T cell tracking

Abstract

The disclosure concerns a polypeptide that comprises (a) a binding domain specific for histamine-succinyl-glycine (HSG) and (b) a membrane anchor that is capable of anchoring the binding domain in a plasma membrane. The disclosure also concerns a cell expressing the polypeptide of the disclosure of the surface. The disclosure further concerns a nucleic acid encoding the polypeptide of the disclosure, a vector comprising the nucleic acid, and a method for producing a cell of the disclosure. In addition, the disclosure concerns a method for treating a disease in an individual comprising administering the cell of the disclosure to the individual and a related medical use, a method of tracking the cell in vivo, and related medical uses.

Description

[0001] T CELL TRACKING

[0002] FIELD OF THE DISCLOSURE

[0003] The disclosure concerns a polypeptide that comprises (a) a binding domain specific for histamine-succinyl-glycine (HSG) and (b) a membrane anchor that is capable of anchoring the binding domain in a plasma membrane. The disclosure also concerns a cell expressing the polypeptide of the disclosure of the surface. The disclosure further concerns a nucleic acid encoding the polypeptide of the disclosure, a vector comprising the nucleic acid, and a method for producing a cell of the disclosure. In addition, the disclosure concerns a method for treating a disease in an individual comprising administering the cell of the disclosure to the individual and a related medical use, a method of tracking the cell in vivo, and related medical uses.

[0004] BACKGROUND

[0005] Cell-based therapies and immunotherapies (such as adoptive T cell transfer, tumour infiltrating lymphocyte (TIL) reinfusion and CAR T cell therapy) are becoming important components in the toolkit against diseases such as cancer. However, there are several barriers to effective treatment, such as the potential for life-threatening toxicities, poor efficacy, antigen escape, restricted trafficking, and limited tumour penetration. To understand and address these barriers, it is important to non-invasively track therapeutic cells administered to an individual over an extended period of time to capture the kinetics of their distribution, expansion, and survival. Nuclear imaging methods such as positron emission tomography (PET) or single photon emission tomography (SPECT) are ideally suited to such tracking: due to the unlimited tissue penetration of gamma radiation, the whole-body distribution of radioactively labelled cell populations can be visualized and quantified with very high sensitivity.

[0006] To image therapeutic cells using methods such as PET and SPECT, it is necessary to radiolabel the cells. Two approaches are employed in the art. In the first approach, a radiolabel is directly introduced to the cell ex vivo. For instance, radiometal complexes such asn iIn- or89Zr-oxine may passively accumulate inside the cell. However, the radiolabel disadvantageously becomes diluted as labelled cells divide, such that the label is lost. Furthermore, cellular efflux of the radiolabel in vivo can result in its uptake and retention in other tissues and organs, leading to an unwanted non-specific background signal.

[0007] The second approach uses so-called indirect labelling, in which the cell is modified to express a reporter gene. Reporter-expressing cells can then be specifically targeted using a radioligand that binds specifically to the reporter. Stable expression of the reporter gene allows cells to be observed over their entire lifetime, providing that radioligand is supplied. In addition, as the reporter gene is passed on during cell division, it is possible to image expanding cell populations and the signal intensity increases with growing cell numbers.

[0008] Several reporter genes have been reported for indirect labelling. For instance, physiologically expressed (and thus non-immunogenic) proteins such as the somatostatin receptor 2 (hsst2), prostate specific membrane antigen (PSMA) and the human sodium iodide symporter (hNIS) have been used as reported. However, in each case, physiological expression of the reporter leads to unwanted “on-target-off-site” tracer accumulation and thus considerable imaging background, which interferes with accurate cell detection and quantification. Bacterial or viral proteins have also been used as reporter genes, but their immunogenicity is associated with safety concerns. The same applies to a radiohaptenbinding mouse scFv.

[0009] Improved reporters are therefore required for in vivo imaging of cells by nuclear imaging methods.

[0010] SUMMARY OF THE DISCLOSURE

[0011] The present inventors have developed an improved reporter that may be used in the for in vivo imaging of cells by nuclear imaging methods. The reporter is a polypeptide that comprises (a) a binding domain specific for histamine-succinyl-glycine (HSG) and (b) a membrane anchor that is capable of anchoring the binding domain in a plasma membrane. The genome of cells to be imaged is modified to encode the polypeptide. The modified cells are administered to an individual, such as a human individual. The polypeptide is expressed by the cells and their progeny, and localizes to the cell surface. When a radioligand (such as a radiolabeled hapten) comprising HSG is administered to the individual, it binds to the binding domain of the polypeptide. In this way, the modified cells become radiolabeled and can be imaged by nuclear imaging (also known as radionuclide imaging) such as PET or SPECT.

[0012] The reporter of the disclosure is advantageous for a number of reasons. Firstly, the reporter is not expressed physiologically. Therefore, the reporter should only be expressed in the cells that it is desired to image. The reporter should not be expressed elsewhere in the body. Therefore, there should not be any “on-target-off-site” accumulation of the radiolabelled hapten. Imaging background is therefore minimised.

[0013] Secondly, the reporter is non-immunogenic. For instance, the reporter may be non- immunogenic in humans. To achieve this, both the binding domain and the membrane anchor may be formed from human or humanised components. Suitable components are already well-known in the art, and include the h679 scFv which is a humanised binding domain specific for HSG.

[0014] Thirdly, radioligands capable of binding to the binding domain are already known and established in the clinic. They are therefore ready for use in the approach of the disclosure. Such radioligands include, for example, IMP -288.

[0015] Fourthly, use of a binding domain specific for HSG allows numerous different radioligands to be used interchangeably, should this be desired. A radioligand may therefore be selected by a user to suit their particular needs, for instance based on convenience, radionuclide characteristics and availability, radionuclide-specific coordination chemistry, cost-effectiveness (SPECT vs PET), or clinical factors such as patient dosimetry. This is because any radioligand comprising HSG is compatible with the reporter. The binding domain will bind to any radioligand that comprises HSG. The identity of the radiolabel comprised in the radioligand, or of a chelator used to couple the radiolabel to HSG does not adversely affect the affinity with which the binding domain binds to HSG comprised in the radioligand. Indeed, certain HSG-comprising radioligands such as TRAP and DOTPI are capable of multimerization, which may boost the sensitivity of target cell detection by providing a dramatically higher apparent affinity for the reporter.

[0016] Accordingly, the disclosure provides:

[0017] - a polypeptide that comprises (a) a binding domain specific for histamine-succinyl-glycine (HSG) and (b) a membrane anchor that is capable of anchoring the binding domain in a plasma membrane;

[0018] - a cell expressing the polypeptide of the disclosure on its surface; - a nucleic acid encoding the polypeptide of the disclosure;

[0019] - a vector comprising the nucleic acid of the disclosure;

[0020] - a method for producing the cell of the disclosure, comprising: (i) transfecting or transducing a cell with the nucleic acid of the disclosure; and (ii) expressing the polypeptide encoded by the nucleic acid in the cell;

[0021] - a method of treating a disease in an individual, comprising administering the cell of the disclosure to the individual;

[0022] - a method of tracking a cell in vivo, comprising: (i) administering the cell of the disclosure to an individual; (ii) administering radioligand comprising HSG to the individual; and subsequently (iii) using nuclear imaging to determine the location of cells binding the radioligand within the individual; and

[0023] - the cell of the disclosure for use in a method of the disclosure.

[0024] DESCRIPTION OF THE FIGURES

[0025] Figure 1: Structure of IMP -288. The ligand is based on a metabolically stable D- peptide backbone with a C-terminal amide; the two HSG-units attached to the backbone are responsible for scFv binding, and the DOTA chelator allows radiolabelling with68Ga3+.

[0026] Figure 2: cross-bridged DOTA-dimer, showing use of DOTA as a bridging unit between two monomers. An analogue of IMP -288.

[0027] Figure 3: TRAP-trimer (TRAP(IMP-288)3). A further analogue of IMP-288.

[0028] Figure 4: mas3-IMP288. A further analogue of IMP-288.

[0029] Figure 5: Schematic representation of the retroviral vector design for cell transduction with HSG-recl.

[0030] Figure 6: A: Detection of surface expression of HSG-recl as well as c-Myc and Thy 1.1 transduction markers in WT Jurkat cells and Jurkat cells transduced with HSG-recl using different amount of virus (300, 100, 30, 10 and 3 pL) using flow cytometry. Data are given in MFI (mean fluorescence intensity) and are means ± SD (n=2). B: Binding of [68Ga]IMP-288 (0.5 nM) after 30 min incubation at RT, 37° and 4°C to WT Jurkat cells and Jurkat cells transduced with HSG-recl using different amount of virus (300, 100, 30 and 10 pL) in the absence (total binding) and presence of IpM unlabeled IMP-288 (nonspecific binding). Data are given in % of added dose, normalized to 100.000 live cells (mean ± SD, n=3). Figure 7: A: Small animal PET / CT phantom study (96-well-plate, maximum intensity projection) of different numbers of Jurkat cells (Y scale) with different transduction levels (X scale) preincubated with 1 nM [68Ga]IMP-288 for 30 min at 37° C and resuspended in a standard volume of 100 pL PBS. B: Binding of [68Ga]IMP-288 (1 nM) after 30 min incubation at 37° C to WT Jurkat cells and Jurkat cells transduced with HSG-recl using different amount of virus (10, 30 and 100 pL). Data are given in % of added dose, normalized to 500.000 live cells (mean ± SD, n=3).

[0031] Figure 8: Schematic representation of the lentiviral vector design for cell transduction with HSG-rec2.

[0032] Figure 9: A: Flow cytometry analysis of HSG-rec2 transduction efficiency in Jurkat cells using different amounts of lentivirus for transduction. Expression of HSG-rec2 is presented in % of NGFR expression. B: Binding of [68Ga]IMP-288 (1 nM) to Jurkat cells transduced with HSG-rec2 are shown. Here, different levels of HSG-rec2 expression were obtained by “diluting” HSG-rec2 transduced Jurkat cells with WT Jurkat cells to the given percentage of transduced cells. All cells were incubated for 30 min at 37°C. Data are given in % of added dose, normalized to 500.000 live cells (mean ± SD, n=3).

[0033] Figure 10: Schematic representation of the design of the in vivo study using the subcutaneous HSG-rec2 Jurkat tumor model.

[0034] Figure 11: A: [68Ga]IMP-288 small animal PET / CT (MIP (maximum intensity projection), Ih p.i., 0.5 nmol ligand) of an NSG mouse on day 1 after subcutaneous injection of 10 Mio HSG-rec2 transduced Jurkat cells on the right shoulder (left) in comparison to a naive NSG mouse (right). B: [68Ga]IMP-288 small animal PET / CT (MIP, Ih p.i., 0.5 nmol ligand) of NSG mice on day 5 after subcutaneous injection of 10 Mio HSG-rec2 transduced Jurkat cells on the right shoulder. C: [68Ga]IMP-288 small animal PET / CT (MIP, Ih p.i., 0.5 nmol ligand) of NSG mice on day 14 after subcutaneous injection of 5 Mio WT Jurkat cells on the right shoulder (negative control).

[0035] Figure 12: [68Ga]IMP-288 PET / CT (MIP, Ih p.i., 0.5 nmol ligand) of two NSG mice (mouse 1 = ml, mouse 2 = m2) 28 days after i.v. tail vein injection of 10 Mio HSG- rec2 transduced Jurkat cells (left). The corresponding transversal and sagittal slices of the liver and spine regions of mouse 2 are shown on the right.

[0036] Figure 13: [68Ga]Ga-IMP-288 PET (MIP, Ih p.i., 0.5 nmol ligand) of a NSG mouse (mouse 2, also see Figure 12) 28 days after i.v. tail vein injection of 10 Mio HSG- rec2 transduced Jurkat cells (left). The corresponding coronal, transversal and sagittal slices of the liver region and photography of the resected liver are shown on the right. Black arrows indicate only some of the multiple HSG-rec2 positive Jurkat liver lesions responsible for the inhomogeneous hepatic uptake of [68Ga]Ga-IMP-288.

[0037] Figure 14: A: Flow cytometry analysis of cMyc transduction efficiency in primary T cells after transduction for 24h. Expression of cMyc is expressed in percent of positive cells (of total). B: Flow cytometry analysis of Thy 1.1 transduction efficiency in primary T cells after transduction for 24h. Thy 1.1 expression is expressed in percent of positive cells (of total). C: Binding of [68Ga]Ga-IMP-288 (0.5 nM) to HSG-recl transduced CD8+ and CD4+ human primary T cells from three different human donors and to the corresponding untransduced control donor T cells. All cells were incubated for 30 min at 37°C. Data are given in % of added dose, normalized to 1 Mio live cells (mean ± SD, n=3).

[0038] Figure 15: Binding of [68Ga]Ga-IMP-288 (0.5 nM) to HSG-recl transduced CD8+ and CD4+ human primary T cells from a human donor and to the corresponding untransduced control donor T cells. All cells were incubated for 30 min at 37°C. Data are given in % of added dose, normalized to 1 Mio live cells (mean ± SD, n=3).

[0039] Figure 16: In vivo [68Ga]Ga-IMP288 PET / CT (0.5 nmol [68Ga]Ga-IMP288, Ih p.i.) of NSG mice 18h after i.v. injection of 7 Mio HSG-rec-1 transduced human donor T cells (CD4 and CD8, respectively; left panels). The right panels show the respective mice injected with the same number of untransduced primary donor T cells (negative controls).

[0040] Figure 17: Schematic representation of the retroviral vector design for cell transduction with HSG-rec3.

[0041] Figure 18: Binding of [68Ga]Ga-IMP-288 (0.5 nM) to HSG-rec3 transduced CD8+ and CD4+ human primary T cells from three different human donors and to the corresponding untransduced control donor T cells. Two different HSG-rec3 analogs were tested, i.e. the construct with (CH2CH3 + EGFR) and without (CH2CH3) truncated EGFR tag, respectively. All cells were incubated for 30 min at 37°C. Data are given in % of added dose, normalized to 1 Mio live cells (mean ± SD, n=3).

[0042] Figure 19: Serial [68Ga]IMP-288 PET / CT (MIP, Ih p.i., 0.5 nmol ligand) of representative PC3-Pip xenograft bearing NSG mice up to 27 days after i.v. tail vein injection of 2 Mio CTTR-T cells (group 4; upper panel) and PZ1-CAR T cells (group 5; lower panel). Mice receiving CTTR-only T cells had to be sacrificed due to tumor growth at day 14 post ACT (humane endpoint).

[0043] Figure 20: Serial [68Ga]IMP-288 PET / CT (MIP, Ih p.i., 0.5 nmol ligand) of representative PC3-Pip xenograft bearing NSG mice up to 27 days after i.v. tail vein injection of 2 Mio CTTR-PZ1-CART cells (group 3, upper panel). Upon complete regression on the tumors, mice were rechallenged by s.c. injection of 5 Mio PC3-Pip cells on the contralateral side and again imaged with [68Ga]IMP-288 PET / CT up to 14 days post rechallenge (lower panel).

[0044] Figure 21: A: Schematic structure of the new CTTR variants in a retroviral vector backbone. B: Flow cytometry analysis of cMyc and Thyl.l transduction efficiency in primary T cells (n=3) after transduction with the different CTTR variants at day 12. Both cMyc and Thyl. l expression are expressed in percent of positive cells (of total), with CD4+ T cells on the left and CD8+ T cells on the right respectively. C: Representative dot plot of orginal CTTR and NGFR-based CTTR, to show higher MFI and homogeneity on CTTR expression level in both CD4+ (top) and CD8+ (bottom) respectively.

[0045] Figure 22: Cellular update of68Ga-IMP288 by CD8+ and CD4+ human donor T cells transduced with the original CTTR construct or with NGFR-CTTR.

[0046] Figure 23: Comparative biodistribution of [68Ga]IMP-288 (white bars) and [68Ga]TRAP(IMP-288)3 (grey bars) in NSG mice (n=4 per group) bearing subcutaneous HSG-rec2 Jurkat xenografts. Data are given in %iD / g and represent means ± SD.

[0047] Figure 24: [68Ga]IMP-288, [68Ga]TRAP(ZMP-288)3and [68Ga]PentixaTher PET (MIP, Ih p.i., 0.08-0.1 nmol ligand) of NSG mice bearing HSG-rec2 -transduced Jurkat xenografts. Mice on the left side of panels (control) received the respective radioligand only, whereas mice on the right side (blocked) were coinjected with a 100-fold molar excess of unlabeled competitor ([natGa]IMP-288, [natGa]TRAP(IMP-288)3 and AMD3100, respectively). For all compounds, bladder activity was masked manually for all compounds to allow comparable scaling of images without spillover from the bladder.

[0048] DETAILED DESCRIPTION

[0049] It is to be understood that different applications of the disclosed methods and products may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0050] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0051] General definitions

[0052] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

[0053] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a steroid” includes “steroids”, reference to “an agonist” includes two or more such agonists, and the like.

[0054] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “polypeptide comprising a binding domain” should be interpreted to mean that the polypeptide contains the binding domain, but may also contain additional components.

[0055] In some aspects of the disclosure, the word “comprising” is replaced with the phrase “consisting of’. The term “consisting of’ is intended to be limiting. For example, the phrase “polypeptide consisting of a binding domain” should be understood to mean that the polypeptide contains the binding domain and no additional components.

[0056] The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0057] For the purpose of this disclosure, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide residues at nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, then the nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100). Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence has a certain percentage identity to SEQ ID NO: X, SEQ ID NO: X would be the reference sequence. For example, to assess whether a sequence is at least 80% identical to SEQ ID NO: X (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: X, and identify how many positions in the test sequence were identical to those of SEQ ID NO: X. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: X. If the sequence is shorter than SEQ ID NO: X, the gaps or missing positions should be considered to be nonidentical positions.

[0058] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0059] Polypeptide

[0060] The disclosure provides a polypeptide that comprises: (a) a binding domain specific for histamine-succinyl-glycine (HSG); and (b) a membrane anchor that is capable of anchoring the binding domain in a plasma membrane. The polypeptide is essentially an artificial receptor for HSG. Expression of the polypeptide on the surface of a cell allows the binding domain to engage with extracellular HSG. In this way, HSG becomes bound to the surface of the cell. Accordingly, when the HSG is comprised in a radioligand, the cell becomes tagged with the radioligand. This allows the cell to be tracked in vivo using nuclear imaging. The polypeptide may therefore be considered as a reporter for a cell type that expresses it. There are numerous advantages associated with use of the polypeptide as a reporter, as set out above.

[0061] The polypeptide may be of any length. For example, the polypeptide may be from 200 to 600 amino acids in length. The polypeptide may, for example, be from 250 to 550 amino acids in length, such as from 300 to 500, 300 to 400, 350 to 550, 320 to 370, or 500 to 550 amino acids in length.

[0062] Binding domain The binding domain may be any domain that is capable of binding to HSG. Preferably, the binding domain is a domain that specifically binds to HSG. In the context of the present application, specific binding for HSG refers to an ability to bind HSG with greater affinity than any other naturally-occurring protein.

[0063] The binding domain may be any type of binding domain. Numerous types of binding domain are known in the art. The binding domain may, for example, comprise or consist of an antibody or an antigen-binding fragment thereof. For instance, the binding domain may, for example, comprise or consist of a monoclonal antibody. The binding domain may comprise or consist of a single chain variable fragment (scFv). The binding domain may, for example, comprise or consist of a scFv-Fc. The binding domain may, for example, comprise or consist of a single-domain antibody, such as a camelid antibody, an artificial VHH fragment or an IgNAR.

[0064] Preferably, the binding domain comprises or consists of a single chain variable fragment (scFv). scFvs are known in the art, and are fusion proteins of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. In the context of the present disclosure, the scFv is typically in the VL-VH orientation. In other words, the scFv has the following arrangement from N-terminus to C- terminus: VL-linker-VH. However, the scFv may alternatively be in the VH-VL orientation. In other words, the scFv may have the following arrangement from N-terminus to C-terminus: VH-linker-VL.

[0065] Any linker may be used in the scFv. The linker may be of any length. The linker may, for example, be from about 8 to about 30 peptides in length, such as about 10 to about 25, about 12 to about 23, about 15 to about 20 peptides in length. The linker may, for example, be a GS linker, such as a GS linker of SEQ ID NO: 10 or SEQ ID NO: 63 (GEGGSGGGGSGGGES).

[0066] The binding domain may, for example, be human. That is, the binding domain may comprise an amino acid sequence that occurs in humans. The binding domain may, for example, be humanised. That is, the binding domain may comprise a modified non-human amino acid sequence, in which the modifications increase the similarity of the sequence to one that occurs in humans. Preferably, the binding domain comprises or consists of a humanised scFv. Use of a human or humanised binding domain advantageously avoids immunogenicity when the polypeptide is expressed in humans.

[0067] The binding domain may, for example, be capable of binding to isolated HSG and / or to HSG in the context of a larger molecule. That is, the binding domain may be capable of binding to isolated HSG. The binding domain may be capable of binding to HSG that exists as part of a larger molecule. The binding domain may be capable of binding to isolated HSG and to HSG that exists as part of a larger molecule. The larger molecule may, for example, be a hapten. The larger molecule may, for example, be IMP- 288 or an IMP-288 analogue. Thus, the binding domain may be capable of binding to IMP -288 or an IMP -288 analogue.

[0068] IMP -288 is a well-known bivalent hapten that is based on the HSG pseudopeptide. IMP -288 may be labelled using a variety of radionuclides. The structure of IMP-288 is shown in Figure 1. IMP-288 is based on a metabolically stable D-peptide backbone with a C -terminal amide (NH2-Tyr-Lys-Glu-Lys-CONH2). Two HSG units are attached to the backbone and may be bound by a binding domain that binds to HSG. A DOTA chelator is attached at the N-terminal of the peptide backbone, which allows radiolabelling with a radionuclide such as68Ga3+.

[0069] In the context of the present application, an IMP-288 analogue is a molecule that comprises the backbone and two HSG units of IMP-288. The presence of the HSG units permits the analogue to be bound by a binding domain that binds to HSG. An IMP-288 analogue may therefore be bound by a binding domain that is capable of binding to IMP- 288.

[0070] An IMP-288 analogue may, for example, be a multimer of IMP-288 such as a dimer that uses DOTA as a bridging unit (Figure 2), a TRAP-trimer (Figure 3) or a DOTPI-tetramer. In a dimer that uses DOTA as a bridging unit, the two monomers comprised in the dimer are bridged (i.e. linked or coupled) by DOTA. Further IMP-288 analogues include mas3-IMP288 (Figure 4), and versions of IMP -288 in which the DOTA chelator is switched for a different chelator. Chelators of use in IMP -288 analogues include copper chelators (such as NOTA, NOD AGA, NODA and TECB2A) and other radionuclide-specific chelators (such as DOTAM and Macropa).

[0071] Binding domains capable of binding to IMP -288 (and IMP-288 analogues) are known in the art and include h679. Details of h679, including its VH and VL sequences, are disclosed in Rossi et al. (Development of new multivalent-bispecific agents for pretargeting tumor localization and therapy. Clin Cancer Res (Suppl) 2003;9:3886S- 96S) and in WO 2016 / 201300, for example. h679 is a humanised scFv that comprises a light chain variable region of SEQ ID NO: 7, and a heavy chain variable region of SEQ ID NO:8. The light chain variable region of SEQ ID NO: 7 comprises a light chain CDR1 (LCDR1) of SEQ ID NO: 1 (LFNSRTRK), a light chain CDR2 (LCDR2) of SEQ ID NO: 2 (WASTRES) and a light chain CDR3 (LCDR3) of SEQ ID NO: 3 (TQVYYLC). The heavy chain variable region of SEQ ID NO:8 comprises a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4 (DLVKPGGSLK), a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 (LRQTPGKGLEWVATLSGDGDDI) and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6 (VRLGDWDFDVWGQ).

[0072] Accordingly, the binding domain may, for example, comprise or consist of an scFv that comprises (i) a light chain variable region that comprises a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2 and a light chain CDR3 (LCDR3) of SEQ ID NO: 3, and (ii) a heavy chain variable region that comprises a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6. The amino acid sequences between the various CDRs represent framework regions. Generally speaking, it is understood in the art that framework regions do not contribute to the antigen specificity of a binding domain. One or more of the VL framework regions may be modified with respect to those comprised in the light chain variable region of SEQ ID NO: 7, for instance to improve cell-surface stability of the polypeptide. One or more of the VH framework regions may be modified with respect to those comprised in the heavy chain variable region of SEQ ID NO: 8, for instance to improve cell-surface stability of the polypeptide.

[0073] The binding domain may, for example, comprise or consist of an scFv that comprises (i) a light chain variable region having at least 90% (such as at least 95%, at least 97%, at least 98% or at least 99%) identity to SEQ ID NO: 7, and (ii) a heavy chain variable region having at least 90% (such as at least 95%, at least 97%, at least 98% or at least 99%) SEQ ID NO:8. In this case, the light chain variable region preferably comprises a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2 and a light chain CDR3 (LCDR3) of SEQ ID NO: 3, and the a heavy chain variable region preferably comprises a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6. The binding domain may, for example, comprise or consist of an scFv that comprises (i) a light chain variable region of SEQ ID NO: 7, and (ii) a heavy chain variable region of SEQ ID NO:8.

[0074] Membrane anchor

[0075] The membrane anchor serves to anchor the binding domain in a plasma membrane. The membrane anchor may be any structure that fulfils this function. This allows the polypeptide to be expressed on the surface of a cell, where it can bind to extracellular HSG such as HSG comprised in a radioligand. Such binding decorates the cell with the radioligand, allowing it to be detected by nuclear imaging.

[0076] Typically, the membrane anchor may, for example, comprise one or more domains of one or more transmembrane proteins. A transmembrane protein is a protein which is inserted into and spans the width of the phospholipid bilayer that forms the plasma membrane. A transmembrane protein comprises one or more transmembrane domains, which are domains that locate within the phospholipid bilayer. A transmembrane protein may additionally comprise one or more extracellular domains and / or one or more intracellular domains. A transmembrane protein may be a transmembrane glycoprotein.

[0077] Any domain of a transmembrane protein may be comprised in the membrane anchor. The membrane anchor may, for example, comprise (i) one or more transmembrane domain of a transmembrane protein, (ii) one or more intracellular domain of a transmembrane protein, and / or (iii) one or more extracellular domain of a transmembrane protein. For instance, the membrane anchor may comprise (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii) and (iii).

[0078] Typically, the membrane anchor comprises at least a transmembrane domain of a transmembrane protein. For instance, the membrane anchor may comprise a transmembrane domain and an intracellular domain. The membrane anchor may comprise a transmembrane domain and an extracellular domain. The membrane anchor may comprise a transmembrane domain, an intracellular domain and an extracellular domain. An intracellular domain may also be known as an endodomain. An extracellular domain may also be known as an exodomain. The one or more domains comprised in the membrane anchor may be from any one or more transmembrane proteins. In one aspect of the disclosure, all of the domains comprised in the membrane anchor may be from the same transmembrane protein. In another aspect of the disclosure, the membrane anchor may comprise domains from two or more different transmembrane proteins.

[0079] One or more domains comprised in the membrane anchor may, for example, be from a transmembrane protein that is expressed by immune cells. For instance, the transmembrane protein may be from a T cell, a B cell, an NK cell, a dendritic cell, a macrophage, or a monocyte. Preferably, one or more domains comprised in the membrane anchor are from a transmembrane protein that is expressed by a T cell. The transmembrane protein may, for example, be a co-receptor for the T cell receptor (TCR). Such co-receptors include CD3, CD4 and CD8, for instance. Therefore, one or more domains comprised in the membrane anchor may be from CD3. One or more domains comprised in the membrane anchor may be from CD4. One or more domains comprised in the membrane anchor may be from CD8. The transmembrane protein may, for example, be T cell co-stimulatory molecule. Numerous T cell co-stimulatory molecules are known in the art. These include molecules belonging to the immunoglobulin superfamily (such as CD28, B7, ICOS, CD226 or CRTAM) and molecules belonging to the TNF receptor superfamily (such as 41-BB, 0X40, CD27, GITR, HVEM, CD40, BAFFR, BAFF and others). One or more domains from any of these co-stimulatory molecules may be comprised in the membrane anchor. In one aspect of the disclosure, the membrane anchor comprises one or more domains from CD28.

[0080] The membrane anchor may, for example, comprise (a) one or more domains from CD3, (b) one or more domains from CD4, (c) one or more domains from CD8, and / or one or more domains from CD28. For example, the membrane anchor may comprise: (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d).

[0081] When the membrane anchor comprises more than one domain from a transmembrane protein, two or more of the domains may be from the same transmembrane protein. For instance, the membrane anchor may comprise three domains from the same transmembrane protein. Alternatively, each of the domains comprises in the transmembrane anchor may be from a different transmembrane protein. By way of illustration, the membrane anchor may comprise: a transmembrane domain only; a transmembrane domain and an intracellular domain from the same transmembrane protein; a transmembrane domain and an extracellular domain from the same transmembrane protein; a transmembrane domain, an intracellular domain and an extracellular domain from the same transmembrane protein; a transmembrane domain from a first transmembrane protein and an intracellular domain from a second transmembrane protein; a transmembrane domain from a first transmembrane protein and an extracellular domain from a second transmembrane protein; a transmembrane domain and an intracellular domain from a first transmembrane protein and an extracellular domain from a second transmembrane protein; a transmembrane domain and an extracellular domain from a first transmembrane protein and an intracellular domain from a second transmembrane protein; or a transmembrane domain from a first transmembrane protein, an intracellular domain from a second transmembrane protein and an extracellular domain from a third transmembrane protein; wherein the first and second (and, if relevant, third) transmembrane proteins differ from each other. In these cases, each transmembrane protein may be any transmembrane protein defined above. By way of non-limiting example, each transmembrane protein may be CD3 (such as CD3Q, CD4, CD8 or CD28, for instance.

[0082] In one aspect of the disclosure, the membrane anchor may comprise an extracellular domain from a transmembrane protein, a transmembrane domain from a transmembrane protein and an intracellular domain from a transmembrane protein. In this case wherein two or three of the exodomain, transmembrane domain and intracellular domain are from the same transmembrane protein. The transmembrane protein may, for example, be CD8, CD28, CD3 or CD4. Preferably, the transmembrane protein is CD8 or CD28. In addition to one or more domains from a transmembrane protein, the membrane anchor may comprise one or more domains from an immunoglobulin, such as IgG. The membrane anchor may, for example, comprise: a transmembrane domain from a transmembrane protein, and a domain from an immunoglobulin; a transmembrane domain from a transmembrane protein, an intracellular domain from a transmembrane protein, and a domain from an immunoglobulin; a transmembrane domain from a transmembrane protein, an extracellular domain from a transmembrane protein, and a domain from an immunoglobulin; or a transmembrane domain from a transmembrane protein, an intracellular domain from a transmembrane protein, an extracellular domain from a transmembrane protein, and a domain from an immunoglobulin;

[0083] The membrane anchor may, for example, comprise a CD8a exodomain, such as the CD8a hinge (SEQ ID NO: 11). The membrane anchor may, for example, comprise a CD8 transmembrane domain. The membrane anchor may, for example, comprise a CD8 endodomain. The membrane anchor may, for example, comprise a CD28 exodomain. The membrane anchor may, for example, comprise a CD28 transmembrane domain. The membrane anchor may, for example, comprise a CD28 endodomain. The membrane anchor may, for example, comprise a CD4 exodomain. The membrane anchor may, for example, comprise a CD4 transmembrane domain. The membrane anchor may, for example, comprise a CD4 endodomain. The membrane anchor may, for example, comprise a CD3 (e.g. CD3Q exodomain. The membrane anchor may, for example, comprise a CD3 (e.g. CD3Q transmembrane domain. The membrane anchor may, for example, comprise a CD3 (e.g. CD3Q endodomain.

[0084] The membrane anchor may, for example, comprise a CD8a exodomain, a CD8 transmembrane domain, and a CD8 endodomain. This is the case for the HSG-recl polypeptide disclosed herein. The membrane anchor may, for example, comprise a CD8a exodomain, a CD28 transmembrane domain, and a CD28 endodomain. This is the case for the HSG-rec2 polypeptide disclosed herein. The membrane anchor may, for example, comprise an IgG CH2CH3 region, a CD8 transmembrane domain, and a CD8 endodomain. This is the case for the HSG-rec3 polypeptide disclosed herein. The membrane anchor may, for example, comprise a GSlong linker, a CD8a exodomain, a CD8 transmembrane domain, and a CD8 endodomain wherein, in the polypeptide, the linker lies in between the binding domain and the CD8a exodomain of the membrane anchor. This is the case for the construct 3 polypeptide disclosed herein. The membrane anchor may, for example, comprise a tNGFR exodomain, NGFR transmembrane domain, and a tNGFR endodomain. This is the case for the construct 5 polypeptide disclosed herein. NGFR refers to nerve growth factor receptor.

[0085] The membrane anchor may, for example, comprise or consist of a CD8a exodomain that comprises or consists of SEQ ID NO: 11. The membrane anchor may, for example, comprise or consist of a CD8 transmembrane domain that comprises or consists of SEQ ID NO: 12. The membrane anchor may, for example, comprise or consist of a CD8 endodomain domain that comprises or consists of SEQ ID NO: 13, which is a truncated CD8 endodomain. The membrane anchor may, for example, comprise or consist of a CD28 transmembrane domain that comprises or consists of SEQ ID NO: 15. The membrane anchor may, for example, comprise or consist of a CD28 endodomain domain that comprises or consists of SEQ ID NO: 16. The membrane anchor may, for example, comprise or consist of a CH2CH3 region that comprises or consists of SEQ ID NO: 18. The membrane anchor may, for example, comprise or consist of a GS long linker that comprises or consists of SEQ ID NO: 10. The membrane anchor may, for example, comprise or consist of a GS long linker that comprises or consists of SEQ ID NO: 63. The membrane anchor may, for example, comprise or consist of a tNGFR exodomain that comprises or consists of SEQ ID NO: 45. The membrane anchor may, for example, comprise or consist of a NGFR transmembrane domain that comprises or consists of SEQ ID NO: 46. The membrane anchor may, for example, comprise or consist of a tNGFR endodomain that comprises or consists of SEQ ID NO: 47.

[0086] In one aspect of the disclosure, the membrane anchor comprises a single-pass transmembrane region from a transmembrane protein. In this case, the transmembrane protein may be EGFR for example. The membrane anchor may further comprise a juxtamembrane sequence. The juxtamembrane sequence is typically short, for instance 3 to 10, 3 to 7 or 3 to 5 amino acids in length. The juxtamembrane sequence may, for example, comprise or consist of 3xArg.

[0087] The polypeptide of the disclosure provides means for decorating the surface of a cell that expresses the polypeptide with a radioligand, such that the cell may be detected by nuclear imaging. Accordingly, it is not important that the polypeptide triggers any intracellular signalling event when the binding domain binds to HSG. Indeed, it is preferable that the polypeptide does not lead to intracellular signalling when HSG is engaged. Preferably, therefore, the polypeptide is devoid of signalling function. This may, for example, be conferred by use of a membrane anchor that is itself devoid of signalling function. That is, the membrane anchor may be incapable of triggering intracellular signalling events. The membrane anchor may be inert.

[0088] One or more domains comprised in the membrane anchor may be modified relative to a wild-type counterpart in order to render the membrane anchor inert. For example, one or more of the domains may be a non-functional substitution variant of a wild-type counterpart. One or more of the domains may be a non-function truncation mutant of a wild-type counterpart.

[0089] Linker

[0090] The polypeptide may comprise a linker between the binding domain and the membrane anchor. Any type or length of linker may be used. Suitable linkers are known in the art.

[0091] The linker may, for example, link a transmembrane domain comprised in the membrane anchor to part of the binding domain (e.g. a VH or VL of a scFv).

[0092] The linker may, for example, be part of the membrane anchor. Thus, the linker may comprise one or more domains from a transmembrane protein. For instance, the linker may comprise an exodomain comprised in the membrane. Exodomains that may function as a linker include a CD8a exodomain (e.g. CD8a hinge), a CD28 exodomain (e.g. CD28 hinge), a CD4 exodomain (e.g. CD4 hinge), and CD3 exodomain. Preferably, a CD8a exodomain (e.g. CD8a hinge) present in the polypeptide links the membrane anchor to the binding domain. However, a CD28 exodomain (e.g. CD28 hinge), a CD4 exodomain (e.g. CD4 hinge), or CD3 exodomain present in the polypeptide may link the membrane anchor to the binding domain.

[0093] Alternatively, the linker may be derived from a different type of molecule. For instance, the linker may be from an immunoglobulin such as IgG. The linker may, for example, comprise an IgG CH2CH3 region (CH2CH3 hinge) such as that of SEQ ID NO: 18. The linker may, for example, comprise an IgG CH3 region (CH3 hinge). The linker may, for example, comprise a GS linker, such as a GS linker of SEQ ID NO: 10 or SEQ ID NO: 63.

[0094] Additional components

[0095] One or more additional components may be present in the polypeptide.

[0096] The polypeptide may, for example, comprise one or more components that promote stability. For instance, the polypeptide may comprise an extracellular DNAX-activating protein 10 (DAP 10) region. DAP 10 has previously been applied in this way in the STOPCAR design (Giordano- Attianese et al., A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol. 2020; 38: 426-432).

[0097] The polypeptide may, for example comprise a truncated cell surface protein that is exogenous with respect to a cell expressing the polypeptide. In other words, the polypeptide may comprise a truncated cell surface protein that is not normally expressed by cells of the type into which the polypeptide is introduced. By including such truncated cell surface protein, the cell can be decorated with marker. The marker may, for example, be used to identify or isolate the cells, for instance by using a binding molecule that is specific for the marker. Truncation of the cell surface protein may ensure that the protein is inert i.e. does not have its usual biological function. The truncated cell surface protein may, for example, be from CD 19, CD20, CD34 or NGFR. The truncated cell surface protein may, for example, be fused to the binding domain of the polypeptide.

[0098] Exemplary polypeptides

[0099] The production and function of certain exemplary polypeptides is considered in the Examples. Exemplary polypeptides of the disclosure include HSG-recl, HSG-rec2 and HSG-rec3. The amino acid sequences of HSG-recl, HSG-rec2 and HSG-rec3 are set out in Table 1 below.

[0100] Table 1

[0101]

[0102]

[0103] The polypeptide of the disclosure may, for example, comprise or consist of a sequence comprising, from N-terminal to C-terminal, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. For instance, the polypeptide may comprise or consist of SEQ ID NO: 9. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 9. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 9 and comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0104] The polypeptide of the disclosure may, for example, comprise or consist of a sequence comprising, from N-terminal to C-terminal, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 15 and SEQ ID NO: 16. For instance, the polypeptide may comprise or consist of SEQ ID NO: 14. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 14. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 14 and comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0105] The polypeptide of the disclosure may, for example, comprise or consist of a sequence comprising, from N-terminal to C-terminal, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 12 and SEQ ID NO: 13. An intervening A residue may be found between SEQ ID NO: 8 and SEQ ID NO: 18, but this is optional. For instance, the polypeptide may comprise or consist of SEQ ID NO: 17. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 17. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 17 and comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6. In any of these cases, the polypeptide may or may not comprise an intervening A residue may be found between sequences corresponding to SEQ ID NO: 8 and SEQ ID NO: 18.

[0106] The polypeptide of the disclosure may, for example, comprise or consist of a sequence comprising, from N-terminal to C-terminal, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. For instance, the polypeptide may comprise or consist of SEQ ID NO: 43. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 43. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 43 and comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0107] The polypeptide of the disclosure may, for example, comprise or consist of a sequence comprising, from N-terminal to C-terminal, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47. For instance, the polypeptide may comprise or consist of SEQ ID NO: 44. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 44. The polypeptide may comprise or consist of an amino acid sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 44 and comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0108] Cell The disclosure provides a cell expressing the polypeptide of the disclosure on its surface. The cell may be any type of cell. Preferably, the cell is a therapeutic cell.

[0109] The cell may, for example, be an immune cell. For instance, the cell may be a T cell, B cell, NK cell, dendritic cell, macrophage, or monocyte. Preferably, the cell is a T cell. The T cell may be a therapeutic T cell, such as a CAR T cell.

[0110] The cell may, for example, be a cell that is capable of repairing damaged tissue. The cell may, for example, be a cell that has regenerative capacity. For instance, the cell may be a stem cell, or another regenerative cell type. Stem cells include pluripotent stem cells, induces pluripotent stem cells, embryonic stem cells, somatic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells.

[0111] The cell may be of any species. Preferably, the cell is a mammalian cell. Most preferably, the cell is a human cell.

[0112] In any case, the cell may comprise a nucleic acid that encodes the polypeptide. Exemplary nucleic acids are described below. The nucleic acid may, for example, be incorporated to the genome of the cell. The nucleic acid may, for example, be located elsewhere in the cell. For instance, the nucleic acid may be comprised in a vector that is comprised in the cell. Vectors are described in detail below.

[0113] Nucleic acid

[0114] The disclosure provides a nucleic acid encoding the polypeptide of the disclosure. The nucleic acid may, for example, be provided to or comprised in a cell, to allow the cell to express the polypeptide of the disclosure.

[0115] The nucleic acid may comprise DNA and / or RNA. For instance, the nucleic acid may comprise DNA. The nucleic acid may comprise RNA. The nucleic acid may comprise DNA and RNA.

[0116] The nucleic acid may comprise any nucleotide sequence that encodes any polypeptide of the disclosure. Due to degeneracy in the genetic code, a single polypeptide of the invention may be encoded by multiple nucleotide sequences. The identity of the particular nucleotide sequence of the nucleic acid is not important. What is important is that the nucleotide sequence encodes the polypeptide of the disclosure. The nucleic acid may, for example, encode one of HSG-recl, HSG-rec2 and HSG- rec3 disclosed herein. Exemplary nucleic acid sequences encoding HSG-recl, HSG-rec2 and HSG-rec3 are provided in Table 2 below. Table 2

[0117]

[0118] The nucleic acid of the disclosure may, for example, comprise or consist of a sequence comprising, from 5’ to 3’, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25. For instance, the nucleic acid may comprise or consist of SEQ ID NO: 19. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 19. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 19 and comprising a nucleotide sequence that encodes a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a nucleotide sequence that encodes a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a nucleotide sequence that encodes a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a nucleotide sequence that encodes a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a nucleotide sequence that encodes a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a nucleotide sequence that encodes a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0119] The nucleic acid of the disclosure may, for example, comprise or consist of a sequence comprising, from 5’ to 3’, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 28. For instance, the nucleic acid may comprise or consist of SEQ ID NO: 26. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 26. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 26 and comprising a nucleotide sequence that encodes a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a nucleotide sequence that encodes a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a nucleotide sequence that encodes a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a nucleotide sequence that encodes a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a nucleotide sequence that encodes a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a nucleotide sequence that encodes a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0120] The nucleic acid of the disclosure may, for example, comprise or consist of a sequence comprising, from 5’ to 3’, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 24 and SEQ ID NO: 25. An intervening GCT sequence may be found between SEQ ID NO: 22 and SEQ ID NO: 30. For instance, the nucleic acid may comprise or consist of SEQ ID NO: 29. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 29. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 29 and comprising a nucleotide sequence that encodes a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a nucleotide sequence that encodes a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a nucleotide sequence that encodes a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a nucleotide sequence that encodes a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a nucleotide sequence that encodes a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a nucleotide sequence that encodes a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0121] The nucleic acid of the disclosure may, for example, comprise or consist of a sequence comprising, from 5’ to 3’, SEQ ID NO: 20, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61. For instance, the nucleic acid may comprise or consist of SEQ ID NO: 48. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 48. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 48 and comprising a nucleotide sequence that encodes a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a nucleotide sequence that encodes a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a nucleotide sequence that encodes a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a nucleotide sequence that encodes a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a nucleotide sequence that encodes a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a nucleotide sequence that encodes a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0122] The nucleic acid of the disclosure may, for example, comprise or consist of a sequence comprising, from 5’ to 3’, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52. For instance, the nucleic acid may comprise or consist of SEQ ID NO:49. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 49. The nucleic acid may comprise or consist of an nucleotide sequence having at least 90% (such as at least 95%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 49 and comprising a nucleotide sequence that encodes a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a nucleotide sequence that encodes a light chain CDR2 (LCDR2) of SEQ ID NO: 2, a nucleotide sequence that encodes a light chain CDR3 (LCDR3) of SEQ ID NO: 3, a nucleotide sequence that encodes a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a nucleotide sequence that encodes a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a nucleotide sequence that encodes a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

[0123] Vector

[0124] The disclosure provides a vector comprising the nucleic acid of the disclosure. As the vector comprises the nucleic acid of the disclosure, it encodes the polypeptide of the disclosure.

[0125] The vector may further encode one or more additional components. For example, the vector may encode a signal peptide. The signal peptide may, for example, be a hlFNb signal peptide, such as a hlFNb signal peptide of SEQ ID NO: 32.

[0126] The vector may encode a leader sequence. The leader sequence may, for example, be a CD8 leader sequence, such as a CD8 leader sequence of SEQ ID NO: 41.

[0127] The vector may encode one or more restriction enzyme sites. The restriction enzyme sites may, for example, comprise a BamHI site such as GGATCC, a Xhol site such as CTCGAGGG, a Mlul site such as ACGCGT, and / or a Sall site such as GTCGAC.

[0128] The vector may encode one or more tags. The vector may, for example, encode a cMyc tag such as a cMyc tag of SEQ ID NO: 33 or 62. The vector may, for example, encode a Thy 1.1 tag such as a Thy 1.1 tag of SEQ ID NO: 37. The vector may, for example, encode a NGFR tag, such as a NGFR tag of SEQ ID NO: 39.

[0129] The vector may, for example, encode EGFR. The vector may, for example, encode truncated EGFR, such as the truncated EGFR of SEQ ID NO: 42. The truncated EGFR may function as a marker of transduction efficiency. The truncated EGFR may be used as a suicide switch.

[0130] The vector may encode one or more other components, such as Furine and / or T2A. The furine may, for example, be a furine of SEQ ID NO: 35. The T2A may, for example, be a T2A of SEQ ID NO: 36.

[0131] The vector may, for example, be a viral vector. For instance, the vector may be a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus or a herpes simplex virus. Methods for producing and purifying such vectors are known in the art. Preferably, the vector is a retroviral vector, a lentiviral vector or an adenoviral vector. The lentivirus may be a modified HIV virus suitable for use in delivering genes. The viral vector may comprise a targeting molecule to ensure efficient transduction with the nucleic acid. The targeting molecule will typically be provided wholly or partly on the surface of the viral vector in order for the molecule to be able to target the virus to cells of interest. The viral vector is preferably replication deficient. Exemplary viral vectors are provided in Table 3 below. In any of the exemplary viral vectors, sequences indicated as “plasmid sequence” are optional and may either be included or excluded from the vector.

[0132] Table 3

[0133]

[0134]

[0135] Alternatively, the vector may, for example, be a non-viral vector. For instance, the vector may be a DNA plasmid, a naked nucleic acid, a nucleic acid complexed with a delivery vehicle, or an artificial virion. The non-viral vector may be a human artificial chromosome, as described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). When the non-viral vector is a nucleic acid complexed with a delivery vehicle, the delivery vehicle may be a liposome, virosome, or immunoliposome. Integration of a plasmid vector may be facilitated by a transposase such as sleeping beauty or PiggyBAC.

[0136] Method of producing a cell

[0137] The disclosure provides a method for producing the cell of the disclosure, comprising. The method comprises (i) transfecting or transducing a cell with the nucleic acid of the disclosure and (ii) expressing the polypeptide encoded by the nucleic acid in the cell.

[0138] The cell may, for example, be an immune cell. For instance, the cell may be a T cell, B cell, NK cell, dendritic cell, macrophage, or monocyte. Preferably, the cell is a T cell. The T cell may be a therapeutic T cell, such as a CAR T cell.

[0139] The cell may, for example, be a cell that is capable of repairing damaged tissue. The cell may, for example, be a cell that has regenerative capacity. For instance, the cell may be a stem cell, or another regenerative cell type. Stem cells include pluripotent stem cells, induces pluripotent stem cells, embryonic stem cells, somatic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells.

[0140] The cell may be of any species. Preferably, the cell is mammalian. More preferably, the cell is human.

[0141] In step (i), the cell is transfected or transduced with a nucleic acid of the disclosure. The term “transduction” may be used to describe virus-mediated nucleic acid transfer. The method may, for example, comprises contacting the cell with a vector of the disclosure. The vector may, for example, be a viral vector. Any of the aspects described above in connection with the vector may apply. Transduction may be in vitro or ex vivo.

[0142] The term “transfection” may be used to describe non-virus-mediated nucleic acid transfer. The cell may be transfected using any method known in the art. Transfection may be in vitro or ex vivo. Any vector capable of transfecting the cell may be used, such as conventional plasmid DNA or RNA transfection. Integration of a plasmid vector may be facilitated by a transposase such as sleeping beauty or PiggyBAC. The CRISPR / Cas9 platform may alternatively be used for transfection. A human artificial chromosome and / or naked RNA and / or siRNA may be used to transfect the cell with the nucleic acid sequence or nucleic acid construct. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptorrecognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817- 4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0143] The cell may be transfected or transduced under suitable conditions. The cell and vector may, for example, be contacted for between five minutes and ten days, preferably from an hour to five days, more preferably from five hours to two days and even more preferably from twelve hours to one day.

[0144] The nucleic acid sequence transduced or transfected into the cell gives rise to expression of the polypeptide of the disclosure in the cell.

[0145] Method of treatment and corresponding medical use

[0146] The disclosure provides a method of treating a disease in an individual, comprising administering the cell of the disclosure to the individual. The disclosure further provides the cell of the disclosure for use in a method of treating a disease in an individual, the method comprising administering the cell of the disclosure to the individual. This disclosure also provides use of the cell of the disclosure in a method for manufacturing a medicament for treating disease in an individual. The disease may be any disease that may be treated with a cell therapy. That is, the disease may be any disease that may be treated with a therapeutic cell. For example, the disease may be a disease that may be treated by cellular immunotherapy, i.e. by administration of a therapeutic immune cell. Such diseases are well-known in the art. The disease may, for example, be a cancer. The disease may, for example, be an infectious disease. The disease may, for example, be an immune-mediated disease, such as an autoimmune disease or allergy. Alternatively, the disease may be a disease that may be treated with a regenerative cellular therapy. That is, the disease may be a disease that may be treated by administering cells having regenerative or reparative capacity, such as stem cells. The disease may, for example, be a disease in which tissue is damaged. For example, the disease may be a disease in which cardiac, musculoskeletal, kidney, liver or lung tissue is damaged. The disease may, for example, be a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung disease. The disease may, for example, be a cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung injury.

[0147] Treatment of the disease may further comprise administering a radioligand comprising HSG to the individual. The radioligand may, for example, comprise one or more HSG units, such as two or more, three or more, four or more or five or more HSG units. The structure of an HSG unit is shown in Figure 1. In any case, administration of such a radioligand allows the cell of the disclosure to be tracked in vivo following administration. As explained above, the radioligand binds to the polypeptide of the disclosure, which is expressed on the surface of the cell of the disclosure. In this way, the surface of the cell becomes decorated with the radioligand, and the cell is detectable by nuclear imaging. Accordingly, treatment of the disease may further comprise tracking the cell in vivo by using nuclear imaging to determine the location of cells binding the radioligand within the individual. The nuclear imaging may, for example, be PET or SPECT. PET and SPECT are well-known in the art.

[0148] Administration of a radioligand comprising HSG to the individual may also have a therapeutic effect. In particular, the radioligand may be capable of irradiating a cell to which it binds. When the radioligand binds to the polypeptide of the disclosure, which is expressed on the surface of the cell of the disclosure, the proximity of the radioligand to the cell may be close enough to facilitate irradiation. Such irradiation may affect the function or longevity of the cell. For example, binding of the radioligand to the cell may affect proliferation of the cell. For instance, proliferation may be impaired. Binding of the radioligand to the cell may affect cytotoxicity of the cell. For instance, cytotoxicity may be impaired. Binding of the radioligand to the cell may affect longevity of the cell. For instance, longevity may be impaired. When the cell is a therapeutic cell, such as a therapeutic T cell (e.g. CAR T cell), impaired proliferation, cytotoxicity or longevity resulting from the irradiation may help to mitigate one or more adverse side effects of administering the therapeutic cell. For instance, toxicity associated with administering the therapeutic cell may be reduced.

[0149] The irradiation may alternatively promote the therapeutic function of a therapeutic cell, such as a therapeutic T cell (e.g. CAR T cell). For instance, irradiation may promote T cell effector function. Irradiation may promote proliferation of the cell. Irradiation may promote cytotoxicity of the cell. Irradiation may promote longevity of the cell. Promotion of therapeutic function by irradiation mediated by administration of a radioligand has been described in Yang el al. (2024); Low -dose targeted radionuclide therapy synergizes with CAR T cells and enhances tumor response,' Front. Immunol. , Sec. Cancer Immunity and Immunotherapy, Volume 15 - 2024.

[0150] The radioligand may, for example, comprise or consist of radiolabeled IMP -288. As explained above, IMP -288 is a well-known bivalent hapten that is based on the HSG pseudopeptide. The radioligand may, for example, comprise or consist of a radiolabeled IMP -288 analogue. As explained above, an IMP-288 analogue is a molecule that comprises the backbone and two HSG units of IMP-288. IMP -288 and IMP-288 analogues may routinely be labelled using a variety of radionuclides, such as68Ga3+, "mTc,64Cu,18F,124I,123I,44Sc,43Sc,155Tb,153Tb,67Cu,177Lu,161Tb,149Tb,225Ac or212Pb. Typically,68Ga3+, "mTc,64Cu,18F,124I,123I,44SC,43SC,155Tb or153Tb may be used for diagnostic imaging. Typically,67Cu,177Lu,161Tb,149Tb.225Ac or212Pb may be used for targeted radioligand therapy. The radioligand may, for example, comprise or consist of [68Ga]IMP- 288. The radioligand may, for example, comprise or consist of [99mTc]mas3-IMP288 or [99mTc]N4-IMP288. The radioligand may, for example, comprise or consist of [64 / 67Cu]NOTA-IMP288, [64 / 67Cu]NODAGA-IMP288, [64 / 67Cu]NODA-IMP288 or [64 / 67Cu] TECB2A-IMP288. The radioligand may, for example, comprise or consist of a dimer using DOTA as a bridging unit. The dimer may optionally be labeled with212Pb, or with any other radionuclide that forms stable complexes with a DOTA bridging unit. The radionuclides listed above (e.g.68Ga3+, "mTc,64Cu,18F,124I,123I,44Sc,43Sc,155Tb,153Tb,67Cu,177LU,161Tb,149Tb or225Ac) may, for instance, be suitable. The radioligand may, for example, comprise of a TRAP-trimer. The radioligand may, for example, comprise a DOTPI-tetramer. The radioligand may, for example, comprise a radionuclide-specific chelator, optionally DOTAM or Macropa.

[0151] Preferably, the radioligand does not comprise or consist of a binding molecule, such as an HSG-labelled binding molecule. In this context, a binding molecule refers to an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a scFv, a scFv-Fc, or a single-domain antibody (such as a camelid antibody, an artificial VHH fragment or an IgNAR).

[0152] In vivo cell tracking and corresponding medical use

[0153] The disclosure provides a method of tracking a cell in vivo, comprising: (i) administering the cell of the disclosure to an individual; (ii) administering radioligand comprising HSG to the individual; and subsequently (iii) using nuclear imaging to determine the location of cells binding the radioligand within the individual. The disclosure further provides the cell of the disclosure for use in a method of tracking a cell in vivo, the method comprising: (i) administering the cell to an individual; (ii) administering radioligand comprising HSG to the individual; and subsequently (iii) using nuclear imaging to determine the location of cells binding the radioligand within the individual.

[0154] The method may, for example, be performed for the purposes of research, for instance to determine the properties of a cell of the disclosure. The cell may, for example, be a potentially therapeutic cell. The properties may, for example, be therapeutic properties or potentially deleterious side effects.

[0155] The nuclear imaging may, for example, be PET or SPECT. PET and SPECT are well-known in the art.

[0156] The radioligand may, for example, comprise or consist of radiolabeled IMP -288. The radioligand may, for example, comprise or consist of a radiolabeled IMP -288 analogue. IMP-288 and IMP-288 analogues are described in detail above. Preferably, the radioligand does not comprise or consist of a binding molecule, such as an HSG-labelled binding molecule. In this context, a binding molecule refers to an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a scFv, a scFv-Fc, or a single-domain antibody (such as a camelid antibody, an artificial VHH fragment or an IgNAR).

[0157] EXAMPLES

[0158] The following Examples illustrate the invention.

[0159] EXAMPLE 1

[0160] Materials and methods

[0161] Cell culture

[0162] Human embryonic kidney 293 cells containing the SV40 T-antigen (HEK293T cells) (CRL-3216) and Jurkat cells (E6.1) were purchased from American Type Culture Collection (ATCC). Jurkat cells, both untransduced (WT) and engineered with HSG-rec 1 or HSG-rec2, were cultured in RPMI-1640 with Glutamax medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin 10’000 IU ml '-streptomycin 10’000 pg mF1(100X) (BioConcept), at 37 °C in a 5% CO2 atmosphere. Cell media were refreshed three times per week to maintain the cells in culture by removing half of the volume and replacing it by fresh medium or by doubling the total volume with fresh medium in the case of Jurkat cell expansion.

[0163] HEK293T cell transfection for Retrovirus and Lentivirus production

[0164] Virus production for transduction of Jurkat cells

[0165] Viral supernatant for Jurkat transduction was produced by seeding HEK293T cells in a 12-well plate at 0.65 * 106cells in 2 ml medium / well 5-6 h prior to transfection. HEK293T cell were transfected with 2 ug total DNA mixture of 1 pg pCL-Eco Retrovirus Packaging Vector (pCL ECO) and 1 pg pMSGV-cMyc-h679-Thyl. l tag transgene vector for HSG-recl retrovirus production or with 1 pg VSV glycoprotein vector (pVSVG) / Rev and Gag / Pol vector (R874) in addition to 1 pg pcRRL-cMyc-h679-NGFR tag transgene vector for HSG-rec2 Lentivirus production. DNA mixture was added to a mix of Turbofect (Thermo Fisher) and Optimem media (Invitrogen, Life Technologies) according to the manufacturer’s instructions, then incubated for 10-20 min at room temperature. All plasmids were purified using PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Life Technologies). After 24h, the medium was refreshed, and at 48h post-transfection, the viral supernatant was collected then used directly to transduce Jurkat cells.

[0166] Virus production for transduction of primary human T cells

[0167] For Examples 1 to 3, human primary T cells were transfected using ultracentrifuged retrovirus produced by seeding 107cells per T-150 tissue culture flask in 30 ml medium 24h prior to transfection. HEK293T cells were transfected using a transfection mixture that comprises plasmid DNA mix added to 180 pl of Turbofect and 3 ml of Optimem per each flask, then incubated for 30 min at room temperature. When the HEK293T cells were 80- 90% confluent at 24h, the medium was removed from T-150 flasks, and the transfection mixture was added on top of the cells and incubated for 1 min, followed by the addition of 31 ml of fresh medium. The viral supernatant was harvested at 48 h and at 72h posttransfection. Viral particles for each T-150 flask were concentrated by ultracentrifugation at 24,000 g for 2h at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 400pl of T cell medium. The lentivirus was then immediately aliquoted, snap-frozen, and stored at -80°C.

[0168] For subsequent experiments, human primary T cells were transfected using ultracentrifuged retrovirus produced by seeding 107cells per T-150 tissue culture flask in 20 ml medium 24h prior to transfection. HEK293T cells were transfected using a transfection mixture that comprises plasmid DNA and added to 100 pl of Turbofect and 3 ml of Optimem per each flask, then incubated for 30 min at room temperature. When the HEK293T cells were 80% confluent at 24h, the medium was removed from T-150 flasks, and the transfection mixture was added on top of the cells and incubated for 1 min, followed by the addition of 16.5 ml of fresh medium. The viral supernatant was harvested at 48 h and at 72h post-transfection. Viral particles for each T-150 flask were concentrated by ultracentrifugation at 24,000 g for 2h at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 400pl of T cell medium. The retrovirus was then immediately aliquoted, snap-frozen, and stored at -80°C.

[0169] For Examples 1 to 3, plasmid DNA mix for HSG-recl retrovirus production includes 40 pg of total DNA divided as 5 pg pVSVG, 15 pg of R874, and 20 pg of pMSGV-cMyc-h679-Thy 1.1 tag transgene vector, while the DNA mix for HSG-rec2 lentivirus production consists of 7 pg pVSVG, 18 pg of R874, and 15 pg of pcRRL-cMyc- h679-NGFR tag transgene vector. All plasmids were purified using PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Life Technologies).

[0170] For subsequent experiments, plasmid DNA mix for HSG-recl (and all retro- variants) retrovirus production includes 40pg of total DNA divided as 5 pg pVSVG, 15 pg of R874, and 20 pg of pMSGV-cMyc-h679-Thyl. l tag transgene vector, while the DNA mix for HSG-rec2 lentivirus production consists of 7 pg pVSVG, 18 pg of R874, and 15 pg of pcRRL-cMyc-h679-NGFR tag transgene vector. All plasmids were purified using PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Life Technologies).

[0171] Transduction of Jurkat cells with HSG-recl and HSG-rec2

[0172] To gene-engineer Jurkat cells with the h679 scFv, i.e. both the first-generation HSG-recl and the second-generation HSG-rec2 constructs, cells were resuspended at a concentration of 1 x 105cells per ml in addition to 8 pg / ml of Protamine Sulfate (Sigma- Aldrich) and seeded in 24-well plates of a final volume of 1 ml per well. Increased amounts of viral supernatants were then used to transduce the cells ranging from 3 pl to 10 pl, 30 pl, 100 pl and 300 pl as the highest volume. After 24h post-transduction, cell media volume was doubled for all conditions and then incubated at 37 °C in a 5% CO2 atmosphere.

[0173] Human Primary T cell isolation, activation, transduction and expansion

[0174] Isolation of human primary T cells

[0175] Human primary T cells were isolated from peripheral blood mononuclear cells (PBMCs), in the form of buffycoats, collected from healthy donors that had given their informed consent to the Interregional Blood Transfusion SRC of the Blood Donation Centre (Epalinges, Lausanne) according to the Swiss Federal law on therapeutic products. PBMCs were isolated using the standard centrifugation protocol by Lymphoprep density gradient medium (Axonlab) followed by negative selection of CD4 + and CD8+T cells using antibody complexes and magnetic beads according to the manufacturer’s protocol (EasySep, Stem Cell Technology).

[0176] T cell activation

[0177] CD4+and CD8+T cells were activated separately on the same day using aCD3 / aCD28 monoclonal antibody (mAb) magnetic beads (Gibco, Thermo Fisher Scientific) at a bead-to-cell ratio of 2: 1, in addition to 50 lU / ml of hIL-2 (Peprotech). T cells were then seeded at 1 x 106cells per ml in a 48-well plate in a total volume of 1 ml per well of T cell media (RPMI-1640 with Glutamax, supplemented with 10% heat- inactivated FBS and penicillin 10’000 IU / ml-streptomycin 10’000 pg / ml (100X) (BioConcept)) before transduction.

[0178] Transduction of primary T cells

[0179] CD4+and CD8+T cells were transduced separately with either HSG-recl or HSG- rec2 18-22h post-activation by adding viral particles to T cells.

[0180] For lentiviral transduction, the lentivirus was directly added to the cells, in addition to lentiboost (Sirion Biotec) following the manufacturer’s protocol.

[0181] For retroviral transduction, the retrovirus is added to RetroNectin precoated plates and then spun before adding activated T cells which are subsequently spun according to the following protocol: non-treated cell-culture 48-well plates (Corning Falcon) were precoated with 250 pl of recombinant RetroNectin (Takara Bio) at a final concentration of 20 pg / ml, overnight (O / N) at 4°C one day prior to transduction. On the day of T-cell retroviral transduction, the RetroNectin-precoated plates were washed twice with PBS and then blocked with T-cell media for 30 min at RT. Subsequently, a volume of T-cell media equal to the virus volume to be used is then removed and replaced by the retrovirus for a final volume per well, dependent on the number of T cells to be infected. Later, the plates were spun at 2’000 g for 1.5 to 2h at 32°C. Finally, the 24h-activated cells were transferred to each coated well for a final volume of 500 pl. The plates were centrifuged at 300g for 10 min before incubation at 37 °C in a 5% CO2 atmosphere. For co-transduction protocol, the 24h-activated cells are transduced with lentiviral supernatant as reported above and incubated O / N at 37°C. At 48h from activation and 24h after lentiviral transduction T cells have been transduced with retrovirus using the protocol reported above.

[0182] Expansion of primary T cells

[0183] Transduced and untransduced T cells were expanded in 24-well plates of a final volume of 1 ml by adding T cell medium enriched with 50 lU / ml of hIL-2 (peprotech) at 48h after transduction. At day 5 post-activation, magnetic beads were removed, and T cells were counted using Trypan blue staining (Gibco, Life Technologies) for expansion at 1 * 106cells per ml using T cell media enriched with lOng / ml of both hIL-7 and hIL-15 (Miltenyi Biotec) added every 2 days until the end of cell culture.

[0184] Cell staining and flow cytometric analysis

[0185] On day 7 after transduction, cell viability along with h679 scfv, c-myc, and Thy 1.1 cell surface expression was evaluated by flow cytometric analysis. To evaluate the viability of transduced and untransduced T cells or Jurkats, cells were stained with near-infrared fluorescent reactive dye (APC Cy-7) (Invitrogen, Life Technologies). To determine the expression of h679 scfv, cells were stained with fluorescent anti-human F(ab’) mAb (APC) (BD Biosciences). Cell surface staining with antibodies against c-myc-FITC (MAB4518, Abeam), Thyl. l(CD90)-BV605 (clone OX-7, Biolegend) and NGFR (Anti human NGFR- AP (Clone ME20.4, Biolegend ) was performed to detect the expression level of the reporter genes. Data were acquired with a BD FACS LRSII flow cytometer with FACS DIVA software (BD Biosciences) and analyzed using FlowJo software (10.7.1).

[0186] Precursor synthesis (IMP -288)

[0187] General

[0188] Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogues as well as coupling reagents were purchased from Iris Biotech (Marktredwitz, Germany) or Bachem (Bubendorf, Switzerland). H-Rink resin was obtained from Iris Biotech. Solid phase peptide synthesis was carried out manually using an LP-180A syringe shaker (Kamush, Gdansk, Poland). Analytical reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a MultoKrom 100 C18 (5 pm, 125 x 4.0 mm) column (CS Chromatographic GmbH, Langerwehe, Germany), using a Shimadzu Corp. (Kyoto, Japan) system with a LC-40D gradient pump, a CBM-40 system controller, an CTO-40C column oven and a SPD-M40 UV / VIS photodiode array detector. For semi-preparative HPLC, a MultoKrom 100 RP 18 column (5 pm, 150 x 10 mm, CS Chromatographic GmbH, Langerwehe, Germany) was used. For radioactivity detection during radio-HPLC, the outlet of the UV-photometer was connected to a Nal(Tl) well-type scintillation counter from EG&G Ortec (Munich, Germany). Data analysis was performed using the Shimadzu Lab Solutions software. Peptides were eluted applying different gradients of 0.1% (v / v) trifluoroacetic acid (TFA) in H2O (solvent A) and 0.1% TFA (v / v) in acetonitrile (solvent B) at a constant flow rate of 1 mL / min (or 4 mL / min for semi-preparative HPLC); specific gradients are cited in the text. Retention times tR are cited in the text. Electrospray ionization mass spectrometry (ESI-MS) was performed using an Advion expression CMS mass spectrometer (Advion, Harlow, UK).

[0189] Precursor synthesis

[0190] IMP -288 was synthesized via a combined solid phase peptide synthesis (SPPS) and solution phase fragment condensation approach. The peptide backbone of IMP-288 (Fmoc- D-Tyr(tBu)-D-Lys(Dde)-D-Glu(tBu)-D-Lys(Dde)-NH2) was assembled stepwise on H- Rink amide resin using a standard Fmoc SPPS protocol. After cleavage of the N-terminal Fmoc protecting group, the peptide N-terminus was reacted with BOC2O (2 eq.) in the presence of triethylamine (4 eq.). Then, the Dde-protecting groups of the D-Lys sidechains were cleaved using 2% hydrazine in DMF, and the glycine-succinyl-histamine sequence was assembled stepwise on the D-Lys sidechains. Upon classical coupling of Fmoc-Gly- OH and Fmoc-deprotection, the resin was reacted with succinic anhydride (6 eq.) in the presence of Pyridine (6 eq.). The resin was then preactivated using COMU (4 eq.) and DIPEA (4 eq.) and reacted with a histamine (4 eq.) in the presence of additional 20 eq. of DIPEA. To cleave the peptide from the solid support, the resin was treated with trifluoroacetic acid, washed with dichloromethane, and the combined filtrated were concentrated in vacuo. Upon precipitation from diethyl ether, the crude peptide was obtained in app. 50% yield based on the resin load provided by the manufacturer. RP-HPLC (gradient 5-20 in 15 min): tR = 9.43min

[0191] Calculated monoisotopic mass (C48H71N15O13): 1066.2; found (ESI-MS): 1066.6 [M+H]+, 533.8 [M+2H]2+, 973.5 [M-histamine+H]+.

[0192] The crude peptide was then reacted with DOTA-tris-tBu-ester (Chematech, Dijon, France;

[0193] 2.5 eq.) in the presence of HATU (O-(7-Azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium-hexafluoro-phosphate, 2.5 eq.) and DIPEA (5 eq.), precipitated from diethyl ether and dried in vacuo. Upon removal of the tBu-protecting group in the chelator moiety using TFA, the product was precipitated using diethyl ether and purified via semipreparative RP-HPLC (8% B isocratic). IMP-288 was obtained in > 98% purity (UV at 214 nm) and 9% yield based on crude starting peptide.

[0194] RP-HPLC (gradient 5-20 in 15 min): tR = 10.14 min Calculated monoisotopic mass (C64H97N19O20): 1452.6; found (ESI-MS): 1453.7 [M+H]+.

[0195] Synthesis ofnatGa-IMP-288

[0196] IMP -288 dissolved in a 3 mM solution of GaNCL to yield a 1 mM solution. This mixture was heated to 90°C for 15 min. RP-HPLC analysis confirmed quantitative formation ofnatGa-TRAP(IMP-288)3, and the resulting stock solution was used as such for the preparation of the dilution series for affinity determinations and as stock solution for radioiodination.

[0197] RP-HPLC (gradient 5-20 in 15 min): tR = 7.31 min Calculated monoisotopic mass (C64H9sNi902oGa): 1519.6; found (ESI-MS): 761.0 [M+2H]2+

[0198] Synthesis of alkyne-IMP-288

[0199] The peptide backbone of IMP -288 (Fmoc-D-Tyr(tBu)-D-Lys(Dde)-D-Glu(tBu)-D- Lys(Dde)-NH2) was assembled as described for IMP-288. After cleavage of the N-terminal Fmoc protecting group, the peptide N-terminus was reacted with hexynoic acid (1.4 eq) in the presence of COMU (1.4 eq) and DIPEA (2.8 eq.). Then, the Dde-protecting groups of the D-Lys sidechains were cleaved using 2% hydrazine in DMF, and the glycine-succinyl- histamine sequence was assembled stepwise on the D-Lys sidechains. Upon classical coupling of Fmoc-Gly-OH and Fmoc-deprotection, the resin was reacted with succinic anhydride (6 eq.) in the presence of Pyridine (6 eq.). The resin was then preactivated using COMU (4 eq.) and DIPEA (4 eq.) and reacted with a histamine (4 eq.) in the presence of additional 20 eq. of DIPEA. To cleave the peptide from the solid support, the resin was treated with trifluoroacetic acid, washed with dichloromethane, and the combined filtrated were concentrated in vacuo. Upon precipitation from diethyl ether, the crude peptide was obtained in 37% yield based on the resin load provided by the manufacturer.

[0200] RP-HPLC (gradient 5-20% B in 15 min): tR = 11.64 min Calculated monoisotopic mass (C54H77N15O14): 1159.6; found (ESI-MS): 1160.3 [M+H]+, 581.0 [M+2H]2+.

[0201] Click reaction with TRAP -triazide (TRAP(IMP-288)3)

[0202] TRAP -triazide was dissolved in a 3 : 1 (v / v) mixture of water and tBuOH to yield a 1 mg / 10 pL stock solution. In a separate vial, alkyne-IMP-288 (3.5 eq) and sodium ascorbate (50 eq) were dissolved in 500 pL water / tBuOH (1 : 1, v / v). To this mixture, TRAP -triazide (1 eq) and Cu(OAc)2 (2.5 eq, stock solution of 1 mg / 20 pL in water) were added. A dark brown precipitate formed, which dissolved upon vortexing, providing a light green solution. The mixture was heated to 60°C for 1 h. Since RP-HPLC analysis revealed complete consumption of the starting materials, NOTA (30 eq) suspended in 1.14 mL 0.1 M HC1 were added to the reaction vial. After heating to 60° for 3h, the Cu-free TRAP(IMP-288)3 was immediately isolated via semipreparative RP-HPLC (gradient 5- 22% B in 20 min, flow: 5 mL / min). The final product was obtained in 10% yield based on TRAP -triazide and a purity of 93%.

[0203] RP-HPLC (gradient 5-30% B in 15 min): tR = 10.54 min Calculated monoisotopic mass (C189H285N60O51P3): 4306.7; found (ESI-MS): 1436.3 [M+3H]3+, 1077.2 [M+4H]4+.

[0204] Synthesis ofnatGa-TRAP(IMP-288)3

[0205] A 2 mM stock solution of TRAP(IMP -288)3 was diluted 1 :4 with 3 mM GaNCL in water (final TRAP(IMP-288)3 concentration: 0.5 mM). This mixture was heated to 90°C for 10 min. RP-HPLC analysis confirmed quantitative formation ofnatGa-TRAP(IMP- 288)3, and the resulting stock solution was used as such for the preparation of the dilution series for affinity determinations and as stock solution for radioiodination.

[0206] RP-HPLC (gradient 5-20 in 15 min): tR = 10.32 min

[0207] Calculated monoisotopic mass (Cis^ssNeoOsiPsGa): 4373.7; found (ESI-MS): 1459.1 [M+3H]3+, 1094.3 [M+4H]4+.

[0208] Radioiodination of Ga-IMP-288

[0209] Radioiodination of Ga-IMP-288 was carried out using the lodoGen® method.

[0210] Briefly, 100-200 pg of Ga-IMP-288 were dissolved in 0.5 mL TRIS iodination buffer (25 mM Tris HCl, 0.4 M NaCl, pH 7.5) and transferred to an Eppendorf reaction tube coated with 150 pg of lodoGen®. Upon addition of [125I]NaI (18-20 MBq, Hartmann Analytik, Braunschweig, Germany), the reaction vessel was briefly vortexed and the labeling reaction was allowed to proceed for 15 min at RT. The peptide solution was then removed from the insoluble oxidizing agent. Separation of Ga-[125I]IMP-288 from unlabeled precursor was achieved using gradient RP-HPLC (column: MultoKrom 100 Cl 8 (5 pm, 125 x 4.0 mm; CS GmbH, Langerwehe, Germany), gradient: 5-25% B within 15 min, flow: ImL / min).

[0211] For in vitro binding and uptake studies, the HPLC product fraction was used as such and diluted to the required concentration (1 nM) using the respective assay medium.

[0212] Radio-RP-HPLC (gradient 5-25% B in 15 min): tR = 12.9 min

[0213] 68Ga-labeling of IMP-288

[0214] IMP -288 precursor (10 nmol in 10 mL of water) was added to 1 mL of68Ga- generator eluate (in 0.1 M HC1, eluted from a GalliPharm Generator from Eckert and Ziegler AG, Berlin, Germany), and either 1.6 mL of 0.2 M NaOAc (pH 4) or 0.3 mL of 1 mg / mL solution of HEPES in deionized water (pH 5) were added. The mixture was heated to 95°C for 15 min. Upon cooling, the reaction mixture was diluted with deionized water to a total volume of 10 ml and passed through a SepPak Cl 8 plus Light (Waters, Eschborn, Germany) cartridge (preconditioned with 5 ml methanol and 5 ml deionized water). The cartridge was then washed with 6 ml of deionized water, and dried with air. For elution, 0.5 mL of acetonitrile (0.1% TFA) were used, and fractions of 3-4 drops of eluate were collected in Eppendorf vials. The fractions containing the highest amount of activity were combined, evaporated to dryness at 80°C under a nitrogen stream and reconstituted to the required activity concentration for the respective experiment using PBS. [68Ga]IMP-288 was usually obtained in 55-65% isolated yield (decay corrected) based on total activity at EOS. Radiochemical purity was > 97% for all syntheses.

[0215] Radio RP-HPLC (gradient 5-20 in 15 min): tR = 11.92 min

[0216] Determination of CTTR-affinity (ICso)

[0217] Affinities of Ga-IMP-288 and Ga-TRAP(IMP-288)3 to HSG-rec2-expressing Jurkat cells (100 pL virus) were determined in a competitive binding assay (ICso). Briefly, samples containing 5 - 105HSG-rec2 -Jurkat cells in 200 pL HBSS (1% BSA) were incubated with 25 pL of Ga-[125I]IMP-288 and 25 pL of 10'5-l 0'11M solutions (n=3 samples / concentration) of the respective unlabeled competitor (Ga-IMP-288 or Ga- TRAP(IMP-288)3) in HBSS (1% BSA). The final radioligand concentration in the assay was 0.1 nM. After Ih at RT, samples were centrifuged at 1000 rpm for 5 min. The supernatants were removed and combined with the supernatants of the subsequent washing step (resuspension with cold PBS and centrifugation). Both the radioactivity in this fraction (free radioligand) and in the cell pellets (bound radioligand) were quantified using a y- counter (WALLAC; 1480 WIZARD™ 3”, Turku, Finland). ICso values were calculated by nonlinear regression using GraphPad Prism (GraphPad Prism 10.4.1 Software, Inc., San Diego, CA, USA). Data represent means ± SD of three separate experiments with n=3 for each competitor concentration, respectively.

[0218] Radioligand binding assay

[0219] Prior to the experiment, cells were centrifuged, the culture medium was removed by aspiration, and cells were resuspended in assay medium (DMEM / F-12 with Glutamax (Thermo Fisher Scientific, Reinach. Switzerland), containing 5% BSA) to yield cell suspensions with a final concentration of 2.5-5- 106cells / ml. For cell counting, an automated CytoSMART Lux cell counter (Axion BioSystems, Atlanta, USA) was used.

[0220] Cellular uptake of [68Ga]IMP288 in transduced Jurkat cells or donor T-cells was determined by incubating the respective cells with [68Ga]IMP-288 (1 nM) at 37 °C for 30 min (n=3 per condition, total sample volume: 250 pL). Wild type Jurkat or untransduced donor T cells (n=3 per condition) were incubated in parallel as negative controls to quantify non-specific (i.e. HSG-recl or HSG-rec2 -independent) tracer binding. To crossvalidate binding specificity of [68Ga]IMP-288 to HSG-recl transduced cells, cells were also coincubated with an excess (1 mM) of unlabeled IMP-288 in some cases. After incubation, the tubes were centrifuged (3 min, 1300 ref, Megafuge 1.0, Heraeus Thermo Scientific) and the supernatant was carefully removed. After washing with 200 pL of cold HBSS, the amount of bound radioligand in the cell pellet as well as the amount of free radioligand in the combined supernatants was quantified using a y-counter (WALLAC; 1480 WIZARD™ 3”). Cellular uptake of [68Ga]IMP-288 is given in % of added radioligand dose (mean ± SD), and values were normalized to either 100.000 or 500.000 live cells to ensure comparability of binding data between samples. l68Ga1IMP-288 PET phantom study

[0221] To determine the detection sensitivity of the small animal PET for HSG-rec- expressing cells using [68Ga]IMP-288, a PET-phantom setup using 96-well-plates was designed. Jurkat cells with different HSG-recl transduction levels (obtained by using 10, 30 and 100 mL of virus supernatant for transduction (see section on retroviral transduction of Jurkat cells)) as well as WT Jurkat cells were incubated with [68Ga]IMP-288 (1 nM) at 37 °C for 30 min in DMEM / 5% BSA, centrifuged and washed once with HBSS. Cells were then resuspended in DMEM / 5% BSA to a concentration of 2- 106cells / 100 mL, and this suspension was successively diluted to final concentrations of 1 • 106, 5 - 105, 1 105, 50.000, 10.000 and 5.000 cells / 100 mL for all transduction levels as well as WT Jurkat cells. 100 mL aliquots of the respective cell suspensions were then transferred into 96 well plates (n=3 per cell type per concentration), and static PET images were acquired. In parallel, absolute cellular [68Ga]IMP-288 uptake in transduced and WT Jurkat cells was quantified in separate samples as described above (see “radioligand binding assay”). In vivo tumor models

[0222] All animal experiments were conducted in compliance with the Swiss legislation for care and use of laboratory animals under the license VD-3595.

[0223] HSG-rec2 Jurkat models'. For induction of solid tumor growth, female NSG mice (6-8 weeks) were subcutaneously (s.c.) injected with 1 • 107HSG-rec2 transduced Jurkat cells (transduction efficiency of app. 90%; n=3) or WT Jurkat cells (n=3) in 100 mL HBSS / Matrigel (1 / 1). To establish disseminated disease, female NSG mice (6-8 weeks, n=3) received an intravenous (i.v.) bolus injection of 1 • 107HSG-rec2 transduced Jurkat cells in HBSS.

[0224] PC3-Pip tumor model with adoptive T cell transfer: Male NSG mice were injected subcutaneously with 5 - 106PC3-Pip cells in serum free medium. On day 5 after tumor inoculation, mice (groups of n=4) received either an intravenous injection of saline (untreated controls, group 1), a peritumoral injection of 2 Mio CTTR-PZ1-CART cells (1 : 1 ratio CD4 / CD8, n = 4, group 2), an i.v. injection of 2 Mio CTTR-PZ1-CART cells (1 : 1 ratio CD4 / CD8, n = 4, group 3), an i.v. injection of 2 Mio CTTR-only T cells (1 : 1 ratio CD4 / CD8, n = 4, group 4) or an i.v. injection of 2 Mio PZl-CAR-only T cells (1 : 1 ratio CD4 / CD8, n = 4). l68Ga1IMP-288 PET / CT

[0225] For in vivo tracking of HSG-rec2 transduced Jurkat cells (s.c. and i.v. model), mice were intravenously injected with 4-7 MBq (constant peptide amount of 0.5 nmol, SA = 8- 14 GBq / mmol) [68Ga]IMP-288. After Ih, static [68Ga]IMP-288 PET / CT acquisition (20 min) was performed on an Albira microPET / SPECT / CT scanner (Bruker BioSpin MRI GmbH, Ettlingen, Germany). All mice were anesthetized for the duration of the imaging sequence by inhalation of isoflurane 2% / O2 and warmed on a heating pad during the scan. PET images were reconstructed using an MLEM reconstruction algorithm. Coregistered CT (0.4 mA, 35 kV, 600 projections, 125 pm voxel size) was used for anatomical localization of tracer uptake. PET images were normalized to %ID / mL and fused with CT images using PMOD 4.4.

[0226] Results Design of first-generation reporter tag (HSG-rec l ) and retroviral T-cell transduction

[0227] A schematic representation of the retroviral vector design for cell transduction with HSG-recl is shown in Figure 5. The nucleic acid sequence of the is shown in Table 2, and the amino acid sequence of HSG-recl is shown in Table 1. Transduction efficiency was confirmed using surface staining of HSG-recl Jurkat cells (Figure 6A). In order to assess the efficiency level of Jurkat cell transduction with the HSG-recl construct, cells were stained with an anti-Fab targeting the cell surface expressed h679 ScFv, in addition to cell surface staining for the c-Myc and Thl .1 reporter genes. The data demonstrated an increasing level of HSG-recl expression that was proportional to the increasing volume of the retroviral supernatant used as measured by direct anti-fab surface staining, ranging from 17% of h679+- Jurkat cells when transduced with 30 p1 of virus to 97% when transduced with 300 pl of virus. Similarly, data showed a proportional increase of c-Myc+ cells from 6% to 95%; and of Thl.1+ cells from 18% to 99% when transduced with 3 pl and 300 pl of virus, respectively (Figure 6 A).

[0228] Radioligand binding assay using HSG-recl expressing Jurkat cells

[0229] Based on the flow cytometry data, demonstrating an increasing transduction efficiency ranging from a low level (below 20%) of expression over an intermediate (20 to 90%) to a high level of HSG-recl expression (over 90%), which was proportional increasing amount of virus superanatant used, a radioligand binding assay using [68Ga]IMP-288 (0.5 nM) was performed to verify full functionality of the HSG-recl construct and unbiased IMP-288 binding by the h679 scFv (Figure 6B).

[0230] As shown in Figure 6, [68Ga]IMP-288 binding correlated linearly with the respective transduction level of the Jurkat cells and was highly HSG-recl specific, as demonstrated by efficient blocking of tracer binding by a large molar excess of unlabeled IMP -288 (1 pM). Under these conditions, [68Ga]IMP-288 binding was reduced to the level found for untransduced WT Jurkat cells. Thus, tracer binding to untransduced Jurkat cells accurately reflects non-specific [68Ga]IMP-288 uptake, and consequently, WT Jurkat cells were consistently used to quantify non-specific binding in all subsequent experiments.

[0231] As expected, the amount of specifically bound [68Ga]IMP-288 gradually decreased with decreasing amounts of virus used for the transduction and was slightly lower at 4°C than at RT or 37°C. For Jurkat cells transduced with 10 pL virus, cell surface expression of HSG-recl was still detectable by FC, but only minimal specific binding of [68Ga]IMP-288 (app. 0.8% of added dose) was observed. The cell surface expression level achievable with 10 pL of virus thus represents the “detection limit” for HSG-recl expression by radioligand binding under the given experimental conditions, and suggests that higher expression levels, ensuring higher absolute tracer binding, will be required for sensitive in vivo detection of T-cells using [68Ga]IMP-288.

[0232] Determination of PET imaging sensitivity via a small animal PET phantom study

[0233] To investigate the actual detection limit for a given number of HSG-recl expressing cells using [68Ga]IMP-288 PET, a small animal PET phantom study was performed (Figure 7). Jurkat cells with different HSG-recl transduction levels were preincubated with [68Ga]IMP-288 under standard conditions, washed once, and then diluted to defined cell numbers in 100 pL HBSS, which were then added (in triplicates) to a 96 well plate (phantom). To be able to directly correlate the PET signal with the absolute tracer binding in vitro (in % of added dose), radioligand binding to the same cells (normalized to 500.000 cells) was also quantified in parallel using a y-counter.

[0234] As shown in Figure 7 and as already concluded from the radioligand binding assays (Figure 6), the low HSG-recl expression level and thus [68Ga]IMP-288 binding observed for Jurkat cells transduced with 10 pL virus (and for donor T cells) represents the lower detection limit for small animal PET. Of note, this only holds true, if 500.000 HSG-recl expressing cells are concentrated in a small volume of 100 pl, a setting that is not very likely to occur in vivo, where transduced T-cells are anticipated to home in larger volumes and to be distributed to different tissues. Thus, to obtain a robust PET signal, allowing in vivo tracing of HSG-recl transfected cells, higher cell surface expression of HSG-recl and consequently higher [68Ga]IMP-288 uptake are required. Based on the phantom study, we concluded that the minimum cell surface expression of HSG-recl on T cells needed for a robustly detectable PET signal in vivo equals that of Jurkat cells transduced with 30 pl of virus, corresponding to an in vitro uptake of roughly 7% of added [68Ga]IMP-288 in 500.000 cells (Figure 7, panel B). Design of second-generation reporter tag (HSG-rec2) and lentiviral T-cell transduction

[0235] A novel, second-generation h679-tag, HSG-rec2, was designed and evaluated. A lentiviral transduction approach was chosen for HSG-rec2, in contrast to the adenoviral transduction strategy used for HSG-recl . In contrast to retroviruses that can only infect divided cells, lentiviruses are known for their capacity to transduce both divided and nondivided cells, thus providing another niche of cells for a stable integration of the gene of interest into their genome, which will be translated in a higher transduction efficiency.

[0236] The sequence of the lentiviral vector used for cell transduction with HSG-rec2 is depicted in Figure 8 and shown in Table 2. The amino acid sequence of HSG-rec2 is shown in Table 1. Transduction efficiency in Jurkat cells was confirmed using surface staining of HSG-rec2 (Figure 9) in analogy to the procedures used for staining HSG-recl.

[0237] Radioligand binding assay using HSG-rec2 expressing Jurkat cells

[0238] Jurkat cells were transfected with HSG-rec2, and surface expression levels were investigated using both FC and [68Ga]IMP-288 binding (Figure 9).

[0239] As observed for the HSG-recl construct (Figure 6), HSG-rec2 transduction efficiency in Jurkat cells was high and closely correlated with the amount of virus used for the transduction (Figure 9A). These FC findings were confirmed by a [68Ga]IMP-288 binding assay (Figure 9C). Jurkat cells showed robust and HSG-rec2 density-related tracer binding.

[0240] In vivo proof-of-concept imaging study

[0241] On Jurkat cells, HSG-rec2 expression was excellent and found to be sufficiently stable over time. Therefore, Jurkat cells with a transduction level of app. 90% (100 pl of virus) were used for first in vivo proof-of-concept studies.

[0242] Subcutaneous implantation of HSG-rec2 expressing Jurkat cells was selected as a first model for in vivo T cell tracking using [68Ga]IMP-288 PET. Since our in vitro data had hinted towards a slight loss of HSG-rec2 surface expression in the transduced Jurkat cells over time, very early time points post inoculation were selected for the first imaging studies (1 and 5d post cell inoculation). A schematic representation of the design of the in vivo study using the subcutaneous HSG-rec2 Jurkat tumor model is shown in Figure 10. As shown in Figure 11 (panel A), high [68Ga]IMP-288 accumulation (Ih p.i.) was observed at the injection site 1 day after cell injection, indicating specific accumulation of the HSG-rec2 targeted tracer in the transduced Jurkat cells. Both a naive control mouse (Figure 11, panel A), as well as mice bearing solid WT Jurkat tumors on the right shoulder (Figure 11, panel C), only showed [68Ga]IMP-288 uptake above background in the kidneys and the bladder, which is occasioned by the renal clearance of the tracer. Since the WT Jurkat tumors did not display any [68Ga]IMP-288 accumulation, the signal obtained for the freshly injected HSG-rec2 expressing Jurkat cells is clearly HSG-rec2 specific.

[0243] What makes the high intensity of the [68Ga]IMP-288 signal at the injection site even more remarkable is the fact, that at Id post inoculation there is no vascular supply to the “tumor” (cells in Matrigel matrix). Apparently, due to the particularly high affinity of [68Ga]IMP-288 for its target, tracer accumulation in the Matrigel-supported cells is even efficient enough via passive diffusion. A second [68Ga]IMP-288 PET scan (Ih p.i.) 5 days after cell inoculation demonstrated decreased tracer uptake at the inoculation site, which reflects the expected initial reduction in viable HSG-rec2 transduced Jurkat at the injection site (cell death occasioned by a lack of vascularization, followed by selection of the most robust cells, their expansion and growth of a solid tumor mass). Unfortunately, for the solid tumor xenografts which developed from the subcutaneously injected HSG-rec2 transduced Jurkat cells app. 2 weeks after s.c. inoculation, no follow-up [68Ga]IMP-288 PET could be performed. Tumor size had reached a humane endpoint and animals had to be sacrificed before the scan could be performed.

[0244] However, encouraged by the excellent detectability of the subcutaneously implanted HSG-rec2 transduced Jurkat cells using [68Ga]IMP-288 PET (Figure 11), another proof-of-concept T cell tracking experiment was performed using a model of disseminated T cell leukemia, generated via intravenous injection of HSG-rec2 expressing Jurkat cells. After app. 3 weeks, when animals presented with signs of sickness (fuzzy coat, hunched posture), [68Ga]IMP-288 PET was performed in these animals (n=2, Figure 12), and they were sacrificed immediately after imaging.

[0245] In these mice, high [68Ga]IMP-288 PET accumulation was observed in the liver and in the bone marrow, primarily of the skull and in the lower part of the spine (Figure 12). This finding is fully in accordance with the predicted homing of the highly CXCR4 positive Jurkat cells to mouse tissues with high CXCL12 expression, namely liver and bone marrow. Surprisingly, virtually no renal tracer uptake was observed (as in the subcutaneous model (Figure 11), probably due to efficient [68Ga]IMP-288 PET binding to the large number of disseminated HSG-rec2 expressing Jurkat cells in liver and bone marrow, alongside rapid excretion via the urine.

[0246] Interestingly, while [68Ga]IMP-288 uptake in the skull and the vertebrae is quite focal and homogenous (with the slight blurring being the result of the long range in tissue of the high-energy positrons emitted by68Ga, limiting resolution), this is not the case for the liver, where tracer accumulation seems more heterogenous and blotchy. This is particularly well discernible in the coronal, transversal and sagittal planes of the liver region of the [68Ga]IMP-288 PET of mouse 2 shown in Figure 13.

[0247] Upon dissection of the animal after the PET scan, the inhomogeneous hepatic uptake of [68Ga]IMP-288 was found to be caused by focal tracer accumulation in a multitude of small HSG-rec2 -positive Jurkat tumors, as shown in photographs of the resected liver in Figure 13. This confirms once more the highly specific uptake of [68Ga]IMP-288 in HSG-rec2-expressing Jurkat cells, with no background accumulation whatsoever in any other tissue (except the excretion organs kidney and bladder).

[0248] Evaluation of HSG-recl for retroviral primary T-cell transduction

[0249] Despite the highly encouraging results achieved with lentiviral vector above, retroviral transduction remains the preferred transduction method for engineering cell products for clinical applications. Therefore, the retroviral transduction of primary T cells with the HSG-recl construct was evaluated (Figure 14). Transduction of primary CD4+ and CD8+ T cells with the downstream marker Thy 1.1 was equally efficient to transduction with the upstream marker cMyc, suggesting reasonable expression levels of the HSG-recl construct. This was confirmed by a radioligand binding assay in the presence of 1 nM [68Ga]Ga-IMP-288, showing excellent and specific tracer binding to the transduced vs untransduced primary T cells. Of note, the radioligand uptake corresponds very closely to the uptake of [68Ga]Ga-IMP-288 observed in Jurkat cells with the same cMyc- and Thy 1.1 transduction efficiency (transduced with 30 pl of virus, Figure 6A and 6B), underlining the reliability of the radioligand binding assay to accurately depict HSG- recl expression density. The cMyc- and Thy 1.1 expression levels on CD4+ and CD8+ primary T cells were found to remain stable over 18 days (end of observation period), suggesting excellent stability of the retrovirally introduced HSG-recl construct and thus encouraging in vivo translation in the context of T cell tracking after adoptive T cell transfer.

[0250] EXAMPLE 2

[0251] First in vivo evaluation primary T-cells transduced with HSG-recl

[0252] CD4+ and CD8+ primary T cells from a human donor were transduced with HSG- recl. Binding of [68Ga]Ga-IMP-288 (0.5 nM) to transduced and corresponding untransduced control donor T cells was investigated. Results are shown in Figure 15. Tracer was found to bind to transduced T cells with excellent specificity.

[0253] The transduced and control untransduced T cells were administered intravenously to mice and PET / CT performed after 18 hours. Results are shown in Figure 16. This initial pilot experiment did not include an ex vivo study after cell injection to quantify the activity accumulation in each organ. It is therefore difficult to exactly allocate the HSG- recl positive T cells to a specific tissue. However, in the image obtained for the CD4- HSG-recl mouse (left upper panel), the cells appear to home to the spleen. Most importantly, however, in both mice who received the transduced cells, there is a tracer accumulation where there is none in the control animals. In the control animals injected with untransduced cells, tracer is only seen in the kidneys, indicative of renal elimination. The results therefore show that CD4+ and CD8+ primary T cells transduced with HSG- recl can be detected in the tissues to which they home after intravenous administration.

[0254] EXAMPLE 3

[0255] Design of third-generation reporter tag (HSG-rec3) and retroviral T-cell transduction

[0256] A novel, third-generation h679-tag, HSG-rec3, was designed and evaluated. A retroviral transduction approach was chosen for HSG-rec3. The sequence of the vector used for cell transduction with HSG-rec3 is depicted in Figure 17 and shown in Table 2. The amino acid sequence of HSG-rec3 is shown in Table 1. Transduction efficiency in human primary T cells was confirmed using [68Ga]Ga-IMP-288 binding to two HSG-rec3 variants, one with and one without a truncated EGFR downstream marker (Figure 18) in analogy to the procedures used for the radioligand binding assays performed for HSG-recl and HSG-rec2.

[0257] EXAMPLE 4

[0258] Cotransduction of primary human T cells with an anti-PSMA CAR (PZ1) and HSG-recl

[0259] Based on the results in Example 1, primary human T cells were first transduced lentivirally with an established anti-PSMA CAR (PZ1) (Liu, H., Moy P., Kim S., Xia Y., Rajasekaran A., Navarro V., Knudsen B., Bander N.H. Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium. Cancer Res. 1997, 57, 3629-3634) and then transduced retrovirally with HSG-recl (subsequently termed Chimeric T cell Tracking Receptor, CTTR). Flow cytometry analysis revealed efficient T cell transduction with the PZ1 CAR only (15 and 4.5% in CD4+ and CD8+ T cell populations, respectively). CTTR-only transduction led to transduction efficiencies of 38 and 21% in CD4+ and CD8+ T cells. Surprisingly, PZ1- CTTR co-transduction provided comparably low levels of doubly positive CD4+ (20%) and CD8+ (8%) T cells. Nevertheless, these cells were used for a first proof-of-ccwce / ?t in vivo imaging study.

[0260] In vivo evaluation of PZ1-CCTR co-transduced human T cells

[0261] For the in vivo evaluation of the PZ1-CTTR co-transduced T cells, PC3-Pip xenograft bearing mice were divided into 5 experimental groups:

[0262] 1. untreated controls (n = 4)

[0263] 2. peritumoral injection of 2 Mio CTTR-PZ1-CART cells (1 : 1 ratio CD4 / CD8, n = 4)

[0264] 3. i.v. injection of 2 Mio CTTR-PZ1-CART cells (1 : 1 ratio CD4 / CD8, n = 4)

[0265] 4. i.v. injection of 2 Mio CTTR-only T cells (1 : 1 ratio CD4 / CD8, n = 4)

[0266] 5. i.v. injection of 2 Mio PZl-CAR-only T cells (1 : 1 ratio CD4 / CD8, n = 4)

[0267] All mice were then sequentially imaged using [68Ga]IMP-288 PET / CT up to 27 post adoptive T cell transfer (ATC). As shown in Figure 19, CTTR-only transduced T cells only show very weak infiltration into the PC3-Pip xenografts, as illustrated by the faint [68Ga]IMP-288 PET signal observed at the tumor site. The low tumor infiltration and lack of tumor-specificity of the CTTR-only T cells also explains the uninhibited growth of the tumor xenografts in group 4, which was identical to the tumor expansion observed in the untreated control mice (group 1). All mice in both groups thus had to be sacrificed at day 15 after ACT. In contrast, the mice having received the PZ1 -CAR-only T cells (group 5) initially showed tumor progression up to day 14 post ACT, which was then efficiently controlled by the PZ1-CART cells, leading to complete response at 27 days post ACT (Figure 19, lower panel). Since the PZ1-CART cells do not bear the CTTR tag, the complete lack of [68Ga]IMP-288 uptake at the tumor site was expected and conclusive, highlighting once more the CTTR-specificity of [68Ga]IMP-288. The very faint tracer non-specific tracer accumulation at the tumor site on day 14 post ACT may be explained by enhanced perfusion of the highly inflamed tumor tissue due to CART cell-mediated tumor cell killing.

[0268] The [68Ga]IMP-288 PET / CT imaging results obtained after ATC of the cotransduced CTTR-PZ1-CART cells (group 3) are summarized in Figure 20. Already at day 1 post ACT, the intravenously injected CTTR-PZ1-CART cells efficiently home to the PC3-Pip tumor xenograft, as demonstrated by the intense [68Ga]IMP-288 signal at the tumor site (Figure 14, upper panel). [68Ga]IMP-288 uptake in the tumor remains high up to 7 days post ACT and then slowly decreases up to day 22 post ACT, which is accompanied by progressive tumor control by the CTTR-PZ1-CART cells, leading to complete remission at day 27 post ACT, as observed for the PZ1-CART cells (Figure 19). Of note, identical imaging results and tumor control were observed for the animals in group 2, who had received peritumoral injection of 2 Mio CTTR-PZ1-CART cells.

[0269] To investigate if there is a memory population of CTTR-PZ1-CART cells after tumor remission, mice were rechallenged with 5 Mio PC3-Pip tumor cells (s.c. injection on the contralateral side) on day 33 post ACT. [68Ga]IMP-288 PET / CT revealed very rapid recruitment of resident CTTR-PZ1-CART cells to the injection site (Figure 20, lower panel) and efficient clearance of reinjected tumor cells within < 7 days, prohibiting tumor regrowth. Identical results were obtained for the mice in group 5 (PZ1-CART cells) upon rechallenge. In summary, the results from this pilot in vivo study using CTTR-mediated T cell tracking with [68Ga]IMP-288 PET / CT in the context of CART cell therapy, demonstrated that, despite non-optimized co-transduction levels of the administered CTTR-CART cell population, a) CTTR-PZ1-CART cells can be sensitively tracked in vivo using [68Ga]IMP-288 PET / CT, allowing a precise assessment of the extent and kinetics of CART-cell infiltration in the tumor, b) the tumor cell killing capacity of CTTR-PZ1-CART seems uncompromised compared to PZ1-CART cells, indicating that CTTR co-transduction does not affect the efficacy of CART cell treatment, and c) CTTR-PZ1-CART cells apparently form a memory population that can be rapidly remobilized to clear PSMA-positive tumor cells upon rechallenge.

[0270] These results underscore the general utility and power of the CTTR / [68Ga]IMP-288 based T cell tracking strategy, but also demonstrate some potential for improvement of the CTTR design, especially in terms of co-transduction efficiency with a (any) CAR. Therefore, the design of the CTTR was revised, and several variants were investigated in vitro.

[0271] Further optimizations of retroviral construct for CTTR transduction

[0272] Generally, higher target expression provides higher imaging sensitivity in nuclear imaging. Enhancing the transduction efficiency of the CTTR construct, both in terms of copies per cell and in terms of % CTTR-positive cells, respectively, will allow to detect lower cell numbers on the one hand and to achieve higher absolute tracer uptake in CTTR- expressing T cells and thus higher target / b ackground ratios on the other hand. Therefore, we attempted to achieve improved cell surface expression of the CTTR tag by modifying different segments of the CTTR construct (transmembrane domain, intracellular domain, hinge region, linker without modifying the scFv component) (Figure 21 A).

[0273] In construct 1 (“CD4”), CTTR-CD4, in frame with the scFv, CD4 D3-D4 ectodomain, CD4 TM and truncated (t)CD4 endodomain were included. In construct 2 (“CD45”), CTTR-CD45, in frame with the scFv, tCD45 ectodomain, CD45 TM and truncated (t)CD45 endodomain were included. In construct 3 (“GS-CD8”), CTTR-GS, a GS long linker was cloned in between scFv and the CD8a Hinge, TM and endodomain. In construct 4 (“DAP 10”), CTTR-DAP10, in frame with the scFv, a tDAPIO ectodomain, DAP 10 TM and tDAPIO endodomain were included, and in construct 5 (“NGFR”), CTTR- NGFR, in frame with the scFv, tNGFR ectodomain, CD4 TM and tNGFR endodomain were included.

[0274] Interestingly, only the original (“ORI”) CTTR construct and the new constructs 3 and 5 showed detectable expression of the upstream-marker cMyc and thus the h679 scFv (Figure 21 B), with only the NGFR-CTTR (construct 5) showing comparable expression levels in both CD4+ and CD8+ T cell populations. Surprisingly, FACS analysis (Figure 21 C) revealed a substantially improved transduction with NGFR-CTTR in terms of copies per cell, with the NGFR-CTTR transduced cells representing a much more defined and clearly distinguishable cell population.

[0275] NGFR-CTTR was further found to have improved expression density on CD8+ and CD4+ human donor T cells, as reflected by substantially increased binding of Ga-IMP288 as compared to the original CTTR construct in a radioligand binding study (Figure 22).

[0276] The properties of NGFR-CTTR (construct 5) may therefore be improved over those of the original CTTR construct.

[0277] EXAMPLE 5

[0278] Optimization of l68Ga1IMP-288 affinity by trimerization

[0279] An alternative approach towards improving the sensitivity of [68Ga]IMP-288-based PET imaging lies in enhancing the affinity of [68Ga]IMP-288 to the h679 scFv in the CTTR construct, i.e. its molecular target. Since the [68Ga]IMP-288 structure is already specifically tailored for high-affinity binding to the h679 scFv, modifications in the pharmacophore to achieve improved binding affinity were precluded. However, the inventors hypothesis that ligand multimerization could instead be used to increase the (apparent) affinity of the radioligand. There is a large body of data available on the multimerization of radiolabeled RGD-peptides, demonstrating up to more than tenfold enhanced affinities for the tri- or tetrameric peptides compared to their monomeric counterparts. This, however, can only be achieved for molecular targets that cluster at the cell membrane (e.g. integrins), facilitating simultaneous tracer binding to more than one receptor (cooperative binding). For molecular targets such as the CTTR in this study, this cannot be expected. However, the inventors considered that multimerization of IMP-288 may lead to a higher apparent concentration at the cell surface, which would favor re- binding to the target after dissociation and could thus lead to an enhanced apparent receptor affinity.

[0280] Therefore, the trifunctional high-affinity Ga-chelator TRAP

[0033] was used as a trimerization platform to generate a trimeric IMP-288 analog, TRAP(IMP-288)3 (Figure 3). As anticipated and in accordance with the above theory, [natGa]TRAP(IMP -288)3 showed a slightly more than 3-fold higher affinity to the h679 scFv than the monomer [natGa]IMP- 288 (IC50 = 0.13 ± 0.02 vs 0.62 ± 0.24 nM, respectively).

[0281] To assess the influence of this improved affinity on ligand biodistribution, both a comparative biodistribution study and PET / CT imaging were performed for [68Ga]IMP- 288 vs [68Ga]TRAP(IMP-288)3 in NSG mice bearing subcutaneous HSG-rec2-Jurkat xenografts (Figures 23 and 24).

[0282] As expected, given the outstanding selectivity of the IMP-288-targeting vector for the h679 scFv in HSG-rec2 (and CTTR), [68Ga]IMP-288 and [68Ga]TRAP(ZMP-288)3show virtually no uptake in any other tissue than the HSG-rec2 positive Jurkat tumor and the kidney (due to renal excretion of the tracers). One observation, however, is highly interesting, namely the identical background clearance of the two compounds despite the considerably increased molecular weight of [68Ga]TRAP(IMP-288)3 (4373 Da vs 1519 Da for [68Ga]IMP-288). In previous studies using TRAP-trimerized RGD peptides, this had led to prolonged tracer circulation and thus enhanced background accumulation. Apparently, however, this effect does not apply to [68Ga]TRAP(IMP-288)3.

[0283] The results of the biodistribution study were further confirmed via a comparative PET imaging study (Figure 24). [68Ga]IMP-288 displayed its usual biodistribution pattern with the only organ with relevant tracer uptake being the HSG-rec2 transduced Jurkat tumor, followed by a faint renal activity signal due to tracer excretion. HSG-rec2 specificity of tumor accumulation was further confirmed by coinjection of a 100-fold molar excess of [natGa]IMP-288, which reduced tracer uptake in tumor to background levels. In the case of [68Ga]TRAP(IMP -288)3, blocking with a 100-fold molar excess of [natGa]TRAP(IMP -288)3 was even more efficient, demonstrating an advantage of the trimer over the monomer in terms of binding specificity.

[0284] To highlight the particular selectivity of the [68Ga]IMP-288 / CTTR pair as a reporter system (with no endogenous receptor expression whatsoever), an additional imaging study was performed using [68Ga]PentixaTher. This tracer binds with high affinity to human CXCR4 and with moderate affinity to mouse CXCR4. Since Jurkat cells express relatively high levels of CXCR4, this target may also serve to visualize the (HSG- rec2 transduced) subcutaneous Jurkat tumors used in this experiment. As shown in Figure 24, [68Ga]PentixaTher also shows high and specific accumulation in the Jurkat xenografts, but due to delayed blood clearance of this radiopharmaceutical (compared to [68Ga]IMP- 288) and additional tracer binding to endogenously expressed CXCR4 (liver, spleen), imaging quality is greatly inferior to both [68Ga]IMP-288 and [68Ga]TRAP(IMP-288)3 PET.

[0285] Summary of the Examples

[0286] Overall, by generating two different constructs, one for retroviral (HSG-recl) and the other for lentiviral cell transduction (HSG-rec2), Jurkat cells were effectively gene engineered to express the h679 ScFv on their cell surface, as shown by flow cytometric analysis (Figure 6A and 9A) and confirmed by the radioligand binding assay using [68Ga]IMP-288 (Figure 6B and 9C).

[0287] In vivo, HSG-rec2 expressing Jurkat cells could be tracked longitudinally with high sensitivity using [68Ga]IMP-288 PET, both in a subcutaneous (Figure 11) and an intravenous tumor model (Figures 12 and 13). Thus, T-cell tracking using the HSG-rec2 construct is not only feasible shortly after cell implantation, as shown for the subcutaneous model (Figure 11, 1 and 5 days after inoculation). In contrast, HSG-rec2 transduced Jurkat cells maintain high levels of surface expression of the reporter-tag over extended periods of time (> 28 days) and show the anticipated expansion and homing to CXCL12 expressing tissues in vivo (Figures 12 and 13). This allows longitudinal, robust tracking of HSG-rec2 expressing cells over time and thus fully supports the feasibility of the underlying reporter tag concept.

[0288] Of note, the transduction of primary donor T cells with the retroviral HSG-recl vector was found to be feasible in a set of confirmatory experiments (Figure 14 displays one of three highly reproducible transduction experiments), providing robust HSG-recl expression in primary CD4+ and CD8+ T cells and thus the basis for in vivo tracking of adoptively transferred donor T cells using [68Ga]IMP-288 PET. Although the co-transduction of primary human donor T cells with an anti PSMA-CAR (PZ1) and CTTR provided relatively low co-transduction efficiencies in a pilot experiment, adoptively transferred CTTR-PZ1-CART cells (both peritumorally and intravenously) could be sensitively tracked via [68Ga]IMP-288 PET PC3-Pip tumor bearing mice over a period of > 4 weeks (Figure 20). Of note, the antitumor efficacy of the CART cells was not compromised by the co-transduction with CTTR, providing unbiased tumor control by the CTTR-PZ1-CART cells in the PC3-Pip model. Additionally, a rechallenge experiment demonstrated that even after complete tumor remission, a memory population of CTTR- PZ1-CART cells persisted in the treated mice, as demonstrated by high [68Ga]IMP-288 uptake at the injection site (on days 1 and 3 post rechallenge; Figure 20) and rapid clearance of the injected tumor cells.

[0289] The further optimization of the CTTR design (modifications of the transmembrane domain, intracellular domain, hinge region and linker without modifying the scFv component) provided a novel variant (NGFR-CTTR) with substantially improved expression levels in primary human T cells.

[0290] To further improve the affinity of [68Ga]IMP-288 towards the h679 scFv in the CTTR construct, a trimeric analog, [68Ga]TRAP(IMP-288)3 was synthesized and evaluated in vitro and in vivo. A reduction in background was demonstrated for [68Ga]TRAP(IMP- 288)3.

[0291] What makes the present [68Ga]IMP-288 / CTTR-based T cell tracking approach particularly unique and powerful, is the established complete lack of “on-target-off-site” binding of [68Ga]Ga-IMP-288, which is a result of the h679 ScFv being exclusively expressed on the respective transduced cells, with no endogenous expression whatsoever. Furthermore, the fact that [68Ga]Ga-IMP-288 is already used clinically in the context of immune PET using bispecific antibodies, considerably lowers the translational hurdles for clinical translation of our approach, at least with regard to the tracer component.

Claims

Claims1. A polypeptide that comprises:(a) a binding domain specific for histamine-succinyl-glycine (HSG); and(b) a membrane anchor that is capable of anchoring the binding domain in a plasma membrane.

2. The polypeptide of preceding claim 1, wherein the membrane anchor is devoid of signalling function.

3. The polypeptide of claim 1 or 3, wherein the binding domain comprises or consists of a single chain variable fragment (scFv), a scFv-Fc, a single-domain antibody, or a monoclonal antibody.

4. The polypeptide of any one of the preceding claims, wherein the binding domain comprises or consists of an scFv, optionally a humanised scFv.

5. The polypeptide of any one of the preceding claims, wherein the binding domain comprises or consists of an scFv that is capable of binding to IMP -288 or an analogue thereof.

6. The polypeptide of any one of the preceding claims, wherein the binding domain comprises or consists of an scFv that comprises (i) a light chain variable region that comprises a light chain CDR1 (LCDR1) of SEQ ID NO: 1, a light chain CDR2 (LCDR2) of SEQ ID NO: 2 and a light chain CDR3 (LCDR3) of SEQ ID NO: 3, and (ii) a heavy chain variable region that comprises a heavy chain CDR1 (HCDR1) of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 5 and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 6.

7. The polypeptide of any one of the preceding claims, wherein the binding domain comprises or consists of an scFv that comprises (i) a light chain variable region of SEQ IDNO: 7, and (ii) a heavy chain variable region of SEQ ID NO: 8.

8. The polypeptide of any one of the preceding claims, wherein the membrane anchor comprises one or more domains of one or more transmembrane proteins.

9. The polypeptide of claim 8, wherein the one or more transmembrane proteins are expressed by immune cells, optionally by T cells.

10. The polypeptide of claim 8 or 9, wherein one or more of the transmembrane proteins is a co-receptor for the T cell receptor (TCR), or a T cell co-stimulatory molecule.

11. The polypeptide of any one of claims 8 to 10, wherein the one or more transmembrane proteins comprise CD3(^, CD4, CD8 and / or CD28.

12. The polypeptide of any one of claims 8 to 11, wherein each of the one or more domains is an exodomain, a transmembrane domain or an intracellular domain of a transmembrane protein.

13. The polypeptide of any one of claims 8 to 12, wherein the one or more domains comprise an exodomain, a transmembrane domain and an intracellular domain.

14. The polypeptide of any claim 13, wherein two or three of the exodomain, transmembrane domain and intracellular domain are from the same transmembrane protein, optionally CD8 or CD28.

15. The polypeptide of any one of claims 8 to 14, wherein the membrane anchor comprises:(i) a tNGFR exodomain, a NGFR transmembrane domain, and a tNGFR endodomain;(ii) a CD8a exodomain, a CD8 transmembrane domain, and a CD8 endodomain, optionally wherein a GS linker links the CD8a exodomain to the binding domain;(iii) a CD8a exodomain, a CD28 transmembrane domain, and a CD28 endodomain; or(iv) an IgG CH2CH3 region, a CD8 transmembrane domain, and a CD8 endodomain.

16. The polypeptide of any one of the preceding claims, comprising or consisting of SEQ ID NO: 44, SEQ ID NO: 9, SEQ ID NO: 43, SEQ ID NO: 14; or SEQ ID NO: 17.

17. A cell expressing the polypeptide of any one of claims 1 to 16 on its surface.

18. A nucleic acid encoding the polypeptide of any one of claims 1 to 16.

19. A vector comprising the nucleic acid of claim 18.

20. The vector of claim 19, wherein the vector is a viral vector, optionally a retroviral vector, a lentiviral vector or an adenoviral vector.

21. A method for producing the cell of claim 17, comprising:(i) transfecting or transducing a cell with the nucleic acid of claim 18; and(ii) expressing the polypeptide encoded by the nucleic acid in the cell.

22. The method of claim 21, wherein transduction in step (i) comprises contacting the cell with the vector of claim 19 or 20.

23. The cell of claim 17, or the method of claim 21 or 2, wherein the cell is (a) an immune cell, optionally a T cell or (b) a cell having regenerative capacity, optionally a stem cell.

24. A method of treating a disease in an individual, comprising administering the cell of claim 17 or 23 to the individual.

25. The method of claim 24, wherein the disease is cancer.

26. The method of claim 24 or 25, further comprising administering a radioligand comprising HSG to the individual.

27. The method of claim 26, further comprising tracking the cell in vivo by using radionuclide imaging to determine the location of cells binding the radioligand within the individual.

28. The method of claim 26 or 27, wherein binding of the radioligand to the cell controls proliferation and / or cytotoxicity of the cell.

29. A method of tracking a cell in vivo, comprising:(i) administering the cell of claim 17 or 23 to an individual;(ii) administering a radioligand comprising HSGto the individual; and subsequently(iii) using nuclear imaging to determine the location of cells binding the radioligand within the individual; optionally wherein the radioligand does not comprise a binding molecule.

30. The method of any one of claims 26 to 29, wherein the radioligand comprises radiolabeled IMP-288 or a radiolabeled IMP-288 analogue.

31. The method of claim 30, wherein the radiolabeled IMP -288 is [68Ga]IMP-288.

32. The method of claim 30, wherein the radiolabeled IMP -288 analogue comprises: a) [99mTc]rnas3-IMP288 or [99mTc]N4-IMP288; b) [64 / 67CU]NOTA-IMP288, [64 / 67CU]NODAGA-IMP288, [64 / 67CU]NODA-IMP288 or [64 / 67Cu]TECB2A-IMP288; c) a dimer using DOTA as bridging unit, optionally labeled with212Pb; d) a TRAP-trimer (TRAP(IMP-288)3); e) a DOTPI-tetramer; or f) a radionuclide-specific chelator, optionally DOTAM or Macropa.

33. The method of any one of claims 26 to 32, wherein radionuclide imaging comprisesPET or SPECT.

34. The cell of claim 17 or 23 for use in the method of any one of claims 24 to 33.

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