Patent Application
20230365636
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named Sequence_Listing and is 36 kilobytes in size.
The present invention relates to a nucleic acid molecule encoding a fusion protein comprising (i) a secretory signal peptide; (ii) a lipocalin-derived binding protein that specifically binds to an exogenous ligand; and (iii) a glycosylphosphatidylinositol (GPI) anchored and/or transmembrane domain.
In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The life sciences have greatly benefited from the development of reliable reporter gene systems such as fluorescent proteins, luciferases or enzymes. These systems enabled the time-resolved quantification of transcription, analysis of cis-acting genetic elements, understanding of developmental processes, determining protein localization, construction of synthetic biosensors and will in the future be applied in synthetic biology to monitor the dynamics of artificial biological systems (Ghim et al., 2010).
First steps towards the use of engineered cells in a medical context are exemplified by the clinical studies and recent FDA approval of chimeric antigen receptors (CARs) in T-cells, which harness the immune system to specifically combat cancer. Furthermore, therapeutic interventions by gene and cell therapy vectors such as Adenovirus- or AAV-based vectors have become established therapies today. Generally, there is desire to monitor or image such genetically modified cells in the body of the patient during and after therapy.
A reporter protein that can be used in various animal models as well as in human patients requires good tissue penetration of the resulting signal, which is unfortunately not the case for conventional, light-based reporter proteins. In contrast, radioactive imaging technologies such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are routinely applied in nuclear medicine and medical care and allow the highly sensitive, spatiotemporally resolved and quantitative monitoring of signals within patients.
In this context, there is an ongoing need for a reporter protein system suitable to reliably monitor these gene/cell therapies and/or engineered/transformed cells within a patient (Serganova and Blasberg, 2005, Serganova et al., 2008, Brader et al., 2013). This need is addressed by the provision of the embodiments characterized in the claims.
Accordingly, the present invention relates in a first aspect to a nucleic acid molecule encoding a fusion protein comprising (i) a secretory signal peptide; (ii) a lipocalin-derived binding protein that specifically binds to an exogenous ligand; and (iii) a glycosylphosphatidylinositol (GPI) anchored and/or transmembrane domain.
The term “nucleic acid molecule”, also referred to a nucleic acid sequence or polynucleotide herein, as used herein includes DNA, such as cDNA or genomic DNA, synthetic DNA and RNA. It is understood that the term “RNA” as used herein comprises all forms of RNA including mRNA. Both, single-strand as well as double-strand nucleic acid molecules are encompassed by this term. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2′-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see (Braasch and Corey, 2001)).
The nucleic acid molecules of the invention can e.g. be synthesized by standard chemical synthesis methods or isolated from natural sources or produced semi-synthetically, i.e. by combining chemical synthesis and isolation from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods, such as restriction digest, polymerase chain reaction (PCR), ligation and molecular cloning.
The term “(poly)peptide” in accordance with the present invention describes a group of molecules which comprises the group of peptides, consisting of up to 50 amino acids, as well as the group of polypeptides, consisting of more than 50 amino acids. Also encompassed by the term “(poly)peptide” are proteins as well as fragments of proteins. (Poly)peptides may further form dimers, trimers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. (Poly)peptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. Homo- or heterodimers etc. also fall under the definition of the term “(poly)peptide”. The terms “polypeptide” and “protein” are used interchangeably herein and also refer to naturally modified polypeptides wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
The “fusion protein” as used herein refers to a protein, wherein (i) a secretory signal peptide; (ii) a lipocalin-derived binding protein specifically binding to an exogenous ligand; and (iii) a glycosylphosphatidylinositol (GPI) anchored and/or transmembrane domain are fused to each other directly or via a peptide-linker. The term “peptide-linker”, as used in accordance with the present invention, preferably relates to peptide or polypeptide linkers, i.e. a sequence of amino acids.
A peptide-linker as envisaged by the present invention is at least 1 amino acid in length. Preferably, the peptide-linker is 1 to 100 amino acids in length. More preferably, the peptide-linker is 5 to 50 amino acids in length and even more preferably, the peptide-linker is 10 to 20 amino acids in length. Preferably, the peptide-linker is a flexible peptide-linker using e.g. the amino acids glycine and/or serine. Preferably, the peptide-linker sequences are (Gly4Ser)3, or (Gly4Ser)2. The length and sequence of a suitable peptide-linker depends on the composition of the respective specific fusion protein. Methods to test the suitability of different peptide-linkers are well known in the art and include e.g. the comparison of the binding affinity to the exogenous ligand or the protein stability or the production yield (surface density of the functional protein) of the fusion protein with and without peptide-linkers.
In the case of a “fusion protein”, conjugation may be carried out by recombinant DNA technology using well established techniques. As a result, the conjugate is created as one continuous polypeptide chain through the joining of two or more genes that originally code for separate molecules. Translation of this fusion gene results in a fusion protein with functional properties derived from each of the original molecules (i) to (iii). Suitable vectors for the recombinant production of the fusion protein are known in the art and will be described herein below.
The term “secretory signal peptide” designates a short peptide (e.g. 15-50 amino acids long) that can be found naturally at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. A typical secretory signal peptide comprises three distinct regions: a polar N-terminal end (n-region) that may have a net positive charge, a central hydrophobic core (h-region) that consists of 6-15 hydrophobic amino acids, and a polar C-terminal (c-region) end that contains amino acids with small side chains. Such a signal peptide that can also be found on the N-terminus of a type I membrane-bound protein. Type I membrane-bound proteins are anchored to the lipid membrane with a stop-transfer anchor sequence and have their N-terminal domains targeted to the endoplasmic reticulum (ER) lumen during biosynthesis and to the extracellular space if mature forms are located on the cell membrane as anticipated in this invention.
In some embodiments, the hydrophobic C-terminal sequence of the translocated protein may be cleaved and replaced by a glycosylphosphatidylinositol (GPI) anchor such that the protein finally remains attached to the outer leaflet of the cell membrane (Paulick and Bertozzi, 2008).
The term “that specifically binds to an exogenous ligand” relates to a molecule that is capable of specifically binding to (also referred to herein as “specifically interacting with”) an exogenous ligand but is not capable of or essentially not capable of cross-reacting with a different compound. Cross-reactivity of a panel of molecules under investigation may be tested, for example, by assessing binding of said panel of molecules to the exogenous ligand as well as to a number of more or less (structurally and/or functionally) closely related compounds. Only those molecules that bind to the exogenous ligand but do not or do not essentially bind to any of the other compounds are considered specific for the exogenous ligand.
The term “a molecule that essentially does not cross-react”, as used herein, refers to a molecule that binds to the exogenous ligand with at least 5-times higher affinity as compared to a different exogenous ligand of similar structure, more preferably at least 10-times higher affinity, such as e.g. at least 50-times higher affinity, more preferably at least 100-times higher affinity, such as e.g. at least 250-times higher affinity. Even more preferably, it binds with at least 500-times higher affinity to the exogenous ligand than to a different compound of similar structure and most preferably with at least 1000-times higher affinity.
The term “ligand” as used herein refers to the compound which is specifically bound by a lipocalin-derived binding protein according to the invention. As discussed, a naturally-occurring lipocalin can be engineered into a lipocalin-derived binding protein that is capable of specifically binding virtually any desired compound. The nature of the ligand is, thus, not particularly limited. The ligand can be, for example, a (poly)peptide, a carbohydrate or a small organic compound. As is evident from the appended examples, a lipocalin-derived binding protein according to the invention may, for example, specifically bind to Bn-CHX-A″-DTPA⋅Me, colchicine or a demethylated colchicine, petrobactin, dig(it)oxigenin or fluorescein. As will be further discussed herein below, the ligand according to the present invention is preferably linked to a radionuclide. Also, the ligands as used in the appended examples are linked to a radionuclide. A radionuclide is a label allowing the detection of the ligand, for example, in vivo within a subject.
The term “exogenous” as used in connection with the ligand indicates that the ligand is an exogenous factor that does no naturally occur in the cell wherein the fusion protein of the invention is to be incorporated and, if used for any medical or diagnostic application, also does not naturally occur in the subject to be treated or diagnosed. The subject is preferably human. Hence, an exogenous factor originates outside that cell and/or subject, as opposed to an endogenous factor.
The term “a lipocalin-derived binding protein specifically binding to an exogenous ligand” may be one or more than one lipocalin-derived binding protein and is preferably one lipocalin-derived binding protein. In the case of more than one lipocalin-derived binding protein each type of lipocalin-derived binding protein may specifically bind to a distinct exogenous ligand. Hence, in the case of a first and a second lipocalin-derived binding protein the first lipocalin-derived binding protein may specifically bind to a first exogenous ligand and the second lipocalin-derived binding protein specifically may bind to a second exogenous ligand. Alternatively, it is possible to use two or more copies of the same lipocalin-derived binding protein. In this case, more than one ligand can be bound to the fusion protein. This in turn can increase the signal strength or specificity, for instance, in case the ligand is bound to a label, which is preferably a radionuclide as will be described in more detail herein below.
Lipocalin-derived binding proteins, also referred to as Anticalins, represent a class of non-immunoglobulin binding proteins based on the human lipocalin scaffold. Lipocalins comprise a diverse family of small (20 kDa) extracellular proteins that occur in many species ranging from bacteria to humans and serve for the transport or scavenging of physiological compounds. Despite mutually low sequence homology, the three-dimensional fold of lipocalins is highly conserved (Schiefner and Skerra, 2015).
The single chain molecular architecture of lipocalin-derived binding proteins is dominated by a compact eight-stranded anti-parallel β-barrel. At the open end of the barrel there are four loops connecting each pair of β-strands. The four structurally variable loops are referred to herein as “loop regions”, whereas the remainder of the protein makes up the framework or “frame regions”. Thus, similar to the structure of antibodies, the lipocalin-derived binding proteins according to the present invention are essentially made of conserved frame(work) regions that are generally not directly involved in the binding to the exogenous ligand, as well as hypervariable, specificity-determining segments with amino acid residues being involved in the binding to the exogenous ligand (here the loop regions, which might be seen as resembling the CDRs in antibodies).
Hence, lipocalin and also lipocalin-derived binding proteins consist of frame regions and loop regions according to the following scheme:
The lipocalin-derived binding protein is preferably a lipocalin 2 (Lcn2)-derived binding protein. Lipocalin-2 (Lcn2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a protein that in humans is encoded by the LCN2 gene. Human LCN2 mRNA is, for example, represented by the NCBI Reference Sequence: NM_005564.5 (as available on Mar. 12, 2019) and the amino acid sequence of human Lcn2 protein including the signal peptide is, for example, represented by the UniProt ID P80188-2 (as available on Nov. 1, 1995).
The lipocalin-derived binding protein binds the exogenous ligand with increased preference with a KD of 200 nM or lower, 100 nM or lower, 50 nM or lower, 10 nM or lower, 1 nM and 500 pM or lower.
The term “KD” refers to the equilibrium dissociation constant (the reciprocal of the equilibrium binding constant) and is used herein according to the definitions provided in the art.
The KD value with which the lipocalin-derived binding protein binds the exogenous ligand can be determined by well known methods including, without being limiting, fluorescence titration, competition ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), flow cytometric titration analysis (FACS titration), radioligand binding assays and surface plasmon resonance spectroscopy (BIAcore). Such methods are well known in the art and have been described e.g. in De Jong, L. A. A. et al. [2005] J. Chromatogr. B 829(1-2):1-25; Heinrich, L. et al. [2010] J. Immunol. Methods 352(1-2):13-22.
Preferably, ELISA or competition ELISA or surface plasmon resonance (BIAcore) is employed to ensure that the KD of the lipocalin-derived binding protein binds the exogenous ligand with the required KD. Even more preferably, the KD is determined by surface plasmon resonance (BIAcore).
A “glycosylphosphatidylinositol (GPI) anchored domain” as used herein relates to a domain comprising a GPI anchor. GPI is composed of a phosphatidylinositol group linked through a carbohydrate-containing linker (glucosamine and mannose glycosidically bound to the inositol residue) and via an ethanolamine phosphate (EtNP) bridge to the C-terminal amino acid of a mature protein. The two fatty acids within the hydrophobic phosphatidyl-inositol group anchor the protein to the cell membrane.
The term “transmembrane domain” as used herein designates any cell membrane-spanning protein domain. Transmembrane domains (TMDs) generally consist predominantly of nonpolar amino acid residues and may traverse the cell membrane bilayer once or several times. The transmembrane domain is preferably a single-pass transmembrane domain. TMDs usually consist of α-helices. The peptide bond is intrinsically polar and can form internal hydrogen bonds between carbonyl oxygens and amide nitrogens. Within the lipid bilayer, where water is essentially excluded, peptides usually adopt the α-helical configuration that maximizes their internal hydrogen bonding. A helix length of 18-21 amino acid residues is sufficient to span the usual width of a lipid bilayer.
It is, thus, to be understood that the fusion protein being encoded by the nucleic acid molecule comprises (i) a secretory signal peptide; (ii) a lipocalin-derived binding protein specifically binding to an exogenous ligand; and (iii) a glycosylphosphatidylinositol (GPI) anchored and/or transmembrane domain in that particular order (i) to (iii) from the N-terminus to the C-terminus of the fusion protein or, alternatively, from the C-terminus to the N-terminus, in this case without a secretory signal peptide, depending on the type of transmembrane domain to be used. The orientation has to be selected—based on the type of transmembrane domain to be used—such that after the incorporation of the transmembrane domain into the cell membrane a lipocalin-derived binding protein specifically binding to an exogenous ligand is found on the extracellular side.
It is evident from the examples herein below that once the nucleic acid molecule of the invention is expressed in a cell the encoded fusion protein of the invention is processed into the mature fusion protein (lacking the signal peptide (i)) and incorporated into the plasma membrane of the cell via the transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor, so that the lipocalin-derived binding protein specifically binding to an exogenous ligand is anchored and present on the extracellular surface of the cell. Once the exogenous ligand comes into contact with a cognate lipocalin-derived binding protein the lipocalin-derived binding protein and the exogenous ligand firmly bind to each other. This binding can be detected (e.g. visualized) by labelling the exogenous ligand with a detectable label as will be described in greater detail herein below.
The anchored lipocalin-derived binding protein encoded by a nucleic acid molecule of the invention and the exogenous ligand preferentially form a reporter system. These compounds making up the reporter system can be administered to a patient or subject (preferably a human patient or subject) either simultaneously or sequentially (wherein in the latter case the nucleic acid molecule of the invention is generally to be administered first).
The exogenous ligand is preferably administered systemically, e.g. via intravenous transfusion or injection. Upon administration the exogenous ligand is distributed among the blood compartment and is selectively bound by the lipocalin-derived binding protein. This in turn allows the monitoring of the cells, e.g. with a living organism with minimal functional impairment and, on the other hand, with a minimal background signal.
As discussed, the lipocalin-derived binding proteins as used herein are also also-known as Anticalins. Anticalins constitute an emerging class of artificial binding proteins obtained by combinatorial design based on the compact and robust human lipocalin scaffold. Due to their human origin, the lipocalin-derived binding proteins according to the present invention have low immunogenic potential, and in several clinical trials Anticalins with different target specificities have demonstrated safety. Moreover, these lipocalin-derived binding proteins according to the present invention show remarkable target specificity, with dissociation constants in the nanomolar and even picomolar range (Table 1). The lipocalin-derived binding protein is preferably selected from SEQ ID NOs 3/4 and 5/6, noting that the lipocalin-derived binding protein of SEQ ID NO: 3/4 is capable of specifically binding DTPA and the lipocalin-derived binding protein of SEQ ID NO: 5/6 is capable of specifically binding colchicine.
As will be further detailed herein below, the Anticalin-based reporter system as provided herein is in particular suitable for therapeutic and diagnostic purposes. The Anticalin-based reporter system is particularly well suited for applications in CAR T-cell anti-cancer therapy, gene therapy and diagnostics.
An antibody-based reporter system which is similar to the Anticalin-based reporter system as provided herein is described in (Krebs et al., 2018). To design a reporter system based on an Anticalin instead of an antibody is technically advantageous because an Anticalin by nature is a single polypeptide chain molecule whereas an antibody comprises heavy and light chains. The less complicated and less interference-sensitive folding process of the Anticalin as compared to an antibody or antibody fragment ensures efficient folding within the secretory pathway and, thus, allows for high expression on the cell surface. Especially when used to genetically tag CAR T-cells, the use of an immunoglobulin folding motif with light and heavy chain may prove disadvantageous as the light chains of i) the antibody-based reporter gene and ii) the chimeric antigen receptor (CAR) may mutually interfere with correct folding. While based on an antibody, a single-chain fragment (scFv) may be designed as was done by (Krebs et al., 2018),
Anticalins display an improved binding affinity to their cognate exogenous ligands as compared to a single-chain fragment of an antibody. (Krebs et al., 2018) designed an antibody-based reporter system wherein the binding between the single-chain fragment and the exogenous ligand is irreversible (i.e. infinite). Due to the superior binding affinity of an Anticalin the Anticalin-based reporter system as provided herein is stable in vivo without the need of an irreversible covalent bond between the Anticalin and the cognate exogenous ligand. Furthermore, the antibody used in (Krebs et al., 2018) was of murine origin and only of limited value for clinical translation. As can be taken from the examples, the system according to the invention allows the clinical monitoring of emerging therapies such as CAR T-cells or gene therapy. The reversible binding between the Anticalin and the cognate exogenous ligand makes the Anticalins-based reporter system as provided herein more flexible and easier to use for such applications.
In summary, the disadvantages of antibody-based reporter system and in particular the scFv fragment-based reporter system of Krebs et al., 2018 are 1) the tendency of the scFv fragment to form oligomers (e.g. Hudson & Kortt, 1999), 2) low surface expression of the system, 3) the murine origin of the system and the resulting potential immunogenicity in humans, and 4) the cross-reactivity of the radioligand (lanthanoid (S)-2-(4-acrylamidobenzyl)-DOTA (AABD)). As an acrylamide derivative the radioligand may unspecifically bind to structures other than the DOTA antibody reporter 1 (DAbR1) reporter protein. On the other hand, the advantages of the lipocalin-based reporter proteins according to the invention include, but are not limited to: 1) the human origin of the protein scaffold, 2) the smaller size of the reporter protein, 3) the single-chain protein architecture, 4) the high in vivo stability of the folded protein and 5) the absence of oligomerisation tendency.
In addition, Example 7 and
Thus, the Anticalin-based reporter system provided herein is in several aspects superior to antibody or antibody fragment-based reporter systems, including the treatment and diagnosis of tumors, and in particular for the in vivo imaging of diseases and therapies.
Means and methods for introducing and expressing the nucleic acid molecule of the invention in a cell, such that the encoded fusion protein is anchored in the cell membrane, will be discussed herein below.
In accordance with a preferred embodiment of the first aspect of the invention the exogenous ligand is linked to a radionuclide, wherein the radionuclide is preferably selected from C-11, F-18, Sc-44, Sc-47, Cu-64, Ga-68, Y-86, Y-90, Zr-89, Tc-99m, In-111, I-123, I-124, I-131, Tb-152 and Lu-177, Bi-213, Ac-225.
The radionuclide may be directly or indirectly linked (i.e. via linker) to the exogenous ligand. The linker is preferably an organic compound. Also, the linker preferably links the exogenous ligand and the radionuclide via 5 to 30 connecting atoms. As illustrated in the examples the linker is most preferably a polyethylene glycol (PEG)-linker.
As is illustrated in the appended examples, the radiolabeling of the exogenous ligand allows for detecting the exogenous ligand, e.g. within a living subject, and also allows for the detection and quantification of cells carrying the fusion protein of the invention in the cell membrane, wherein these cell have bound the exogenous ligand. Each cell comprises a plurality of fusion proteins within its cell membrane, such that also a plurality of the exogenous ligand is bound to the cell. Thereby labelled exogenous ligands come into close proximity on the cell surface which can be distinguished from unbound, free exogenous ligands, which are rapidly excreted via the kidneys or the hepato-biliary route. For this diagnostic application the exogenous ligand needs to be administered to a subject comprising such cells. As a consequence, the location of the cells, transfected with the nucleic acid according to this invention and having bound the ligand, can be visualized and quantified in the subject.
A radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide and is preferably a diagnostic radionuclide. A therapeutic radionuclide is a radionuclide that can be used for the treatment or prevention of a disease. For instance, a therapeutic radionuclide may be used to specifically kill cells that accumulate the radionuclide. The radionuclides being best suited for therapy are generally those emitting high energy ionizing radiation with short penetration into the tissue, e.g. α (alpha) or β− (beta) emitters. This ionizing radiation causes damage to the DNA and, thus, harm the cells. On the other hand, the radiation emitted by diagnostic radionuclides (e.g. β+ (positron) radiation for PET or γ (gamma) radiation for SPECT) essentially does not kill cells. Diagnostic radionuclides used, for example, for imaging procedures are generally radionuclides emitting γ (gamma) radiation, directly or after positron annihilation. γ (gamma) radiation is able to penetrate tissue.
The following lists of radionuclides are non-limiting but preferred examples of radionuclides that are used in the art for diagnostic (C-11, F-18, Sc-44, Cu-64, Ga-68, Y-86, Zr-89, Tc-99m, In-111, I-123, I-124, Tb-152, Tb-155) or therapeutic (Sc-47, Y-90, 1-131, Tb-149, Tb-161, Lu-177, Bi-213, Ac-225) purposes.
While a (1) diagnostic radionuclide such as Fluorine-18 is the preferred label it is also possible to link the Anticalin-bound exogenous ligand moiety to (2) (super)paramagnetic (nano)beads for Magnetic resonance imaging (MRI) detection or magnetic-activated cell sorting (MACS), (3) pharmaceutically active substances, such as growth factors, toxins or immunomodulators, or (4) therapeutic radionuclides to harm the cells.
In accordance with another preferred embodiment of the first aspect of the invention the encoded fusion protein further comprises a peptide affinity tag, preferably a V5, Strep-tag II, Flag, myc, HA, Spot, T7 or NE tag.
Affinity tags are peptide sequences genetically incorporated into the fusion protein of the invention, preferentially at the outer side of the cell membrane. This affinity tag can be used together with a cognate antibody or another binding protein, e.g. streptavidin, for the detection of the invention by various methods such as flow cytometry, immunofluorescence microscopy, western blot detection or immunohistochemistry (IHC). Furthermore, the affinity tag can be used together with a cognate antibody, or streptavidin/Strep-Tactin, and/or superparamagnetic microbeads for the isolation of cells labelled with the invention via magnetic-activated cell sorting (MACS) (Miltenyi and Schmitz, 2000, Grutzkau and Radbruch, 2010)
In the examples herein below the V5-epitope tag is used as a peptide affinity tag, so that the peptide affinity tag is most preferably a V5-epitope tag (Southern et al., 1991, Dunn et al., 1999).
The affinity tag generally is located on the extracellular part of the fusion protein of the invention. It is preferably inserted between (ii) the lipocalin-derived binding protein specifically binding to an exogenous ligand; and (iii) a glycosylphosphatidylinositol (GPI) anchored and/or transmembrane domain. The affinity tag is most preferably inserted at this position in the form of the linker of SEQ ID NO: 7/8, which linker comprises a V5-epitope tag. This has the advantage that the tag is available on the outside of a cell but very likely does not interfere with the ability of a lipocalin-derived binding protein to specifically bind its exogenous ligand.
In accordance with a further preferred embodiment of the first aspect of the invention the encoded fusion protein further comprises a fluorescent protein, preferably a monomeric autofluorescent protein such as mRuby3, GFP, eGFP, sfGFP, UnaG, miRFP703 or miRFP720.
The fluorescent protein is used to enable a visual or spectrophotometric readout on the fusion protein of the invention, on a cellular and sub-cellular level.
GFP (green fluorescent protein) and its variants (e.g. eGFP and sfGFP) are the most commonly used fluorescence tags. More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not).
mRuby3 (Bajar et al., 2016) is a monomeric bright red fluorescent protein derived from Entacmaea quadricolor which is has a low acid sensitivity.
UnaG is a basic (constitutively fluorescent) green fluorescent protein derived from Anguilla japonica. It has low acid sensitivity. It requires the cofactor bilirubin for fluorescence (Kumagai et al., 2013).
MiRFP703 (Shcherbakova et al., 2016) or miRFP720 (Shcherbakova et al., 2018) are monomeric near-infrared (NIR) fluorescent proteins that also require the cofactor bilirubin for fluorescence. Due to the low absorbance of different tissues for light of this wavelength and the low autofluorescence, these fluorescent proteins can be used for in vivo imaging experiments.
In the examples herein below mRuby3 and miRFP720 are used as the fluorescent protein, so that the fluorescent protein is most preferably the mRuby3 of SEQ ID NO: 17/18 or the miRFP720 of SEQ ID NO: 19/20.
The fluorescent protein preferably is located on the intracellular part of the fusion protein of the invention, such that it may not interfere with the ability of the lipocalin-derived binding protein to specifically bind its exogenous ligand.
The fluorescent protein is preferably separated from the reporter protein using a 2A self-cleaving peptide sequence. 2A self-cleaving peptide sequences (or 2A peptides) are a class of 18-22 residue peptides, which can induce ribosomal skipping during translation of a protein in a cell (see, for example, Liu et al. (2017)), Sci Rep., 7:2193).
The exemplified fusion protein of the invention without a fluorescent protein but comprising an anti-colchicine Anticalin, a linker with a V5-tag SEQ ID NO: 7/8 and the transmembrane domain of CD4 is shown in SEQ ID NO: 9/10. Said exemplified fusion protein with a mRuby3 label is shown in SEQ ID NO: 21/22 and said exemplified fusion protein with a miRFP720 label is shown in SEQ ID NO: 27/28. The fusion proteins comprising any one of SEQ ID NOs 21/22 and 27/28 are particularly preferred examples of the fusion protein of the invention. These SEQ ID NOs comprise the fluorescent protein within the intracellular part of the fusion protein.
The detection of the fluorescent protein can be used to detect the presence and location of the encoded fusion protein, for example in vivo. In case the fusion protein sits in a cell and that cell is to be administered to a subject and presence, location and movement of the cells in the subject can be detected. In case the exogenous ligand comprises a radionuclide as will be discussed herein below, the co-localization of the radionuclide and the fluorescent protein may indicate the binding of a lipocalin-derived binding protein of the fusion protein to the exogenous ligand.
In accordance with a preferred embodiment of the first aspect of the invention the exogenous ligand comprises a small molecule, wherein the small molecule is preferably selected from (i) a chelator, preferably a metal chelator complex, more preferably [(R)-2-amino-3-(4-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid (p-NH2-Bn-CHX-A″-DTPA), (ii) an alkaloid, preferably colchicine or a demethylated variant thereof, (iii) an iron-chelating siderophore, preferably petrobactin, enterobactin or bacillibactin, (iv) a plant steroid, preferably digoxigenin or digitoxigenin, and (v) an organic dye, preferably fluorescein or aminofluorescein, or the exogenous ligand is a peptide with at least 2 and less than 10 amino acid residues, preferably VFFAED (SEQ ID NO: 29).
It is to be understood that in accordance with a preferred embodiment the lipocalin-derived binding protein specifically binds a small molecule. Hence, the small molecule comprises an “epitope” which is recognized and specifically bound by the lipocalin-derived binding protein.
A small molecule is a low molecular weight compound which is by definition not a polymer. The upper molecular weight limit for a small molecule is preferably 2000 Da, more preferably 1500 Da and most preferably 1200 Da. Libraries of small organic molecules and high-throughput techniques for screening such libraries with a binding protein, such as an antibody or an Anticalin, are established in the art. The small molecule is preferably an organic small molecule. Particularly, preferred examples of small molecules comprised by the exogenous ligand are shown in
The present invention is also directed to an exogenous ligand comprising a small molecule selected from the small molecules as shown in
Chelators are small molecules with two or more separate interacting groups that can bind to a single central metal ion, forming a chelator complex. The chelator p-NH2-Bn-CHX-A″-DTPA is used in the art together with a radionuclide (e.g. Y-90) as a therapeutic radiometal (Price and Orvig, 2014, Price et al., 2016).
Alkaloids are a class of nitrogenous organic compounds of plant origin which have pronounced physiological actions on humans. They include many drugs (morphine, quinine) and poisons (atropine, strychnine). The alkaloid colchicine is a drug that is used mainly in gout to treat an acute attack or to prevent an attack while starting uric acid-lowering therapy.
Iron-chelating siderophores are small, high-affinity iron-chelating compounds that are secreted by microorganisms such as bacteria and fungi and serve primarily to transport iron across cell membranes, although a broader range of siderophore functions is now being appreciated. Siderophores are among the strongest soluble Fe3+ binding agents known. Non-limiting but preferred examples of iron-chelating siderophores are petrobactin, enterobactin and bacillibactin.
Organic dyes possess a colour because they 1) absorb light in the visible spectrum (400-700 nm) or the near-infrared spectrum (700-950 nm), 2) have at least one chromophore (colour-bearing group), 3) have a conjugated system, i.e. a structure with alternating double and single bonds, and 4) exhibit resonance of electrons. Non-limiting but preferred examples of organic dyes are fluorescein and aminofluorescein. Fluorescein is commonly used in microscopy, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494 nm and also an emission maximum of 512 nm (in water). The excitation wavelength of aminofluorescein (5-AF) is 490 nm and the emission wavelength is 515 nm.
Peptides have been defined herein above. The peptide VFFAED (SEQ ID NO: 29) is a fragment of the Alzheimer amyloid peptide.
In accordance with a further preferred embodiment of the first aspect of the invention the secretory signal peptide is the signal peptide of a lipocalin, preferably of lipocalin 2 (Lcn2).
As discussed above, the binding proteins according to the invention are lipocalin-derived and in particular derivatives of Lcn2. For this reason, it is preferred to use as the secretory signal peptide the naturally occurring signal peptide of a lipocalin, preferably of Lcn2. In the examples herein below the natural signal peptide of lipocalin 2 (Lcn2) is used, which is shown in SEQ ID NO: 1/2. The secretory signal peptide therefore most preferably comprises or consists of SEQ ID NO: 1/2.
In accordance with a still further preferred embodiment of the first aspect of the invention the transmembrane domain is the transmembrane domain of CD4 or CD28 or a sequence stimulating the attachment of a GPI-anchor.
The transmembrane domains of CD4 or CD28 are single-span a-helical transmembrane domains. Moreover, CD4 and CD28 are both expressed on T-cells and can be found in the cell membrane of T-cells. The choice of a protein sequence that is already expressed in the desired cell type (like CD4 or CD8 TMD in T-cells) is expected to decrease the risk of unintended immunogenicity caused by the membrane protein of the invention. As will be further discussed herein below, the fusion protein being encoded by the first aspect to is to be preferably incorporated into the cell membrane of T-cells. The transmembrane domains of CD4 or CD28 preferably have the sequences of SEQ ID NO: 9/10 and 11/12, respectively.
Alternatively, a sequence that stimulates the attachment of a glycosylphosphatidylinisotol (GPI) membrane anchor to the fusion protein of the invention may be used. To this end, the last 1 to 20 amino acid residues of this protein together with the downstream propeptide sequence of a human protein that is naturally anchored to the cell membrane via a GPI-anchor may be fused to the lipocalin-derived binding protein or the V5-tag. Sequences may be derived from every GPI-anchored protein, preferentially from human Complement decay-accelerating factor (CD55; UniProtKB-P08174 (DAF_HUMAN) as available on Jul. 31, 2020) SEQ ID NO: 13/14 or CD59 glycoprotein (UniProtKB-P13987 (CD59_HUMAN) as available on Jul. 31, 2020) SEQ ID NO: 15/16.
The present invention relates in a second aspect a vector comprising the nucleic acid molecule of the first aspect, wherein the vector is preferably a retroviral vector, an adenoviral vector or adeno-associated vector (AAV).
The definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second aspect of the invention.
The vector is typically an expression vector and can be a linear nucleic acid, plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g. in genetic engineering.
Generally, vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. In addition, the coding sequences cloned on the vector can be ligated to transcriptional regulatory elements and/or to other coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) (Owens et al., 2001), and, optionally, regulatory elements ensuring termination of transcription and stabilization of the transcript. Non-limiting examples for such regulatory elements ensuring the initiation of transcription comprise promoters, a translation initiation codon, enhancers, insulators and/or regulatory elements ensuring transcription termination. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. The vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding. Vectors can also contain a sequence encoding a 2A self-cleaving peptide or 2A peptide (Kim et al., 2011). These peptides are a class of 18-22 amino acids long peptides, which can induce cleavage of a recombinant protein in a cell. This can be used to express a functional protein such as a chimeric antigen receptor (CAR) together with the membrane protein of the embodiment under the control of a single promoter.
Vector elements that have been optimized for the expression of Anticalins in bacteria have been described in the art, e.g. in (Gebauer and Skerra, 2012) and include the tetracycline promoter/operator (tetp/o), which is chemically inducible with anhydrotetracycline, an affinity tag, such as e.g. Strep-tag II or the A3C5 tag, the rho-independent Ipp terminator as well as an ampicillin-resistance gene (β-lactamase), a truncated ColEI origin of replication, and, optionally, the intergenic region of the filamentous phage f1 for the biosynthesis of phagemid particles upon co-infection of E. coli with a helper phage.
Additional examples of suitable origins of replication include, for example, the full length ColE1, the SV40 viral and the M13 origins of replication, while additional examples of suitable promoters include, without being limiting, the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, EF-1α promoter, chicken β-actin promoter, CAG-promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer), the gai10 promoter, human elongation factor 1α-promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoter, the T7 or T5 promoter, the lacUV5 or ara promoter, the Autographa califomica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or a globin intron in mammalian and other animal cells. One example of an enhancer is e.g. the SV40-enhancer or the Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). Non-limiting additional examples for regulatory elements ensuring transcription termination include the SV40-poly-A site, the tk-poly-A site or the AcMNPV polyhedral polyadenylation signals. Further non-limiting examples of selectable markers include dhfr, gpt, neomycin, hygromycin, blasticidin or geneticin.
Preferably, the vector of the present invention is an expression vector. An expression vector according to this invention is capable of directing the expression of the nucleic acid molecule of the invention and, accordingly, of the fusion protein of the present invention encoded thereby.
The nucleic acid molecules and/or vectors of the invention as described herein above may be designed for introduction into cells by e.g. non-chemical methods (electroporation, sonoporation, optical transfection, gene electrotransfer, hydrodynamic delivery or naturally occurring transformation upon contacting cells with the nucleic acid molecule of the invention), chemical-based methods (calcium phosphate, liposomes, DEAE-dextrane, polyethylenimine, nucleofection), particle-based methods (gene gun, magnetofection, impalefection) phage vector-based methods and viral methods including infection. For example, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, adeno virus, herpes viruses, Semliki Forest Virus or bovine papilloma virus, may be used for delivery of the nucleic acid molecules into targeted cell population. Additionally, baculoviral systems can also be used as vectors in a eukaryotic expression system for the nucleic acid molecules of the invention.
The vector is preferably a retroviral vector, an adenoviral vector or adeno-associated vector (AAV) because these vectors are commonly used in gene therapy (Lundstrom, 2018). The most applied viral vectors are adenoviruses or viruses being based on adenoviruses. Naked dsDNA adenoviruses possess a packaging capacity of 7.5 kb of foreign DNA, providing short-term episomal expression of the gene of interest in a relatively broad range of host cells. The original adenovirus vectors generated strong immune responses, whereas the so-called gutted second and third generation vectors containing deletions have proven to elicit substantially reduced immunogenicity.
AAV vectors carry a small ssRNA genome, which allows packaging of only 4 kb inserts. Generally, AAV vectors are considered to generate low pathogenicity and toxicity and provides long-term episomal expression of the gene of interest. One limitation of using AAV relates to the immune response triggered by repeated administration and the seroprevalence against some serotypes. This problem has been addressed by applying a different AAV serotype for each re-administration. Another issue relates to the limited packaging capacity of foreign DNA into recombinant AAV particles. This shortcoming has been addressed by engineering dual AAV vectors.
Retroviruses possess a ssRNA genome with an envelope structure. Typically, retroviruses are randomly integrated into the host genome, resulting in transduced cells with a certain range of transgene expression strength. However, this shortcoming has triggered the development of safer vectors showing targeted integration and also of improved helper cell lines. Retroviruses can accommodate up to 8 kb of foreign inserts and have been considered the gold standard vectors for long-term gene therapy applications. One drawback of retroviruses is their incapability to infect non-dividing cells.
Retroviral vector system are known in the art and, for example reviewed in (Vargas et al., 2016). Also, adenoviral vector and adeno-associated vector (AAV) are known in the art; for review see, for example (Li and Samulski, 2020),
The present invention relates in a third aspect to a fusion protein being encoded by the nucleic acid molecule of the first aspect of the invention.
The definitions and preferred embodiments of the above aspects of the invention apply mutatis mutandis to the third aspect of the invention.
As discussed herein above, the fusion protein comprises (i) a secretory signal peptide; (ii) a lipocalin-derived binding protein specifically binding to an exogenous ligand; and (iii) a glycosylphosphatidylinositol (GPI) anchored and/or transmembrane domain. As also discussed herein above, said signal peptide ensures that the fusion protein is sorted towards the secretory pathway.
After protein translocation of the ER membrane, a secretory signal peptide is cleaved off. The fusion protein of the invention is thereby processed to its mature form and, after protein sorting, incorporated into the plasma membrane of a cell via its transmembrane domain or glycosylphosphatidylinositol (GPI) anchor.
Hence, the present invention also discloses a fusion protein being encoded by the nucleic acid molecule of the first aspect of the invention which only comprises the (ii) a lipocalin-derived binding protein specifically binding to an exogenous ligand and (iii) a transmembrane domain or glycosylphosphatidylinositol (GPI) anchor. Once said mature fusion protein is located in the plasma membrane of a cell, a lipocalin-derived binding protein specifically binding an exogenous ligand can be found on the outside of the cell (i.e. on the extracellular side).
The present invention relates in a fourth aspect to a cell being transduced or transfected with (or comprising) the nucleic acid molecule of the first aspect or the vector of the second aspect of the invention and/or comprising the fusion protein of the third aspect of the invention.
The definitions and preferred embodiments of the above aspects of the invention apply mutatis mutandis to the fourth aspect of the invention.
The cell is also referred to as a host cell. The host cell is preferably a human host cell. A host cell is preferably an ex vivo cell; i.e. a cell outside of a multi-cellular organism. It will be appreciated that the term “host cell or a non-human host transformed or transduced with the vector”, in accordance with the present invention, relates to a host cell or a non-human host that comprises the vector of invention.
Suitable prokaryotic hosts comprise e.g. bacteria of the species Escherichia, Corynebacterium (glutamicum), Pseudomonas (fluorescens), Lactobacillus, Streptomyces, Salmonella or Bacillus.
Typical mammalian host cell lines include, Hela, HEK293, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS0 and C127 cells, COS 1, COS 7 and CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.
Also within the scope of the present invention are primary mammalian cells or cell lines. Primary cells are cells which are directly obtained from an organism. Suitable primary cells are, for example, embryonic fibroblasts (EF), primary hepatocytes, cardiomyocytes and neuronal cells as well as muscle stem cells (satellite cells), dermal and pulmonary fibroblasts, epithelial cells (nasal, tracheal, renal, placental, intestinal, bronchial epithelial cells), secretory cells (from salivary, sebaceous and sweat glands), endocrine cells (thyroid cells), adipose cells, smooth muscle cells, skeletal muscle cells, leucocytes such as peripheral blood mononuclear cell (PBMC), B-cells, T-cells, NK-cells, macrophages, neutrophils or dendritic cells and stable, immortalized cell lines derived thereof (for example hTERT or oncogene immortalized cells).
Appropriate culture media and conditions for the above described host cells are known in the art.
The host cells in accordance with this embodiment may, e.g., be employed to produce sufficient amounts of the fusion protein of the present invention suitable for detection.
The transfer of the nucleic acid molecule of the first aspect of the invention into the cell of the invention can be established, for example, by: i) CRISPR/Cas 9 mediated targeted genome integration after plasmid transfection or nucleofection, ii) retroviral gene transfer with stable random integration into the genome or iii) adenoviral or adeno-associated viral vectors that lead to transient gene expression without genomic integration.
In accordance with a preferred embodiment of the fourth aspect of the invention the cell is a lymphocyte, preferably a T-cell, more preferably a human T-cell.
A lymphocyte is a subtype of a white blood cell. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T-cells (for cell-mediated, cytotoxic adaptive immunity), and B-cells (for humoral, antibody-driven adaptive immunity).
The T-cells may be CD4 T-cells, CD8 T-cells or both and are preferably both. In initial T-cell therapies CD8 T-cells were primarily administered. These cells had optimal cytotoxicity but did not have sufficient replicative capacity after infusion, and with rare exceptions, the infused T-cell products had poor persistence in the patients (DeRenzo and Gottschalk, 2019, Shah and Fry, 2019, Labanieh et al., 2018). It is now widely accepted that mixtures of CD4 and CD8 T-cells are often preferred, likely because the CD4 T-cells provide growth factors and other signals to maintain function and survival of the infused CD8 T-cells.
In accordance with another preferred embodiment of the fourth aspect of the invention the cell further comprises a chimeric antigen receptor or a transgenic T-cell receptor.
T-cells are used for therapy in humans and in particular in anti-cancer treatments (Miliotou and Papadopoulou, 2018).
The currently most frequently used type of T-cell therapy is a Chimeric Antigen Receptor (CAR) T-cell therapy type of treatment in which a patient's T-cells (a type of immune system cell) are changed in the laboratory, so that they will attack cancer cells independent of the specificity of its T-cell receptor (DeRenzo and Gottschalk, 2019, Shah and Fry, 2019, Labanieh et al., 2018). In brief, T-cells are taken from a patient's blood. Then the gene for a special receptor that binds to a certain protein on the patient's cancer cells, often incorporating an scFv antibody fragment directed against a tumor antigen, is added in the laboratory. The special receptor is called a chimeric antigen receptor (CAR). Large numbers of the CAR T-cells are grown in the laboratory and given to the patient by infusion. CAR T-cell therapy is also called chimeric antigen receptor T-cell therapy in the art. Clinical trials have shown very promising results in end-stage patients with a full recovery of up to 92% in Acute Lymphocytic Leukemia (Miliotou and Papadopoulou, 2018). The transgenic T-cell receptor is generally comprised in a T-cell receptor modified T-cells.
The present invention relates in a fifth aspect to a kit comprising (i) the nucleic acid molecule, the vector, the fusion protein, and/or the cell of the above aspects of the invention, and (ii) an exogenous ligand.
The definitions and preferred embodiments of the above aspects of the invention apply mutatis mutandis to the fifth aspect of the invention.
In particular, also in connection with the kit, the exogenous ligand preferably comprises a radionuclide, wherein the radionuclide is preferably selected from C-11, F-18, Sc-44, Sc-47, Cu-64, Ga-68, Y-86, Y-90, Zr-89, Tc-99m, In-111, I-123, I-124, I-131, Tb-152 and Lu-177.
Yet further in accordance with a preferred embodiment the exogenous ligand is a small molecule, wherein the small molecule is preferably selected from (i) a chelator, preferably a metal chelator complex, more preferably [(R)-2-amino-3-(4-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid (p-NH2-Bn-CHX-A″-DTPA), (ii) an alkaloid, preferably colchicine or a demethylated colchicine, (iii) an iron-chelating siderophore, preferably petrobactin, enterobactin or bacillibactin, (iv) a plant steroid, preferably digoxigenin or digitoxigenin, (v) an organic dye, preferably fluorescein or aminofluorescein, or the exogenous ligand is a peptide, preferably a peptide with at least 2 and less than 10 amino acid residues, preferably VFFAED (SEQ ID NO: 29). Particularly preferred examples of small molecules are shown in
The various components of the composition may be packaged as a kit with instructions for use. For instance, the components of the kit may be packed into one or more vials. The instructions for use preferably provide guidance for using the kit in accordance with the sixth aspect of the invention.
The present invention relates in a sixth aspect to the cell of the fifth aspect for use in the treatment of a disease by a cell-based therapy or a gene therapy, preferably a T-cell based therapy.
The definitions and preferred embodiments of the above aspects of the invention apply mutatis mutandis to the sixth aspect of the invention.
A cell-based therapy is any treatment of prevention of a disease comprising administering a cell of a patient. The cell-based therapy is preferably a T-cell based therapy as has been described herein above. For instance, in the case of CAR T-cell therapy a cell of the fifth aspect is used which expresses in addition the CAR. An example of a cell-based therapy is a therapy in which a viral vector is used in order to temporarily express a gene of interest in a cell type in order to treat the disease phenotype. This may be a growth factor, a shRNA to knock down a gene or a CRISPR/Cas 9 pair that changes the genome.
Gene therapy is designed to introduce genetic material into cells to compensate for an abnormal gene or to produce a beneficial protein. For instance, if a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. In the case of a gene therapy the cell to be used in the therapy is therefore the cell of the fifth aspect, wherein the cell in addition expresses the genetic material to compensate for an abnormal gene or wherein the cell produces the beneficial protein.
The technical advantage of using a cell of the fifth aspect in the treatment of a disease by a cell-based therapy or a gene therapy, preferably a T-cell based therapy and, hence, a cell of the fifth aspect as the therapeutic cell is that upon the administration of an exogenous ligand as described herein above the location of the therapeutic cell in the patient or subject to be treated can be determined. This will be explained in more detail in connection with the following seventh aspect of the invention.
The present invention relates in a seventh aspect to an exogenous ligand for use in an in vivo method of diagnosing the efficacy of a cell-based therapy, preferably a T-cell based therapy in a subject, wherein the subject has been treated with the cell of the fifth aspect, and wherein the method preferably comprises positron emission tomography (PET) or single photon emission computed tomography (SPECT).
The definitions and preferred embodiments of the above aspects of the invention apply mutatis mutandis to the seventh aspect of the invention.
A positron emission tomography (PET) scan is inter alia used as a diagnostic imaging that can help to reveal the activity of therapy in certain tissues and organs. For this imaging, a PET scanner detects the presence of a radionuclide whose positron emission leads to a pair of perpendicularly photons with opposite directions, each with energy of 511 keV. Single-photon emission computed tomography (SPECT, or less commonly, SPET) is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera (that is, scintigraphy) but is able to provide true 3D information. Hence, PET and SPECT both require the administration or the delivery of a radioisotope (a radionuclide) into the patient, preferably through injection into the bloodstream. As described herein above, the exogenous ligand according to the invention may comprise such a radionuclide.
Hence, also in accordance with the seventh aspect the exogenous ligand preferably comprises a radionuclide, wherein the radionuclide is preferably selected from C-11, F-18, Sc-44, Sc-47, Cu-64, G-68, Y-86, Y-90, Zr-89, Tc-99m, In-111, I-123, I-124, I-131, Tb-152 and Lu-177.
Further in accordance with a preferred embodiment the exogenous ligand is a small molecule, wherein the small molecule is preferably selected from (i) a chelator, preferably a metal chelator complex, more preferably [(R)-2-amino-3-(4-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid (p-NH2-Bn-CHX-A″-DTPA), (ii) an alkaloid, preferably colchicine, (iii) an iron-chelating siderophore, preferably petrobactin, enterobactin or bacillibactin, (iv) a plant steroid, preferably digoxigenin or digitoxigenin, (v) an organic dye, preferably fluorescein or aminofluorescein, or the exogenous ligand is a peptide, preferably a peptide with at least 2 and less than 10 amino acid residues, preferably VFFAED (SEQ ID NO: 29). Particularly preferred examples of small molecules are shown in
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the patent specification, including definitions, will prevail.
All amino acid sequences provided herein are presented starting with the most N-terminal residue and ending with the most C-terminal residue (N to C), as customarily done in the art, and the one-letter or three-letter code abbreviations are used to identify amino acids throughout the present invention correspond to those commonly used for amino acids.
Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.
Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.
The above considerations apply mutatis mutandis to all appended claims.
The figures show:
Jurkat cells, either transfected with DTPA-R or Colchi-R were injected s.c. into CD1 nude mice (Charles River Laboratories) and explanted after mice had been sacrificed. Tumor tissue was fixed in 10% (w/v) neutral-buffered formalin solution (Otto Fischar, Saarbrücken, Germany) for 48 h and stored in PBS at 4° C. Tissue samples were dehydrated using an automated system (ASP300S; Leica Biosystems) and subsequently embedded in paraffin. Serial 2 μm sections were prepared with a rotary microtome (HM355S; ThermoFisher Scientific) and subjected to histological and immunohistochemical analysis. Hematoxylin and eosin (H&E) staining was performed on deparaffinized sections with Eosin and Mayer's Haemalaun (Morphisto, Frankfurt am Main, Germany). Immunohistochemistry was performed using a Bond RXm system (Leica Biosystems) with primary antibodies against V5-tag (SV5-Pk1; Biorad) using 1:250 dilutions. Briefly, slides were deparaffinized using deparaffinization solution (Leica Biosystems), pretreated with Epitope retrieval solution 1 (corresponding to citrate buffer, pH 6) for 30 min. Bound antibody was detected with a polymer refine detection kit without post primary reagent and with an intermediate Rabbit anti-mouse IgG secondary antibody (diluted 1:400; Leica Biosystems) and signals were developed with 3,3′-diaminobenzidine (DAB). Representative images were collected on an Aperio AT2 digital pathology slide scanner using ImageScope (ver.12.3) software (both from Leica Biosystems).
The human T-cell line Jurkat was lentivirally transduced with expression cassettes for different reporter proteins and after FACS sorting of the 10% highest expressing clones, individual stable sub-cell lines were created. For these cell lines the doubling time was assessed using the CFSE assay (A) and the number of receptors on the cell surface was quantified using flow cytometry measurements (B).
To this end, cells were cultivated in RPMI medium with 10% FCS and penicillin and streptomycin. In order to further assess the influence of the T-cell activation on cell division rate and reporter protein expression, the cells were unspecifically activated with Phorbol myristate acetate (PMA) and lonomycin (both from InvivoGen, Toulouse, France). Cells were seeded at a density of 0.5×10{circumflex over ( )}6 cells/ml in medium with or without 1 μg/ml PMA and 10 μg/ml lonomycin for 24 h and were then transferred into medium without chemical activators and measurements were performed after 2 days. (A) Exponentially dividing Jurkat cells were counted and a number of 15×10{circumflex over ( )}6 cells was dissolved in 500 μl PBS and labeled with the CFSE Cell Division Tracker Kit (BioLegend, CA, USA) for 20 min at 37° C. The fluorescent probe 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) conjugates to primary amine groups on the cells which subsequently can be quantified using flow cytometry. As the CFSE-signal of cells is reduced by 50% by every cell division, the quantification of the signal after some days allows calculation of the number of cell division that had occurred. In order to quantify the loss of signal, in addition to the stained cells also beads with a known concentration of the same fluorescent dye (Quantum FITC-5 MESF, Bangs Laboratories) were used for flow cytometry on a LSRFortessa (BD Biosciences) instrument. The reduction of the fluorescence signal was analyzed following 4 days of cell growth and the MESF kit was then used to calculate the number of corresponding fluorescein dyes that was then used to calculate the doubling time. (B) The same cells, with and without chemical activation using PMA/lonomycin were counted and a number of 0.5*10{circumflex over ( )}6 cells was stained with 2 μl of a 147 μg/ml SV5-PK1 antibody solution (Bio-Rad Laboratories, Hercules, CA) that had been conjugated with Alexa Fluor488-NHS (Lumiprobe, Hannover, Germany) at a degree of labeling of 3.85 dyes per antibody. Here the Quantum Alexa Fluor 488 MESF kit (Bangs Laboratories, Fishers, In) was used to calculate the number of Molecules of Soluble Fluorochrome (MESF) based on mean fluorescence intensities and it was assumed that one antibody binds with its two paratopes (Fab arms) to two reporter proteins. Error bars indicate standard deviation and represent the variance of the cell populations recorded in flow cytometry.
In order to confirm the absence of negative impact of the reporter protein of the invention on the cellular function of a CAR T-cell, two different expression constructs were compared, both featuring an anti-CD19 chimeric antigen receptor, a self-cleaving 2A peptide followed by either a truncated version of the epidermal growth factor receptor (EGFRt) or the reporter protein DTPA-R. In short, peripheral blood mononuclear cell (PBMC) were isolated from a healthy donor and subsequently transduced with lentiviral vectors encoding the described expression cassettes (CAR/EGFRt or CAR/DTPA-R). After isolation of the cell population expressing the transgene, cells were expanded in RPMI medium with 200 U/mI IL-2, 12.5 ng/ml IL-7 and IL-15, each (all from PeproTech, Rocky Hill, NJ, USA) and maintained at a cell density of 0.25×106 cells/ml. The ability of the CAR T-cells to kill CD19 expressing target cells was assessed by Chrome-51 (51Cr) release assay that works by loading the target cells with the radioactive isotope and the subsequent quantification of the Cr-51 that has been released into the supernatant of the culture. For this assay, the CD19-positive target cell lines Nalm-6 (A) or Raji (B) were incubated together with the respective CD19-CAR T-cells at different ratios of T-cells over target cells. A number of 10 000 target cells was incubated with the respective number of PBMCs in a V-bottom 96-well plate in 150 μl culture medium. The plate was incubated for 4 h with occasional agitation at 37° C. Afterwards, the radioactivity in the supernatant was quantified using a Wizard2 gamma counter (PerkinElmer) and the Percent Specific Lysis was calculated using the following formula: [(experimental release−spontaneous release)/(maximum release−spontaneous release)]*100. In addition, the ability of the CAR T-cells to kill CD19 expressing target cells was assessed by a FACS killing assay (C-F). To this end, GFP expressing NALM-6 target cells were incubated with 1:1 or 5:1 excess of CAR/DTPA-R or CAR/EGFRt CAR T-cells. After 48 h a comparable fraction of the cultured cells (as measured by FACS quantification beads) was analyzed and the number of GFP-positive target cells that remained after the killing assay was quantified.
The precursor +TMA-Py-D-Glu-D-Glu-PEG(4)-Colchicine was used for synthesis of 18F-Py-D-Glu-D-Glu-PEG(4)-Colchicine (called 18F-Colchicine). (A) In short, the radioactive 18F− was produced by a cyclotron in house. Fluorine was eluted from a Sep-Pak Accell Plus QMA Carbonate (Waters) cartridge using 75 mM Tetrabutylammonium (TBA HCO3) solution and was subsequently dried in a Wheaton V-Vial under a continuous flow of dry argon gas at 95° C. by azeotropic distillation. An amount of 0.5 to 2 mg of the precursor was dissolved in DMSO and added into the V-Viral. The exchange reaction of 18F for the Trimethyl ammonium (TMA) leaving group also occurred at 95° C. for 10 minutes. Subsequently, the reaction mixture was purified by a Chromafix PS-H+ (Macherey-Nagel) and a Sep-Pak C18 Classic (Waters) cartridge, which allows the quantitative removal of the unlabeled precursor by its positive charge. Subsequently, the activity was eluted from the C18 cartridge by EtOH and in some cases the radioligand was further purified by HPLC with a Multospher 100 RP 18−5μ 5μ 250×10 mm column with an isocratic elution at 25% Acetonitrile with 0.1% TFA.
(B) Quality control by HPLC of the radiosynthesis of 18F-Py-D-Glu-D-Glu-PEG(4)-Colchicine: i) after radiolabeling reaction, ii) after cartridge purification and iii) after preparative HPLC purification, which were performed on a 100×4.6 mm Chromolith performance RP-C18 column (Merck Millipore) with a gradient of 5 to 55% acetonitrile with 0.1% TFA at a flow rate of 3 ml/min. A single peak was obtained as for both other precursors shown in
Based on the required ligand moiety for the DTPA-R (here NH2-Bn-CHX-A″-DTPA) there are different variants of radioligands that can be synthesized and compared in order to find the lead candidate which is expected to exhibit high affinity for the DTPA-R, in vivo stability and the absence of quantitative structural modifications in the patient and a favorable excretion pattern (ideally only via the renal route).
(A) The ligand moiety alone can serve as the radioligand when used together with an appropriate radiometal. (B) A variant where the amino-group within the NH2-Bn-CHX-A″-DTPA was replaced by the leaving group for radiofluorination (trimethylamine leaving group according to the “minimalist approach for radiofluorination” (Richarz et al., 2014)). (C) A design with a radiofluorination moiety, two D-Glu residues and a PEG(4) linker (called 18F-DTPA) as in
The binding of different radioligands to cells that express the DTPA-R or Colchi-R was shown in binding assays. To this end, Jurkat or PC3 cells that stably express the respective reporter protein of the invention were cultivated, counted and a defined concentration was incubated together with the radioligand in PBS with 2% BSA for 1 h at 37° C. Subsequently, the cells were washed twice with PBS with 2% BSA and finally in the cell-bound fraction and the supernatant fraction the respective radioactivity was detected. (A) NH2-Bn-CHX-A″-DTPA⋅Y-90 binding was competed with a non-radioactive competitor NH2-Bn-CHX-A″-DTPA⋅Y-89. (B) Specific binding of 18F-Py-D-Glu-D-Glu-PEG(4)-Colchicine to Colchi-R Jurkat and Colchi-R PC3 cells was quantified and compared to the reporter gene with another specificity (DTPA-R). Furthermore, the specific binding was blocked by 200 μM Colchicine solution to prove the specificity of the binding. (C) A comparative binding assay with 18F-Py-Ahx-DTPA and 18F-Py-Glu-Glu-PEG(4)-Colchicine to Jurkat cells expressing either DTPA-R or Colchi-R proving the orthogonal functionality of the two reporter protein & reporter probe pairs.
CD1 nude mice were subcutaneously injected with a Colchi-R PC3 xenograft in the right flank and a DTPA-R PC3 xenograft in the left flank. After xenograft tumors had developed the fluorine labeled radioligand was injected i.v. and a dynamic PET/MR was recorded for 90 minutes using a Mediso nanoScan PET/MR scanner. (A) Maximal intensity projections after 18F-Py-D-Glu-D-Glu-PEG(4)-Colchicine i.v. injections are shown for the 5-10 min and 75-90 min time frame and exhibit a hepato-biliary excretion pattern. (B) In contrast, the i.v. injection of 18F-Py-D-Glu-D-Glu-PEG(4)-DTPA⋅Tb (18F-DTPA) resulted in the specific accumulation of the radioligand in the tumor at the opposite shoulder (PC3 DTPA-R) together with a favorable, exclusively renal excretion pattern. Here, the mouse was awake for 60 min ahead of the PET-scan that was recorded von 60-90 min post injection. For the successful detection of this small xenograft tumor, also axial image sections are depicted. (C) Furthermore, Jurkat cells expressing DTPA-R were incubated in vitro with an excess of 18F-DTPA and washed twice. Subsequently, a dilution series was prepared and the cells were scanned within PCR tubes for 60 min to determine the detection limit of the reporter gene system, which was found to be around 5.000 cells.
(A) Comparable to the PET-imaging study in
(A) For the detection of viral transduction events based on the adeno-associated virus, viral vectors were constructed by flanking an expression cassette for the DTPA-R or the Colchi-R with AAV2 inverted terminal repeats (ITRs). This genetic construct was used to produce and purify AAV9 viral vectors. (B) A dose dependent transduction was confirmed for DTPA-R and Colchi-R reporter genes after transduction of HEK cells with respective AAV viral vectors. (C) Anti-V5 immunohistochemistry staining of an axial cryosection of a mouse heart that had been i.v. injected with AAV before. (D) Maximal intensity projection of a PET-scan of a mouse that had been injected 7 days before with AAV9 viral vectors encoding DTPA. Data was recorded 70-90 min post injection of the 18F-DTPA⋅Tb radioligand. Axial sections are depicted showing specific signal in shoulder and dorsal muscle tissue as well as the myocardium.
(A-C) Design of expression constructs for the comparison of Anticalin-based reporter proteins according to this invention versus scFv-based reporter proteins from the state of the art. (A) Construct for CL31d-V5-CD4TMD, (B) construct for scFv(muC825)-V5-CD4TMD and (C) construct for scFv(huC825)-V5-CD4TMD. Jurkat T-cells after stable retroviral transduction with one of the constructs were stained with an anti-VS-tag antibody conjugated with AlexaFluor488, which was detected using flow cytometry. (D) Histogram of the transduced cells analyzed by flow cytometry along with (E) the conversion to absolute numbers of receptors per cells (as explained for
The following Examples illustrate the invention.
A reporter system was developed comprising a genetically encoded, membrane anchored reporter protein and a cognate small-molecule radioactive probe (radioligand) which can be injected intravenously. The radioligand is distributed in the blood compartment and selectively bound by the reporter protein, which is expressed on the genetically modified cell type. After rapid excretion of excess unbound radioligand via the kidneys or the hepato-biliary system, its radioactive decay in the body can be registered and by this way the distribution and location of the reporter gene-tagged cell type can be analyzed. This concept resembles the use of radiopharmaceuticals in nuclear medicine to detect various tumor targets (such as somatostatin, PSMA or integrins). The difference here is that the molecular target in the patient is a synthetic membrane-associated protein, whose coding gene has been incorporated specifically into the tagged cell type, which may serve for cell or gene therapy.
For an ideal reporter gene system, its components need to be bioorthogonal: neither the reporter (membrane) protein nor the radioligand should interfere with the healthy organs or tissues. This allows the monitoring of biological processes with minimal functional impairment and, on the other hand, with a minimal background signal. Furthermore, also the radioligand should be inert and not be modified or cleaved within the organism. A further requirement for clinical application is the lack of immunogenicity. For signal normalization, or for the simultaneous analysis of different biological processes, it is also desirable to have several independent reporter systems at hand that can be used in parallel without mutual interference (called multiplexing), similar to the well-established dual-luciferase systems or fluorescent proteins with distinct spectral properties used in biomedical research.
Anticalins are a class of engineered binding proteins based on the natural lipocalins. Most often, the human lipocalin 2 (Lcn2), also known as neutrophil gelatinase-associated lipocalin, NGAL, is used as scaffold for the selection of specific binding proteins (Richter et al., 2014, Schiefner and Skerra, 2015). The natural ligand of Lcn2 is the iron-chelating siderophore enterobactin that is employed by bacteria to sequester iron ions which are essential for their growth. The plasma protein Lcn2 binds the iron-siderophore-complex (Fe⋅enterobactin) before it can be taken up by the bacterium and, thus, restricts the growth of bacteria in the human body by deprivation of this essential metal. Lcn2 is part of the innate immune system and, in an engineered form, ideally suited for biomedical applications. Furthermore, the calyx-shaped ligand pocket of the lipocalins favors the tight binding of small-molecule ligands in correspondingly engineered Anticalins (
The human origin of many Anticalins lowers immunogenicity after expression on a given cell of interest, e.g. an immune cell. As in current clinical investigations the immunogenicity of CAR receptors limits the persistence of some CAR T-cells in patients and hampers a positive therapy outcome, this is one of the most critical factors. Apart from protein or peptide targets, Anticalins have been selected to recognize several small molecules with high affinity (Table 1), for example petrobactin, a specific siderophore of certain Bacillus species (Dauner et al., 2018), colchicine (Barkovskiy et al., 2018) (
Generally, the reporter gene system of the invention features a modular design in a way that (i) different radionuclides can be incorporated into the radioligand via suitable chelator groups or selective conjugation chemistry to allow PET and/or SPECT imaging and (ii) the membrane-bound reporter protein can be expressed in diverse cellular context driven by different promoters (constitutive, chemically inducible or inducible by biological processes). Furthermore, iii) the reporter protein itself is modular in a way that it features further to mandatory protein domains (secretory signal peptide, Anticalin, membrane anchor) also optional protein domains such as the extracellular V5-epitope tag and the intracellular fluorescent proteins (
18F-Py-PEG(4)-Colchicine
18F-Py-PEG(4)-Digoxigenin
18F-Py-PEG(4)-Fluorescein
Anticalins binding suitable small-molecule radioligands with high affinity (see Table 1) need to be expressed in sufficient density on the surface of the engineered/transformed cell in order to allow complex formation between the Anticalin and its cognate radioligand in reasonable amounts. This design is somewhat similar to a membrane-based reporter gene that binds a DOTA⋅Me complex (Wei et al., 2008, Krebs et al., 2018). This reporter gene was made of the following protein moieties: (i) a signal peptide, (ii) a murine antibody scFv fragment (2D12.5/G54C), (iii) a transmembrane domain from CD4 and (iv) a P2A-separated green fluorescent protein (GFP) (Krebs et al., 2018). Disadvantages of this design, which is based on a single chain variable fragment (scFv) binding protein include: 1) oligomerisation of the antibody fragments (Hudson and Kortt, 1999), 2) low surface expression, 3) murine origin and, hence, potential immunogenicity in humans, 4) the cross-reactive radioligand lanthanoid(S)-2-(4-acrylamidobenzyl)-DOTA (AABD) which, as acrylamide derivative, unspecifically binds also to biological structures other than the DAbR1 reporter protein. In contrast, advantages of the lipocalin-based reporter proteins according to the invention include, but are not limited to: 1) human origin of the protein scaffold, 2) smaller size of the reporter protein, 3) single-chain protein architecture by nature, 4) high stability of the folded protein and 5) the absence of oligomerisation tendency. While antibodies exist in nature both in soluble and in a membrane-anchored form, lipocalins are exclusively found as soluble secretory proteins in mammals. ApoD may be seen as a rare exception as it is found associated with high density lipoprotein particles; however, this lipocalin does not carry a transmembrane domain or GPI anchor (Schiefner and Skerra, 2015).
In one embodiment, the reporter protein of the invention (
Various different expression vectors for the different reporter proteins have been constructed and tested (
Some applications as well as the beneficial properties of the reporter proteins according to this invention will be described and illustrated in the following Examples.
High surface expression of exemplary constructs on transfected cells was verified by flow cytofluorometry (
Finally, the sub-cellular distribution pattern of the fluorescent protein mRuby3 as part of the longer reporter gene construct was investigated in transgenic HEK-(
The incorporation of an epitope-tag into the reporter protein enables the use of antibodies against this tag for the detection of the protein on the cell surface (e.g. flow cytometry, masscytometry or MACS) or in an isolated form (e.g. western blot). For these applications, the V5-epitope tag (Southern et al., 1991, Dunn et al., 1999) was selected because of its high affinity (KD=24 pM for SV5-PK1), the strong denaturing conditions that are required to break this interaction (9 M urea and 1% Tween-20) as well as the hydrophilic and only slightly charged amino acid sequence (GKPIPNPLLGLDST, see SEQ ID NO:7/8) of the tag (Dunn et al., 1999).
The possibility to bind an accessible epitope on the cell surface with a high affine antibody was used for magnetic-activated cell sorting (
Furthermore, the presence of the V5-epitope tag on the cell surface was used to identify the transgenic cells on a cellular level in a histological context (
When the reporter gene system of the invention is used for PET-detection of a cell population of interest, such as infused CAR T-cells or cells transduced with an AAV viral vector, an important prerequisite for the application of the reporter protein is the absence of negative impact on the therapeutic function of this cell population.
In order to check some of the major aspects that may be impaired, stable Jurkat cell lines with different reporter proteins were established and subsequently the doubling time of these cell lines (
The determination of the doubling time for these Jurkat cell lines using the CFSE cell proliferation assay confirmed the absence of a negative effect of the expression of the reporter gene on the cell division rate, as there were no significant differences between the doubling times determined for these cell lines. Although, the unspecific activation of the T-cell using PMA/ionomycin slightly increased the doubling time of all cell lines, there was no detectable difference between all evaluated reporter gene constructs and the untransduced control cells (
The determination of the total cell surface expression of the reporter gene was conducted with anti-V5 antibody that had been labeled with a fluorescent dye for which a kit with calibration beads was available. The results for the total number of receptors on the cell surface reflected the results earlier determined for different reporter gene constructs when quantifying the intrinsic fluorescence of the fluorescent protein (
Thus, it is not only confirmed that the expression of different reporter proteins of the invention does not change the proliferation rate, but also that major events in the physiology of the T-cell only have a minor effect on the surface expression of the reporter gene.
Furthermore, anti-CD19 CAR T-cells created from peripheral blood mononuclear cell (PBMC) of a healthy donor were evaluated for their potential to kill CD19-positive target cells. The cellular toxicity to the target cells was measured by a radioactive Chrome-51 release assay at a 4 h end point (
This result was furthermore confirmed by a FACS based killing assay in which the successful killing of GFP-expressing NALM-6 target cells was confirmed by both CAR T-cells, CAR/EGFRt and CAR/DTPA-R, respectively (
It is crucial for the development of both a preclinically and a clinically useful reporter system to select the best affinity pair composed of a genetically encodable binding protein (here an Anticalin) and its ligand. Relevant criteria for this choice include: (i) the affinity of the binding protein to the small molecule ligand (see Table 1), (ii) the availability of different radiolabeling strategies to generate radioligands with different properties (
Given these criteria, different ligands were synthesized (
Finally, the radioligands for the Colchi-R were injected into nude mice and a dynamic PET-scan was recorded. While the biodistribution of the 18F-Py-PEG(4)-colchicine radioligand is dominated by its lipophilic nature (positive LogP value) and, thus, is only eliminated insufficiently from the organism (
Besides the radioligands for the Colchi-R (
For the sensitive and specific detection of cells that are labeled with the reporter gene of the invention, the binding between the Anticalin binding protein and the radioligand is of utmost importance. For this reason, the binding of different radioligands to cells that were stably modified with the reporter genes was studies in vitro (
The ability to use the reportergene system for in vivo PET-imaging was assessed (
To further analyze the dynamics of the 18F-DTPA⋅Tb radioligand in vivo, a dynamic PET-scan was conducted for 90 min post injection. An equal distribution of the radioligand in the whole animal can be seen in the first 5 minutes, which is then followed by a rapid elimination of the radioligand via the kidneys. Already after 30 minutes, a clear contrast is visible that allows the identification of the tumor that was DTPA-R modified, while the Colchi-R labeled tumor xenograft remained invisible. Again, elimination related PET-signals were visible and somewhat more pronounced because the lack of physical activity of the mouse during anesthesia led to a slower clearance of the radioligand. At the same time, the signal of the radioligand within the tumor remained constant over the whole 90 minutes of the dynamic PET-scan, which is caused by the high affinity of the Anticalin for the DTPA ligand-moiety that efficiently limits the dissociation and subsequent elimination of the radioligand. In addition, it must be pointed out, that the radioligand only accumulated in the vital border regions of the xenograft tumor, while the core region containing secrete and necrotic cells showed no PET-signal above background level (
As explained herein above in Example 1, a murine antibody scFv fragment (2D12.5/G54C) was previously employed as a reporter protein for PET imaging (Krebs et al., 2018). This scFv fragment, binds DOTA:metal complexes and was derived from the murine monoclonal antibody 2D12.5/G54C—It was used to construct the synthetic reporter gene DabR1 (DOTA Antibody Reporter 1) (Krebs et al., 2018). Based on the Antibody 2D12.5, also scFv fragments with improved affinity were developed and proposed as binding proteins which can be used as part of a membrane-anchored reporter protein (improved version of DAbR1). The corresponding muC825 scFv as well as its humanized version, huC825 scFv, were described for example in EP3256164B1 and WO 2019/060750A2. To best knowledge of the inventors, the scFv huC825 represents the most advanced and most appropriate candidate of an antibody-derived binding protein for the construction of a PET reporter gene according to the art (Dacek et al., 2021)(Krebs et al., 2018). Due to the increased affinity of C825 for DOTA:metal complexes, the complicated formation of a covalent bond between the engineered Cys residue G54C and the acrylamidobenzyl group as part of the radioligand (Krebs et al., 2018) was no longer necessary. The free Cys residue in the oxidizing extracellular milieu is prone to chemical modification while the chemical cross-reactivity of the acrylamidobenzyl group leads to undesired background signals. Apart from that, the humanization can be expected to reduce the potential immunogenicity of the murine protein in immunocompetent patients, which is of high importance for clinical translation (Dacek et al., 2021).
Both the murine and the humanized scFv C825 were compared with the Anticalin-based reporter protein according to this invention side by side. To this end, three membrane-anchored reporter proteins were constructed which only differ in the binding protein domain specific for the radioactive chelator:metal complex. These were (a) the DTPA-binding Anticalin CL31d (SEQ ID NO 23/24,
The amino acid sequences for the murine and human scFv, including the signal peptide (MGWSCIILFLVATATG; SEQ ID NO: 32), were taken from WO 2019/060750A2 and codon-optimized for Homo sapiens. The synthesized genes were cloned using Pacl and Clal restriction sites on pMP71-Lcn2_SignalP-CL31d-V5-V5-CD4TMD, that is the plasm id used for the retroviral transduction with Lcn2_SignalP-CL31d-V5-CD4TMD. Sanger sequencing confirmed correctness of the coding regions and, subsequently, the three plasmids were used for the production of retroviral vectors (Engels et al., 2003), followed by transduction of the human T-cell line Jurkat. The amount of virus was chosen to achieve a low transduction rate (20-33%) in order to avoid events of multiple transduction and genomic integration.
Transduced Jurkat cells were then stained with the AlexaFluor488-conjugated V5-binding IgG antibody SV5-PK1. Surface density of the reporter constructs in the three stably transduced cell lines was analyzed under mutually identical conditions by detection of the V5-tag via flow cytometry in two independent experiments (
Furthermore, the MFI values were converted into absolute numbers of detected fluorescent dye molecules (as described in the legend of
M., DREXLER, I., SADELAIN, M., LIU, L., JENSEN, M. C., RIDDELL, S. R. & BUSCH, D. H. 2016. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. J Clin Invest, 126, 4262-4272.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20207716.0 | Nov 2020 | EP | regional |
This patent application is a 35 U.S.C. 371 national phase patent application of PCT/EP2021/081775 filed on Nov. 16, 2021, entitled “BIOORTHOGONAL REPORTER GENE SYSTEM”, naming Volker MORATH, Arne SKERRA, Wolfgang WEBER, and Katja FRITSCHLE as inventors, and designated by attorney docket no. AC1553 PCT, which claims priority to European Application No. 20207716.0 filed on Nov. 16, 2020, entitled “BIOORTHOGONAL REPORTER GENE SYSTEM,” naming Volker MORATH, Arne SKERRA, Wolfgang WEBER, and Katja FRITSCHLE as inventors, and designated by attorney docket no. AD1553 EP. The entire content of the foregoing patent applications is incorporated herein by reference, including all text, tables and drawings.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/081775 | 11/16/2021 | WO |
