbucil, gemcitabine (GEMZAR™), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs, vinblastine (VELBAN™), platinum, etoposide (VP-16), ifosfamide, mitoxantrone, Vincristine (ONCOVIN™), oxaliplatin, leucovovin, Vinorelbine (NAVELBINE™), novantrone, edatrexate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids, capecitabine (XELODA™), pharmaceutically acceptable salts, acids or derivatives as well as combinations thereof.

[0110] In embodiments, chemotherapeutic agents are selected from the group comprising alkylating agents, such as thiotepa and CYTOXAN™ cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins, especially bullatacin and bullatacinone; delta-9-tetrahydrocannabinol, especially dronabinol and MARINOL™; beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin, including the synthetic analogue topotecan (HYCAMTIN™), CPT-II (irinotecan, CAMPTOSAR™), acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; callystatin; CC-1065, including its adozelesin, carzelesin and bizelesin synthetic analogues; podophyllotoxin; podophyllinic acid; teniposide; cryptophycins, particularly cryptophycin 1 and cryptophycin 8; dolastatin; duocarmycin, including the synthetic analogues, KW-2189 and CB I- TMI; eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics, e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-S-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin; epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-Fll); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, catmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSKTM(Polysaccharide- K) polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’, 2"- trichlorotriethylamine; trichothecenes, especially T-2 toxin, verracurin A, roridin A and anguidine; urethan; Vindesine (ELDISINE™, FILDESIN™); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL™, paclitaxel, ABRAXANE™, and TAXOTERETMdocetaxel; chlorambucil; gemcitabine (GEMZAR™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN™); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; Vincristine (ONCOVIN™); oxaliplatin; leucovovin; Vinorelbine (NAVELBINE™); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA™); pharmaceutically acceptable salts, acids or derivatives thereof; as well as combinations thereof.

[0111] In some embodiments, chemotherapeutic agents are selected from the group comprising thiotepa and CYTOXAN™ cyclosphosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimines, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, dronabinol, MARINOL™, beta-lapachone, lapachol, colchicines, betulinic acid, topotecan (HYCAMTIN™), CPT-II (irinotecan, CAMPTOSAR™), acetylcamptothecin, scopolectin, 9-aminocamptothecin, bryostatin, callystatin, CC-1065, CC-1065 adozelesin analogue, CC-1065 carzelesin analogue, CC-1065 bizelesin analogue, podophyllotoxin, podophyllinic acid, teniposide, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW- 2189, CB l-TMl; eleutherobin, pancratistatin, a sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, enediyne antibiotics, calicheamicin, dynemicin, an esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-S-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, 5-fluorouracil (5-Fll), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, catmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfomithine, elliptinium acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSKTM(Polysaccharide-K) polysaccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2’,2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, Vindesine (ELDISINE™, FILDESIN™), dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), thiotepa, TAXOL™, paclitaxel, ABRAXANE™, TAXOTERETMdocetaxel, chloranbucil, gemcitabine (GEMZAR™), 6-thioguanine, mercaptopurine, methotrexate, cisplatin, carboplatin, vinblastine (VELBAN™), platinum, etoposide (VP-16), ifosfamide, mitoxantrone, Vincristine (ONCOVIN™), oxaliplatin, leucovovin, Vinorelbine (NAVELBINE™), novantrone, edatrexate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, capecitabine (XELODA™), pharmaceutically acceptable salts or acids thereof; as well as combinations thereof.

[0112] As used herein, the term “toxin” refers to a naturally occurring organic substance having a detrimental effect on the growth or proliferation of a cell, produced by metabolic activities of living cells or organisms, in particular of bacterial, fungal, plant or animal origin. In embodiments, the toxin is a small molecule, peptide or protein. In embodiments, toxins are selected from exotoxins or endotoxins. In embodiments, toxins are 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, maytansinoids, such as maytansinoid DM1 and DM4; maitansine, mitomycin, paclitaxel, pseudomonas exotoxin, ricin, ricin A and vinblastine.

[0113] In embodiments, the detectable label, therapeutically or pharmacologically active agent is a radioactive isotope selected from the group comprising68Ga,18F,89Zr,99mTc,61Cu,64Cu,67Cu,43Sc,44Sc,47Sc,149Tb,152Tb,155Tb,161Tb,123l,124l,177Lu,225Ac,213Bi,211At,212Pb and223Ra, in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof, optionally the radioactive isotope is68Ga.

[0114] In preferred embodiments, the single domain antibody is associated with at least one chelator and / or a prosthetic group and at least one radionuclide, in particular68Ga,18F,89Zr,99mTc,61Cu,64Cu,67Cu,43Sc,44Sc,47Sc,149Tb,152Tb,155Tb,161Tb,123l,124l,177Lu,225Ac,213Bi,211At,212Pb and223Ra, in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof, optionally the radioactive isotope is68Ga.

[0115] In some embodiments, the radioactive isotope or radionuclide has a radioactive half-life of at most 750 minutes, at most 500 minutes, at most 250 minutes, at most 180 minutes, or at most 100 minutes. Quick distribution of the single domain antibody within a patient allows for the use of short-lived isotopes because imaging may be performed shortly after administration without the need for intense radioactive activity over prolonged periods.

[0116] In an embodiment, this disclosure relates to a single domain antibody which binds to human CD7 for use in the diagnosis and / or therapy of cancer in vivo, the single domain antibody comprising complementarity determining regions (CDR):

[0117] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14 to SEQ ID No. 17,

[0118] CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 to SEQ ID No. 20 and

[0119] CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 25; wherein the single domain antibody comprises a chelator, in particular suitable for chelating one or more radioisotopes selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,67Cu,43Sc,44Sc,47Sc,149Tb,152Tb,155Tb,161Tb,123l,124l,177Lu,225Ac,213Bi,211At,212Pb and223Ra.

[0120] The single domain antibody according to this embodiment may find utility as an intermediate product, i.e. this single domain antibody may be free of a radioactive isotope. The chelator may be suitable for chelating a radioactive isotope so that the single domain antibody according to the eleventh aspect may be used for preparing a diagnostic and / or therapeutic final antibody, i.e. carrying a radioactive isotope, e.g. at the hospital or point of care. This allows for the use of particularly short-lived isotopes. All the properties and features described for the single domain antibody herein apply to the single domain antibody as an intermediate product accordingly. In particular, the chelator may be attached to the single domain antibody directly or indirectly, i.e. with or without a linker.

[0121] This disclosure also relates to a method of preparing a single domain antibody of this disclosure by combining the single domain antibody as an intermediate product disclosed hereinabove with one or more radioisotopes disclosed herein. Afterwards one or more of the methods and medical uses discussed herein may be performed.

[0122] A further aspect of the invention provides a pharmaceutical composition for use in the diagnosis and / or therapy of cancer in vivo comprising at least one of the single domain antibodies according to this disclosure.

[0123] The pharmaceutical composition may be administered parenterally, such as intravenously. In embodiments, the pharmaceutical composition is present in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion or suspension.

[0124] In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable thinner (dilution agent) or carrier. In embodiments, the carrier is selected from water, an aqueous buffer solution, 0.9 % saline solution, 5 % glucose, 5 % xylitol, 0.3 % glycine solution, ringer solutions or amino acid solutions. In further embodiments, the aqueous buffer solution is selected from an aqueous histidine, sodium succinate, sodium acetate, sodium citrate, sodium phosphate or potassium phosphate-buffered solution with a pH value in the range of pH 5.0 to pH 7.0. In embodiments, the aqueous buffer solution has a buffer concentration in the range of 1 mmol / l (mM) to 500 mM, such as in the range of 5 mM to 20 mM, particularly in the range of 5 mM to 10 mM. In embodiments, the carrier comprises sodium chloride, preferably in a concentration in the range of 1 mM to 300 mM, especially about 150 mM.

[0125] In embodiments, the pharmaceutical composition further comprises a stabilizer, such as with a concentration in the range of 1 mM to 900 mM, especially in the range of 50 mM and 600 mM. In embodiments, the stabilizer is sucrose, trehalose or L-methionine.

[0126] In embodiments, the pharmaceutical composition may further comprise a radical scavenger, in particular an antioxidant, optionally with a concentration in the range of 1 mM to 900 mM, especially in the range of 20 mM and 200 mM. In embodiments, the 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, a radical scavenger, in particular an antioxidant, can react with and neutralize free radicals formed by radioactivity, which are highly reactive molecules that can initiate oxidation reactions. Thus, a radical scavenger prevents or delays the oxidative degradation of sensitive substances, in particular the single domain antibodies according to this disclosure.

[0127] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. The term “pharmaceutically acceptable excipients” refers to compounds, which provide approximately physiological conditions and / or increase the stability, such as agents for adjusting the pH value and buffering agents, agents for adjusting the toxicity and the like. In embodiments, pharmaceutically acceptable excipients are selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and polysorbate-80, such as polysorbate-80 in the range of 0.0001 % (w / v) to 1 % (w / v), especially in the range of 0.001 % (w / v) to 0.1 % (w / v).

[0128] In optional embodiments, the pharmaceutical composition comprises the single domain antibody in a concentration in the range of 0.1 pg / ml to 2500.0 pg / ml, such as in the range of 1.0 pg / ml to 1500.0 pg / ml, optionally from 10 pg / ml to 1000.0 pg / ml.

[0129] In further embodiments, the pharmaceutical composition is sterile. The pharmaceutical composition may be sterilized by conventional well-known techniques including, but not limited to, sterile filtration.

[0130] In embodiments, the pharmaceutical composition is used for administration to a subject.

[0131] In embodiments, the pharmaceutical composition is lyophilized prior to storage or stored as solution at ambient temperature or below, including, but not limited to, frozen storage. In embodiments, the pharmaceutical composition is reconstituted and / or diluted in an infusion and stabilizer solution prior to administration to a subject. The solutions used for reconstitution or infusion / stabilization may contain any of the components mentioned for the pharmaceutical composition or similar components.

[0132] Another aspect of this disclosure relates to a kit for use in the diagnosis and / or therapy of cancer in vivo comprising i. at least one single domain antibody according to the invention and ii. at least one pharmaceutically acceptable excipient or a solution thereof.

[0133] In embodiments of the kit, the at least one single domain antibody is associated with at least one chelator and / or a prosthetic group.

[0134] The at least one pharmaceutically acceptable excipient or a solution thereof may be used for reconstitution and / or dilution of the at least one single domain antibody prior to administration to a subject.

[0135] In embodiments, the at least one single domain antibody and / or the at least one pharmaceutically acceptable excipient or a solution thereof is sterile.

[0136] In embodiments, 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, optionally sodium acetate.

[0137] In embodiments, the solution of the at least one pharmaceutically acceptable excipient has a concentration of the excipient in the range of 0.0001 % (w / v) to 1 % (w / v), such as in the range of 0.001 % (w / v) to 0.1 % (w / v).

[0138] In embodiments, the kit further comprises an inorganic acid. Advantageously, the inorganic acid is used for preparing a radioactive isotope, in particular for elution from a generator for a radioactive isotope. In embodiments, the inorganic acid is hydrochloric acid.

[0139] In embodiments, the radioactive isotope is68Ga and the generator is a Ga / Ge generator.

[0140] In embodiments, the kit is used for the preparation of a single domain antibody associated with a radioactive isotope for the use in the diagnosis and / or therapy of cancer. Advantageously, the kit can be used to prepare the diagnostic and / or therapeutic agent immediately before use. Another aspect of this disclosure is the use of the single domain antibody, the pharmaceutical composition and / or the kit according to this disclosure in the diagnosis and / or therapy of cancer in vitro or ex vivo or for the synthesis of radioactive tracers and / or contrast agents.

[0141] A further aspect of the disclosure 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. 13.

[0142] In embodiments, the single domain antibody comprises an amino acid sequence with at least 90% sequence similarity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13, such as an amino acid sequence with at least 95% sequence similarity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13, particularly an amino acid sequence with at least 99% sequence similarity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13.

[0143] In embodiments, the single domain antibody comprises an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13.

[0144] In embodiments, the single domain antibody comprises complementarity determining regions (CDR):

[0145] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14 to SEQ ID No. 17,

[0146] CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 to SEQ ID No. 20 and

[0147] CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 25.

[0148] In embodiments, the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, such as an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9.

[0149] In embodiments, the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 10 and SEQ ID No. 11, such as an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 10 and SEQ ID No. 11. In embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID No. 2, an amino acid sequence with at least 90% sequence similarity with SEQ ID No. 2 or an amino acid sequence with at least 90% (or at least 95%, or at least 99%) sequence identity with SEQ ID No. 2.

[0150] In embodiments, the single domain antibody comprises:

[0151] - CDR1 with SEQ ID No. 14, CDR2 with SEQ ID No. 18 and CDR3 with SEQ ID No. 21,

[0152] - CDR1 with SEQ ID No. 15, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 22,

[0153] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 22,

[0154] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 23,

[0155] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 24, or

[0156] - CDR1 with SEQ ID No. 17, CDR2 with SEQ ID No. 20 and CDR3 with SEQ ID No. 25.

[0157] In embodiments, the single domain antibody comprises:

[0158] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16,

[0159] CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 and SEQ ID No. 19, and

[0160] CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 24.

[0161] In embodiments, the single domain antibody comprises:

[0162] CDR1 with an amino acid sequence according to SEQ ID No. 14, CDR2 with an amino acid sequence according to SEQ ID No. 18 and CDR3 with an amino acid sequence according to SEQ ID No. 21.

[0163] In embodiments, the single domain antibody has a size in the range of 11 kDa to 16 kDa, such as in the range of 13 kDa to 15 kDa, particularly in the range of 14.0 kDa to 14.5 kDa.

[0164] In embodiments, the single domain antibody has a sequence length in the range of 105 amino acids to 140 amino acids, such as in the range of 110 amino acids to 130 amino acids.

[0165] In embodiments, the single domain antibody is in a multivalent form. In embodiments, the multivalent form of the single domain antibody is formed by bonding, chemically or by recombinant DNA techniques, of at least two single domain antibodies according to the invention. In embodiments, the single domain antibody is in a bivalent form (two bound single domain antibodies), a trivalent form (three bound single domain antibodies) or tetravalent form (four bound single domain antibodies). The single domain antibodies comprised within a multivalent construct may be identical or different.

[0166] In embodiments, the single domain antibody is associated with at least one of a detectable label, a chelator, a prosthetic group, a therapeutically and / or pharmacologically active agent.

[0167] In embodiments, the single domain antibody according to this disclosure is used for the ex vivo or in vitro detection of CD7 in a sample.

[0168] In embodiments, the single domain antibody according to this disclosure is used for the detection of immune cells, in particular ? cells and NK cells. Advantageously, the distribution of immune cells, in particular T cells, can be detected in the human body to assess the activity of inflammatory processes in cancer, infection, and chronic inflammatory diseases.

[0169] In embodiments, the single domain antibody according to this disclosure is used in the diagnosis of cancer. In embodiments, the single domain antibody according to this disclosure is used for the diagnosis of cancer by imaging of immune cells, in particular T cells or NK cells, in immunotherapies.

[0170] In embodiments, the diagnosis is carried out before, during and / or after immunotherapy of cancer. In embodiments, the single domain antibody is used as radioactive tracer 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 radioactive tracers and / or contrast agents.

[0171] In embodiments, the single domain antibody according to this disclosure is used for the therapy of cancer or an autoimmune disease.

[0172] In further embodiments, the single domain antibody according to this disclosure is used for theranostics, i.e. the combined diagnosis and therapy, of immune cell related cancer, such as lymphomas, in particular ? cell or NK lymphomas; or leukaemia.

[0173] In embodiments, the single domain antibody according to this disclosure is used for the manufacture of a medicament for the treatment of cancer or an autoimmune disease.

[0174] A further aspect of the invention provides a nucleic acid comprising a nucleic acid sequence encoding for the single domain antibody according to this disclosure. A further aspect of the disclosure provides a pharmaceutical composition comprising at least one single domain antibody according to this disclosure.

[0175] Another aspect of this disclosure is a method of diagnosing a subject in need thereof comprising administering a diagnostically effective dose of a single domain antibody according to this disclosure to the subject in need thereof, preferably a human having cancer.

[0176] Another aspect of the disclosure is a method of treating a subject in need thereof comprising administering a therapeutically effective dose of a single domain antibody according to this disclosure to the subject in need thereof, preferably a human, having cancer or an autoimmune disease.

[0177] For diagnostic or therapeutic applications, a sterile pharmaceutical composition according to this disclosure or a sterile kit according to this disclosure, comprising a single domain antibody according to this disclosure is administered to a subject in order to diagnose or treat the illnesses.

[0178] In embodiments, the pharmaceutical composition is administered parenterally, preferably intravenously.

[0179] In embodiments of the method of diagnosing, the diagnosis is carried out after the administration of the single domain antibody according to the invention, such as in the range of 10 min to 240 min after the administration of the single domain antibody according to this disclosure.

[0180] In embodiments of the method of diagnosing, the diagnosis is carried out by non-invasive medical imaging of T cells in vivo, such as by NIR, PET, PET / CT, PET / MR, SPECT or MR imaging, particularly by PET, PET / CT, PET / MR or SPECT imaging. As used herein “imaging” or “direct imaging” may be selected from imaging by NIR, PET, PET / CT, PET / MR, SPECT and / or MR imaging, particularly by PET, PET / CT, PET / MR and / or SPECT imaging

[0181] In embodiments of the method of treatment, the single domain antibody according to this disclosure is administered as at least one dosage, optionally as at least two dosages.

[0182] In embodiments of the method of treatment, the single domain antibody according to this disclosure is administered as at least two dosages, wherein the period between the two dosages is in the range of 6 weeks to 12 weeks. In embodiments of the method of treatment, the single domain antibody according to this disclosure is administered in combination with at least one further therapeutically and / or pharmacologically active agent and / or with at least one further therapy, in particular surgical removal of a tumor, radiotherapy, chemotherapy, stem cell transplantation, hyperthermia treatment and / or immunotherapy.

[0183] The invention is not limited to the embodiments shown and described, but also includes all embodiments having the same effect within the meaning of the disclosure. Furthermore, the invention is also not limited to the specifically described combinations of features but may also be defined by any other combination of specific features of all the individual features disclosed as a whole, provided that the individual features are not mutually exclusive, or a specific combination of individual features is not explicitly excluded.

[0184] In an embodiment, the single domain antibody is associated with a detectable label and binds human CD7 with an affinity, the single domain antibody having a molecular weight and the detectable label having a radioactive half-life, wherein a figure of merit defined as affinityr[nmol / Lni X molecular weightr[g / molnxradioactive half-life [s] , is at least 0.05 [L / g*sl. Optionally, the figure of merit [L / g*s] may be at least 0.25, at least 0.50, at least 0.75, at least 1.0, at least

[0185] 1.5 or at least 2.0. In some embodiments, the figure of merit is up to 4.0, up to 3.0, or up to 2.5.

[0186] For example, the figure of merit may range from 0.50 to 4.0, from 0.75 to 3.0, or from 1.0 to 2.5.

[0187] The single domain antibody of this disclosure binds human CD7 with high affinity. In an embodiment, the single domain antibody binds to human CD7 with an affinity of less than 15 nM. In an embodiment, the single domain antibody binds to human CD7 with an affinity from about 3.90 nM to about 11.0 nM, such as about 6.34 10'9M, about 5.66 10'9M, about 11.0- 10'9M, about 8.16 10-9M, about 4.04- 10'9M or about 3.90- 10'9M. In certain embodiments, the affinity of the single domain antibody to human CD7 is less than 12 nM, less than 10 nM, or less than 8 nM. In some embodiments, affinity ranges from 1 nM to <15 nM, from 2 nM to <12 nM, or from 3 nM to <10 nM. The term “about” as it relates to affinities may include the indicated value ±20% of the indicated value, or ±10% of the indicated value.

[0188] In an embodiment, this disclosure relates to a single domain antibody for use in the diagnosis and / or therapy of cancer in vivo, which binds to human CD7, the single domain antibody being associated with a detectable label and comprising complementarity determining regions (CDR):

[0189] CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14 to SEQ ID No. 17, CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 to SEQ ID No. 20 and

[0190] CDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 25, wherein the diagnosis and / or therapy of cancer includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0191] In an embodiment, this disclosure relates to a single domain antibody for use in the diagnosis and / or therapy of cancer in vivo, which binds to human CD7, the single domain antibody being associated with a detectable label and comprising complementarity determining regions (CDR):

[0192] - CDR1 with SEQ ID No. 14, CDR2 with SEQ ID No. 18 and CDR3 with SEQ ID No. 21,

[0193] - CDR1 with SEQ ID No. 15, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 22,

[0194] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 22,

[0195] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 23,

[0196] - CDR1 with SEQ ID No. 16, CDR2 with SEQ ID No. 19 and CDR3 with SEQ ID No. 24, or

[0197] - CDR1 with SEQ ID No. 17, CDR2 with SEQ ID No. 20 and CDR3 with SEQ ID No. 25. wherein the diagnosis and / or therapy of cancer includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0198] In a more specific embodiment, this disclosure relates to a single domain antibody for use in the diagnosis and / or therapy of cancer in vivo, which binds to human CD7, the single domain antibody being associated with a detectable label and comprising an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity therewith, wherein the diagnosis and / or therapy of cancer includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0199] In a more specific embodiment, this disclosure relates to a single domain antibody for use in the diagnosis and / or therapy of cancer in vivo, which binds to human CD7, the single domain antibody being associated with a detectable label and comprising an amino acid sequence according to SEQ ID No. 2, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity therewith, wherein the diagnosis and / or therapy of cancer includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0200] In an embodiment, this disclosure relates to single domain antibody for use in therapy of cancer in vivo, the single domain antibody binding to human CD7 with an affinity of less than 15 nM, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with a detectable label, which detectable label is a radioactive isotope with a half-life of at most 750 minutes, or at most 250 minutes, wherein the therapy includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0201] In an embodiment, this disclosure relates to a single domain antibody for use in therapy of cancer in vivo, the single domain antibody binding to human CD7 with an affinity from about 3.90 nM to about 11.0 nM, such as about 6.34 10'9M, about 5.66 10'9M, about 11.0- 10’9M, about 8.16 1 O'9M, about 4.04 10-9M or about 3.90- 10’9M, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with a detectable label, which detectable label is a radioactive isotope with a half-life of at most 750 minutes or at most 250 minutes, wherein the therapy includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0202] In an embodiment, this disclosure relates to a single domain antibody for use in therapy of cancer in vivo, the single domain antibody binding to human CD7 with an affinity from about 3.90 nM to about 11.0 nM, such as about 6.34 10'9M, about 5.66 10'9M, about 11.0- 10’9M, about 8.16 1 O'9M, about 4.04 - IO’9M or about 3.90- 10’9M, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with a detectable label, which detectable label is a radioactive isotope with a half-life of at most 750 minutes or at most 250 minutes, wherein the therapy includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0203] In an embodiment, this disclosure relates to a single domain antibody for use in therapy of cancer in vivo, the single domain antibody binding to human CD7 with an affinity from about 3.90 nM to about 11.0 nM, such as about 6.34 10'9M, about 5.66 10'9M, about 11.0- 10’9M, about 8.16 1 O'9M, about 4.04 - IO’9M or about 3.90- 10’9M, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with a detectable label, which detectable label is a radioactive isotope selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb,123l,124l,213Bi and combinations thereof, in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof, optionally the isotope is68Ga, wherein the therapy includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

[0204] As described in more detail elsewhere in this disclosure, the single domain antibody binds to immune cells that express CD7, thereby facilitating detection of the detectable label by suitable detection methods. The detection methods depend on the detectable label and include immunohistochemistry, optical imaging, near infrared imaging (NIR), positron emission tomography (PET), single photon emission computed tomography (SPECT) or magnetic resonance imaging (MRI). By detecting the immune cells, e.g. T cells, in the tumor environment treatment efficacy of the administered immunotherapy can be determined. For example, if T cell accumulation in the tumor environment is detected, it may be concluded that immunotherapy is efficacious, whereas if no or insufficient T cell accumulation is detected, the therapy may be ineffective and the patient should be switched to a different therapy.

[0205] In an embodiment, this disclosure relates to a single domain antibody, such as a single domain antibody that binds to human CD7 for use in cancer therapy, wherein the single domain antibody is associated with a detectable label, the detectable label being a radioactive isotope, wherein the cancer therapy comprises administering cancer therapy, such as immunotherapy, to a patient in need thereof, administering an effective amount of the single domain antibody to the patient, monitoring a presence and / or amount of immune cells expressing human CD7 at the tumor site, e.g. in the vicinity of the tumor and / or in the tumor, optionally, deciding whether the cancer therapy is effective based on the presence and / or amount of the immune cells at the tumor site, further optionally wherein the single domain antibody binds human CD7 with an affinity, the single domain antibody having a molecular weight and the detectable label having a radioactive half-life, wherein a figure of merit defined as i rn r n, is at least 0.05 [L / g*sl. Optionally, affinity [nmol / L\x molecular weight [g / mol\xradioactive half-life [s] the figure of merit [L / g*s] may be at least 0.25, at least 0.50, at least 0.75, at least 1.0, at least

[0206] 1.5 or at least 2.0. In some embodiments, the figure of merit is up to 4.0, up to 3.0, or up to 2.5.

[0207] For example, the figure of merit may range from 0.50 to 4.0, from 0.75 to 3.0, or from 1.0 to 2.5. The figure of merit provides for rapid distribution of the small single domain antibodies in the patient’s body so that rapid accumulation of sufficient amounts of single domain antibodies at the tumor site can be achieved. Because of the rapid distribution of these molecules, radioactive isotopes with a short radioactive half-life can be employed. Short-lived radioactive isotopes have the benefit that patients may leave the hospital earlier after having undergone the therapy disclosed herein compared to isotopes with longer half-lives. Suitable radioactive isotopes that can be used in this context are68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb, 123| 124| 2i3gjanc| combinations thereof, in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof.

[0208] In an embodiment, the single domain antibody comprises an amino acid sequence of SEQ ID No. 2 with at least 90%, such as at least 95%, particularly at least 99% (including 100%) sequence identity within the framework regions of these sequences (and optionally 100% sequence identity in the CDRs), the single domain antibody having an affinity of less than 10 nM, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with a detectable label, which detectable label is a radioactive isotope with a half-life of at most 750, or at most 250 minutes, such as68Ga.

[0209] In an embodiment, the single domain antibody that binds to human CD7 is used in cancer therapy, wherein the single domain antibody is associated with a radioactive isotope, a chemotherapeutic agent and / or a toxin, wherein the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 less than 10 nM.

[0210] Using the single domain antibody in monomeric form contributes to quick distribution in the subject’s body which - in combination with the high affinity to the target - allows for rapid accumulation of the single domain antibody at the tumor site.

[0211] In an embodiment, the single domain antibody that binds to human CD7 is used in cancer therapy, wherein the single domain antibody is associated with a radioactive isotope, wherein the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 less than 10 nM, wherein the radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0212] In an embodiment, the single domain antibody that binds to human CD7 is used in cancer therapy, wherein the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity therewith, wherein the single domain antibody is associated with a radioactive isotope, wherein the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 less than 10 nM, wherein the radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0213] In an embodiment, the single domain antibody that binds to human CD7 is used in cancer therapy, wherein the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% (including 100%) sequence identity within the framework regions of these sequences (and optionally 100% sequence identity in the CDRs), wherein the single domain antibody is associated with a radioactive isotope, wherein the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 less than 10 nM, wherein the radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0214] In an embodiment, the single domain antibody that binds to human CD7 is used in cancer therapy, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID No. 2, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity therewith, wherein the single domain antibody is associated with a radioactive isotope, wherein the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 less than 10 nM, wherein the radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0215] In an embodiment, the single domain antibody that binds to human CD7 is used in cancer therapy, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID No. 2, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% (including 100%) sequence identity within the framework regions of these sequences (and optionally 100% sequence identity in the CDRs), wherein the single domain antibody is associated with a radioactive isotope, wherein the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and has an affinity to human CD7 less than 10 nM, wherein the radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

[0216] In the following, the disclosure will be explained in more detail by means of an embodiment example. The example is intended to describe the disclosure without limiting it. Implementations of the disclosure will be described, by way of example only.

[0217] If nothing else is indicated, the single domain antibody used in the examples is that of SEQ ID No. 2.

[0218] Fig. 1 shows the analysis of the capability of the anti-CD7 single-domain antibody (CD7-single domain antibody) to bind human CD8+ T cells. Flow cytometry-based determination of binding specificity to human CD8+ T cells of CD7-single domain antibody. A non-targeting irrelevant single domain antibody (R3b23-single domain antibody) was used as a control to exclude unspecific binding of the single domain antibody basic framework-structure (first graph). A monoclonal antibody against CD7 was used as a positive control to verify possible binding of respective target antigens.

[0219] Fig. 2 shows the analysis of anti-CD7 single domain antibody binding to retrovirally transduced LI698M cells. Flow cytometry-based determination of binding specificity to retrovirally transduced B cell lymphoma tumor cell line LI698M with CD7 target antigen. (A) Exemplary binding of R3b23 to CD7-transduced LI698M cells, with wild type LI698M used as a negative control. (B) CD7-single domain antibody binding to CD7-transduced LI698M cells, with wild type LI698M used as a negative control.

[0220] Fig. 3 shows binding affinity of anti-CD7 single domain antibody on human CD8+ T cells. Determined affinity binding curves for CD7-single domain antibody (B-G) at different concentrations on human CD8+ T cells. R3b23-single domain antibody served as a negative control for unspecific binding (A). Binding of CD7-single domain antibody according to SEQ ID No. 2 (B) is shown as a fraction of the maximum specific binding (Bmax) and the binding intensity of R3b23-single domain antibody (A) and CD7-single domain antibodies according to SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13 are shown via the MFI at the different concentrations. MFI was performed with gating on alive lymphocytes. Measurements were performed in triplicates and data is depicted on a semi-logarithmic scale. The dissociation constants (KD) for CD7 were calculated based of one-site specific binding and are in the range of 3.90- nM to 11.0 nM. Fig. 4 shows the analysis of the thermostability of the ability of the anti-CD7 single domain antibody to bind to human CD8+ T cells via flow cytometry analysis. CD7-single domain antibody kept at 4 °C, 37 °C, 60 °C or 90 °C for 0.5 h, 1 h, 2 h, 4 h or 6 h, after which their binding capabilities to human CD8+ T cells were analyzed via flow cytometry. The temperatures of single domain antibody were kept steady at 37 °C, 60 °C and 90 °C and 4° C. Measurements were performed in triplicates and flow cytometry data is depicted as MFI.

[0221] Fig. 5 shows the analysis of the cytokine secretion of activated T cells (CD8+ T cells) after coincubation with the anti-CD7 single domain antibody according to this disclosure by ELISA. IFNy levels were determined by ELISA after 4 h co-culture of human CD8+ T cells, the respective target cell lines and CD7-single domain antibody and R3b23-single domain antibody as a control. Measurements were performed in triplicates.

[0222] Fig. 6 shows cytokine secretion analysis of IL2, GM-CSF and TNF-a by ELISA after coincubation with CD7-single domain antibody. Cytokine secretion levels of IL-2, GM-CSF and TNF-a were determined by ELISA after 4 h co-culture of human CD8+ T cells, the respective target cell lines and CD7-single domain antibody and R3b23-single domain antibody as a control. Used target cell lines were ML2-B7 (A), NB4-B7 (B) and HL60-B7 (C). Measurements were performed in triplicates.

[0223] Fig. 7 shows an in vivo analysis of T cell functionality of T cells after injection of the single domain antibody according to this disclosure. Schematic of the experimental setup of in vivo tumor rejection model for CD8+ T cells. NSG mice were subcutaneously (s.c.) injected with ML2-B7 cells in the right flank and ML2-B15 cells in the left flank. After eight days, TCR- transgenic human CD8+ T cells were injected intravenously (i.v.) through the tail vein, followed three days later by i.v. injection of R3-b23-single domain antibody, OKT 11 , RPA, or CD7-single domain antibody. Tumor growth was monitored from tumor onset until the end of experiment at day twelve.

[0224] Fig. 8 shows monitoring of tumor growth kinetics of ML2-B15 (A) and ML2-B7 tumors (B) in NSG mice. On day 0, eight days after subcutaneous tumor injection, mice were intravenously injected with TOR 2.5D6-transgenic CD8+ T cells and three days later with either PBS, R3b23- single domain antibody, CD2-F(ab')2 (OKT11), CD2-F(ab')2 (RPA-2.10) or CD7-single domain antibody. Kinetics of tumor growth were monitored daily for twelve days post T-cell injection. The experiment was ended at day twelve, at which point all relevant non-control tumors had been fully rejected. Tumor sizes are shown in mm2and mean values and SDs are depicted for each group of mice. PBS n = 4, R3b23-single domain antibody n = 3, CD2-F(ab')2 (OKT 11) n = 6, CD2-F(ab')2 (RPA-2.10) n = 4, CD7-single domain antibody n = 5. Significance was calculated using Mann-Whitney test (* p < 0.05, ** p < 0.01, *** p < 0.001). The application of the CD7-single domain antibody did not impair T-cell cytotoxicity in vivo.

[0225] Fig. 9 shows in vivo imaging using [68Ga]Ga -NOTA-labeled anti-CD7 single domain antibody (CD7-single domain antibody) in NSG mice. A) Schematic of the experimental setup for in vivo imaging. Experimental layout of in vivo imaging study using intravenous injection of CD8+ T cells after ML2-B7 and -B15 tumor injection. Two groups of four mice per group were injected with each condition and 1 h p.i. scanned using PET / MRI. B) In vivo tracking of intravenously injected TCR-transgenic CD8+ T cells in ML2-B7 tumors using [68Ga]Ga-NOTA-CD7-single domain antibody. PET / MRI images acquired 1 h p.i. of mice injected i.v. with [68Ga]Ga-NOTA- CD7-single domain antibody (left) or [68Ga]Ga-NOTA-R3b23-single domain antibody (right). Exemplary mice are shown in coronal, sagittal and axial orientation (A) or just in coronal orientation (B). 2 107TCR-transgenic CD8+ T cells were injected i.v. and the injected dose of applied tracer was 13 ±1 MBq per mouse. Scale bar is represented as percentage of injected dose per gram (%l D / g), 0.4 - 1.5 %l D / g. B = Bladder, K = Kidney. Striped arrow = ML2-B7 tumor, white arrow = ML2-B15 tumor, (below) Biodistribution data of [68Ga]Ga-NOTA-CD7- single domain antibody and [68Ga]Ga-NOTA-R3b23-single domain antibody in mice bearing ML2-B7 and -B15 tumors 1.5 h post injection and after previous PET / MR image acquisition. Mean %l D / g ± SD is depicted for each group of mice. [68Ga]Ga-NOTA-R3b23-single domain antibody n = 4, [68Ga]Ga-NOTA-CD7-single domain antibody n = 4. Significance was calculated using Mann-Whitney test (* p < 0.05).

[0226] The functionality and specificity of the single domain antibody according to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13 was investigated, by examination of the binding to the T cells as well as to the target marker, CD7 (see Fig. 1 to 3). For this purpose, the human tumor cell line LI698M (B-cell lymphoma) was transduced with the specific gene encoding for CD7. Flow cytometry showed specific binding to the transduced cells compared to non-transduced controls. In addition, the strength of the binding affinity was determined for the single domain antibody by binding to human CD8+ T cells and it was 6.34 10-9M (SEQ ID No. 2), 5.66 10-9M (SEQ ID No. 4), 11.0-10-9M (SEQ ID No. 7), 8.16 10-9M (SEQ ID No. 8), 4.04-10’9M (SEQ ID No. 9) and 3.90 10-9M (SEQ ID No. 13). This demonstrates not only the functionality but also the specificity of the single domain antibody to the target. Furthermore, the aspect of thermostability (see Fig. 4), cytokine secretion (see Fig. 5 and 6), functional impairment of cytotoxicity of T cells after co-incubation with the single domain antibody was investigated.

[0227] It was demonstrated that co-incubation had no negative effect on the cytotoxic potential of T cells, i.e., binding of the CD7 surface marker on T cells by the single domain antibody did not lead to impairment of their function in vitro to recognize and destroy tumor cells.

[0228] Regarding the binding of the anti-CD7 single domain antibody, no changes in IFNy secretions could be observed in all tumor cell lines examined (ML-2, NB-4 and HL-60) compared to the control group in which an irrelevant-binding single domain antibody (R3b23) was used. Thus, it can be suggested that the single domain antibody binding has no effect on the secretion of cytokines, which could trigger a cytokine storm syndrome in patients.

[0229] Furthermore, the in vivo effect of the CD2-binding F(ab')2 fragments was compared to the CD7- single domain antibody (see Fig. 7 and 8). CD2-binding F(ab')2 fragments were used as positive controls as they impair the T-cell functionality. The group receiving the single domain antibody showed a picture congruent with the PBS control. The tumors were detected and rejected.

[0230] Thus, it could be shown that the single domain antibody not only has no influence on the functionality of the T cells in vitro, but also has no effect on the function of the T cells in the mouse model in vivo. The use of CD2-binding F(ab')2 fragments (positive control) in vivo showed four days after T cell injection a significant increase in tumor volume and thus, a functional impairment of T cells.

[0231] Based on the in vitro and in vivo data regarding purity, binding strength and specificity, as well as the lack of restriction of T cell functionality after single domain antibody binding, the potential of the CD7 single domain antibody for immuno-PET imaging was then investigated (see Fig. 9). In this mouse experiment, four mice received [68Ga]Ga-NOTA-CD7-single domain antibody and were used for single domain antibody-based PET imaging and four mice were injected with the irrelevant single domain antibody [68Ga]Ga-NOTA-R3b23-single domain antibody to serve as a control. For this purpose, ML-2 tumor cells were transduced with genes coding for HLA-B*07:02 (B7) or HLA-B*15:01 (B15) and injected into the left (B15) and right (B7) shoulders of the mice. Tumors bearing HLA-B15 served as negative controls. After successful growth of the tumor cells eight days after injection, human CD8+ T cells were intravenously injected through the tail vein. Five days later, the radiolabeled single domain antibodies were injected intravenously. To allow imaging by PET imaging, the single domain antibody was previously conjugated with the p-SCN-Bn-NOTA chelator and labeled with the radioactive isotope gallium-68 (68Ga) at room temperature, which with its short half-life of 67 minutes would be very advantageous for potential clinical application. This demonstrated that the single domain antibody targeting CD7 is capable of binding to T cells in vivo and imaging them by PET imaging.

[0232] In addition, the radioactive labeling of the single domain antibody was developed and optimized, which is essential for imaging by PET. The tracer was labeled with the radioactive isotope gallium-68 and then evaluated for its imaging properties. The result of PET imaging showed a clear accumulation in the treated experimental tumor ML2-B7 which was infiltrated by T cells already one hour after injection of the tracer. This underlines the rapid and specific accumulation of the tracer. Furthermore, apart from an expected accumulation in the kidneys (due to excretion), no noticeable accumulation of the tracer in other organs or in the control tumor (ML2-B15) could be observed and therefore could be shown that the radiolabeled single domain antibody is able to bind to T cells in the mouse model without loss of function and to visualize them specifically and clearly by PET imaging.

[0233] Example of therapy

[0234] Two NSCLC cancer patients A and B are administered immunotherapy. Both receive treatment with an anti-PD-1 antibody. After administration of anti-PD-1 antibody, both receive [68Ga]Ga- NOTA-CD7-single domain antibody (SEQ ID No. 2) intravenously. Direct imaging of immune cells is performed on both patients using PET imaging within 30 to 120 minutes post administration. Patient A exhibits strong signals of68Ga in the tumor, indicating that anti-PD-1 effectively counteracts PD-1-mediated tumor immune evasion. In patient B no strong signals are observed in the tumor indicating that anti-PD-1 therapy is ineffective in this patient. Treatment of patient A with anti-PD-1 is continued, whereas patient B is switched to a different therapy.

[0235] Cited non-patent literature

[0236] Auletta S, lodice V, Galli F, Lepareur N, Devillers A, Signore A (2018) Study of Binding Kinetics and Specificity of 99mTc-SSS-Complex and 99mTc-HMPAO to Blood Cells. Contrast Media Mol Imaging 5603902. doi: 10.1155 / 2018 / 5603902.

[0237] Dromain C, Beigelman C, Pozzessere C, Duran R, Digklia A (2020) Imaging of tumour response to immunotherapy. Eur Radiol Exp. 4(1):2. doi: 10.1186 / s41747-019-0134-1. Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N, Deininger MW, Silver RT, Goldman JM, Stone RM, Cervantes F, Hochhaus A, Powell BL, Gabrilove JL, Rousselot P, Reiffers J, Cornelissen JJ, Hughes T, Agis H, Fischer T, Verhoef G, Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich JP, Simonsson B, Taylor K, Baccarani M, So C, Letvak L, Larson RA; IRIS Investigators (2006) Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med. 355(23):2408-2417. doi: 10.1056 / NEJMoa062867.

[0238] Kasbauer T, Gosmann D, Russelli L, Bassermann F, Weber W, D’Alessandria C, Krackhardt AM (2019) In vitro characterisation of nanobodies targeting a pan-T-cell marker as potential universal immunotherapy T-cell trackers, World Molecular Imaging Congress Montreal, 04.- 07.09.2019.

[0239] Tang J, Li J, Zhu X, Yu Y, Chen D, Yuan L, Gu Z, Zhang X, Qi L, Gong Z, Jiang P, Yu J, Meng H, An G, Zheng H, Yang L (2016) Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells. Oncotarget. 7(23):34070-83.

[0240] Weist MR, Starr R, Aguilar B, Chea J, Miles JK, Poku E, Gerdts E, Yang X, Priceman SJ, Forman SJ, Colcher D, Brown CE, Shively JE (2018) PET of Adoptively Transferred Chimeric Antigen Receptor T Cells with89Zr-Oxine. J Nucl Med. 59(10):1531-1537. doi: 10.2967 / jnumed.117.206714.

[0241] Wester HJ, Schottelius M (2007) Fluorine-18 Labeling of Peptides and Proteins. In: Schubiger PA, Lehmann L, Friebe M (eds) PET Chemistry. Ernst Schering Research Foundation Workshop 64. Springer, https: / / doi.org / 10.1007 / 978-3-540-49527-7_4.

[0242] Chalian H, O'Donnell JK, Bolen M, Rajiah P. Incremental value of PET and MRI in the evaluation of cardiovascular abnormalities. Insights Imaging. 2016 Aug;7(4):485-503. doi: 10.1007 / S13244-016-0494-5. Epub 2016 May 25. PMID: 27221975; PMCID:

[0243] PMC4956622. McCarthy CE, White JM, Viola NT, Gibson HM. In vivo Imaging Technologies to Monitor the Immune System. Front Immunol. 2020 Jun 2;11 :1067. doi:

[0244] 10.3389 / fimmu.2020.01067. PMID: 32582173; PMCID: PMC7280489.

[0245] Mayer KE, Mall S, Yusufi N, Gosmann D, Steiger K, Russelli L, Bianchi HO, Audehm S, Wagner R, Braunlein E, Stelzl A, Bassermann F, Weichert W, Weber W, Schwaiger M, D'Alessandria C, Krackhardt AM. T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target. Theranostics. 2018 Nov 28;8(21):6070-6087. doi: 10.7150 / thno.27275. PMID: 30613283;

[0246] PMCID: PMC6299443.

Claims

Claims1. A single domain antibody which binds to human CD7 for use in the diagnosis and / or therapy of cancer in vivo, the single domain antibody comprising complementarity determining regions (CDR):CDR1 with an amino acid sequence selected from the group comprising SEQ ID No. 14 to SEQ ID No. 17,CDR2 with an amino acid sequence selected from the group comprising SEQ ID No. 18 to SEQ ID No. 20 andCDR3 with an amino acid sequence selected from the group comprising SEQ ID No. 21 to SEQ ID No. 25.

2. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 1 comprisingCDR1 with an amino acid sequence according to SEQ ID No. 14,CDR2 with an amino acid sequence according to SEQ ID No. 18 andCDR3 with an amino acid sequence according to SEQ ID No. 21.

3. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 1 or 2 comprising an amino acid sequence according to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13 or at least 90% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13.

4. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the claims 1 to 3, wherein the single domain antibody has a size in the range of 11 kDa to 16 kDa.

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

6. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 5, wherein the detectable label, and / or the therapeutically or pharmacologically active agent is a radioactive isotope selected from the group comprising68Ga,18F,89Zr,"mTc, 6iCUi64Cu,67Cu,43Sc,44Sc,47Sc,149Tb,152Tb,155Tb,161Tb,123l,124l,177Lu,225Ac, 213Bi,211At,212Pb and223Ra.

7. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the claims 1 to 6, wherein the single domain antibody is used as radioactive tracer and / or contrast agent in non-invasive medical imaging in vivo, such as by PET or SPECT imaging.

8. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 1 to 7 , the single domain antibody being associated with a detectable label, wherein the diagnosis and / or therapy of cancer includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

9. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 1 to 8, wherein the single domain antibody is associated with a detectable label and binds human CD7 with an affinity, the single domain antibody having a molecular weight and the detectable label having a radioactive half-life, wherein a figure of merit defined as - - - is at least affinity [nmol / L]x molecular weight [g / mol]xradioactive half-life [s]0.05 or at least 0.50 [L / g*s],10. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 8 or 9, wherein immunotherapy includes immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies binding to specific targets on cancer cells, cancer vaccines, and / or immune system modulators.11 . The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, wherein diagnosis and / or therapy includes: observing whether immune cells are present on and in a tumor.

12. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims wherein the single domain antibody comprises anamino acid sequence according to one of the sequences selected from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity within the framework regions of these sequences and optionally 100% sequence identity in the CDRs.

13. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims wherein the single domain antibody is associated with a detectable label, the detectable label being a radioactive isotope, and wherein the cancer diagnosis and / or therapy comprises administering cancer therapy, such as immunotherapy, to a patient in need thereof, administering an effective amount of the single domain antibody to the patient, monitoring a presence and / or amount of immune cells expressing human CD7 at the tumor site, e.g. in the vicinity of the tumor and / or in the tumor, optionally, deciding whether the cancer therapy is effective based on the presence and / or amount of the immune cells at the tumor site.

14. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims wherein the detectable label is a radioactive isotope selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb,123l,124l,213Bi and combinations thereof, in particular selected from68Ga,18F,61Cu,64Cu and combinations thereof, wherein optionally the isotope is68Ga.

15. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, wherein the radioactive isotope has a radioactive half-life of at most 750 minutes, at most 500 minutes, or at most 250 minutes.

16. The single domain antibody for use in cancer therapy according to one or more of the preceding claims wherein the method comprises administering to a subject having cancer an amount of the single domain antibody which binds to human CD7, wherein the single domain antibody is associated with a radioisotope, a chemotherapeutic agent or a toxin,wherein the cancer cells express human CD7, wherein optionally the single domain antibody has a molecular weight of at most 16 kDa, is present in monovalent form, and / or has an affinity to human CD7 less than 10 nM.

17. The single domain antibody for use in cancer therapy according to claim 16, wherein the radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu,225Ac,211Ac,212Pb,223Ra and combinations thereof.

18. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, wherein the cancer is immune cell-derived, such as T cell-derived cancer.

19. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, wherein the cancer is selected from lymphomas, in particular T cell or NK lymphomas, or leukemia.

20. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, wherein the cancer is metastasized, in particular with solid metastases.21 . The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, the method comprising administering to a subject an amount of the single domain antibody, wherein the single domain antibody is associated with a first radioisotope, performing imaging by detecting the presence of the radioisotope in the subject, administering to the subject an amount of the single domain antibody, wherein the single domain antibody is associated with a second radioisotope, wherein the cancer cells express human CD7.

22. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 21 , wherein the second radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu, 225Ac,211Ac,212Pb,223Ra and combinations thereof, and / or the first radioisotope is selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb,123l,124l,213Bi and combinations thereof.

23. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 21 or 22, wherein the first radioisotope and the second radioisotope are different isotopes of the same element, optionally of Cu, Tb or Sc.

24. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 21 , 22 or 23, wherein- the first radioisotope is61Cu and / or64Cu, and the second radioisotope is67Cu;- the first radioisotope is43Sc and / or44Sc, and the second radioisotope is47Sc; or- the first radioisotope is149Tb,152Tb and / or155Tb, and the second radioisotope is149Tb and / or161Tb.

25. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of the preceding claims, wherein diagnosis and / or therapy includes determining the existence, localization and / or size of a tumor or metastasis of a CD7 positive cancer.

26. A pharmaceutical composition comprising at least one of the single domain antibodies as defined in one of the claims 1 to 25 for use in the diagnosis and / or therapy of cancer in vivo.

27. A kit for use in the diagnosis and / or therapy of cancer in vivo comprising i. at least one single domain antibody as defined in one of the claims 1 to 25 and ii. at least one pharmaceutically acceptable excipient or a solution thereof.

28. The kit for use in the diagnosis and / or therapy of cancer in vivo according to claim 27, wherein the at least one single domain antibody is associated with at least one chelator and / or a prosthetic group.

29. A single domain antibody which binds to human CD7 comprising an amino acid sequence according to one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13 or at least 90% sequence identity with one of the sequences selected from SEQ ID No. 2 to SEQ ID No. 13.

30. The single domain antibody according to claim 29, wherein the single domain antibody comprises an amino acid sequence according to one of the sequences selected from SEQID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 13, or an amino acid sequence with at least 90%, such as at least 95%, particularly at least 99% sequence identity within the framework regions of these sequences and optionally 100% sequence identity in the CDRs.

31. The single domain antibody of claim 30, wherein the single domain antibody is associated with at least one of a detectable label, a chelator, a prosthetic group, or a therapeutically and / or pharmacologically active agent.

32. The single domain antibody of claim 29, 30 or 31 , covalently bound to a chelator which forms a coordination complex with a radioactive isotope, optionally68Ga.

33. The single domain antibody of or more of claims 29 to 32, wherein the single domain antibody is associated with a detectable label and binds human CD7 with an affinity, the single domain antibody having a molecular weight and the detectable label having a radioactive half-life, wherein a figure of merit defined as- affinity [nmol / L]x molecular weigh - -t [ / mol]xradioactive half-life [s] is at least 0.05 or at least 0.50[L / g*s],34. The single domain antibody according to one or more of claims 29 to 31 , wherein the single domain antibody comprises a chelator, and wherein the single domain antibody does not comprise a radioisotope.

35. A nucleic acid comprising a nucleic acid sequence encoding for the single domain antibody according to one or more of claims 29 to 34.

36. A pharmaceutical composition comprising at least one single domain antibody according to one or more of claims 29 or 34.

37. Use of the single domain antibody as defined in one of the claims 1 to 25 or 29 to 33, the pharmaceutical composition as defined in claim 26 or 36 and / or the kit as defined in claim 27 or 28 in the diagnosis and / or therapy of cancer, e.g. in vitro or ex vivo, or for the synthesis of radioactive tracers and / or contrast agents.

38. A single domain antibody for use in therapy of cancer, the single domain antibody binding to human CD7 with an affinity of less than 15 nM, the antibody having a size of at most 16 kDa, such as in the range of 11 kDa to 16 kDa, wherein the antibody is associated with adetectable label, which detectable label is a radioactive isotope with a half-life of at most750 minutes, wherein the therapy includes administering immunotherapy to a patient, administering the single domain antibody to the patient, performing imaging of immune cells, and evaluating a response pattern of the immunotherapy.

39. A single domain antibody, that binds to human CD7 for use in cancer therapy, wherein the single domain antibody is associated with a detectable label, the detectable label being a radioactive isotope, wherein the cancer therapy comprises administering cancer therapy, such as immunotherapy, to a patient in need thereof, administering an effective amount of the single domain antibody to the patient, monitoring a presence and / or amount of immune cells expressing human CD7 at the tumor site, optionally, deciding whether the cancer therapy is effective based on the presence and / or amount of the immune cells at the tumor site, further wherein the single domain antibody binds human CD7 with an affinity, the single domain antibody having a molecular weight and the detectable label having a radioactive half-life, wherein a figure of merit defined as i is at least 0.05 or at least 0.50 affinity [nmol / L]x molecular weight [ / mol]xradioactive half-life [s][L / g*s],40. A single domain antibody for use in the diagnosis and / or therapy of cancer, comprising administering to a subject an amount of the single domain antibody which binds to human CD7, wherein the single domain antibody is associated with a first radioisotope, performing imaging by detecting the presence of the radioisotope in the subject, administering to the subject an amount of the single domain antibody which binds to human CD7, wherein the single domain antibody is associated with a second radioisotope,wherein the cancer cells express human CD7.41 . A single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 40, wherein the second radioisotope is selected from67Cu,47Sc,149Tb,161Tb,177Lu, 225Ac,211Ac,212Pb,223Ra and combinations thereof, and / or the first radioisotope is selected from68Ga,18F,89Zr,99mTc,61Cu,64Cu,43Sc,44Sc,149Tb,152Tb,155Tb,123l,124l,213Bi and combinations thereof42. A single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 40 or 41 , wherein the first radioisotope and the second radioisotope are different isotopes of the same element, optionally of Cu, Tb or Sc.

43. The single domain antibody for use in the diagnosis and / or therapy of cancer according to claim 40, 41 or 42, wherein- the first radioisotope is61Cu and / or64Cu, and the second radioisotope is67Cu;- the first radioisotope is43Sc and / or44Sc, and the second radioisotope is47Sc; or- the first radioisotope is149Tb,152Tb and / or155Tb, and the second radioisotope is149Tb and / or161Tb.

44. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 38 to 43, wherein diagnosis and / or therapy includes determining the existence, localization and / or size of a tumor or metastasis of a CD7 positive cancer.

45. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 38 to 44, wherein the cancer is immune cell-derived, such as T cell- derived cancer.

46. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 38 to 45, wherein the cancer is selected from lymphomas, in particular T cell or NK lymphomas, or leukemia.

47. The single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 38 to 46, wherein the cancer is metastasized, in particular with solid metastases.

48. The single domain antibody according to one or more of claims 29 to 34, or the single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 1 to 25, or 38 to 47, wherein the binding of the single domain antibody to the target molecule CD7 does not affect the function of the immune cells, in particular T cell and / or NK cells.

49. The single domain antibody according to one or more of claims 29 to 34, or the single domain antibody for use in the diagnosis and / or therapy of cancer according to one or more of claims 1 to 25, or 38 to 47, wherein- the single domain antibody does not alter IFNy secretion of activated T cells; - the single domain antibody does not alter IL2, GM-CSF and / or TNF-a secretion of activated T cells; and / or- the single domain antibody does not have an influence on tumor growth.

Images