PNA probes for pre-targeted imaging and therapy

Abstract

The present invention relates to a kit for targeting a diagnostic or therapeutic agent to a target site, the kit comprising: (a) a first conjugate comprising (i) a targeting moiety capable of selectively binding to the target site; and (ii) a first hybridization probe portion comprising a PNA oligomer; and (b) a second conjugate comprising (i) a second hybridization probe portion comprising a complementary PNA oligomer; and (ii) a diagnostic or therapeutic agent portion, wherein the complementary PNA oligomer of the second hybridization probe portion is 14 bases or less in length. The present invention further relates to a method for delivering a diagnostic or therapeutic agent to a target site in a mammal, and to a method for diagnosing or treating a medical condition, such as cancer, in a mammal.

Description

[Technical Field]

[0001] The present invention relates to a kit for targeting a diagnostic or therapeutic agent to a target site, the kit comprising: (a) a first conjugate comprising (i) a targeting moiety capable of selectively binding to the target site; and (ii) a first hybridization probe moiety comprising a PNA oligomer; and (b) a second conjugate comprising (i) a second hybridization probe moiety comprising a complementary PNA oligomer; and (ii) a diagnostic or therapeutic agent moiety. The present invention further relates to a method for delivering a diagnostic or therapeutic agent to a target site in a mammal, and to a method for diagnosing or treating a medical condition, such as cancer, in a mammal. [Background technology]

[0002] Affibody® molecules are small (7 kDa molecular weight) engineered scaffold proteins that can be selected to bind a wide range of biomolecules with high affinity (Stahl 2017) and belong to a class of engineered scaffold proteins with potential for cancer diagnosis and therapy (Weidle 2013). Their small size and high affinity and selectivity for cancer-associated targets make them well suited as radionuclide imaging probes (Stahl 2017). Affibody® molecules can be easily recombinantly produced in prokaryotes with high yields. Affibody-based imaging probes for epidermal growth factor receptor (EGFR or HER1), human epidermal growth factor receptor type 2 (HER2), human epidermal growth factor receptor type 3 (HER3), platelet-derived growth factor receptor β (PDGFRβ), insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor receptor 2 (VEGFR2), and programmed death ligand 1 (PD-L1) have demonstrated very promising characteristics in preclinical experiments (Tolmachev 2020). Furthermore, excellent imaging of HER2 has been demonstrated in the clinic (Sorensen 2014; Sorensen 2016).

[0003] Targeting HER2 using monoclonal antibodies and antibody-drug conjugates extends survival in patients with breast and gastroesophageal cancer, but the emergence of resistance to such treatment is inevitable despite preserved HER2 expression (Kreutzfeldt 2020; Garcia-Alonso 2020). In this case, HER2-targeted radionuclide therapy may offer a solution. However, the primary approach to targeted radionuclide therapy, the use of radiolabeled monoclonal antibodies, is inefficient in solid tumors because their long residence in the blood circulation causes overirradiation of the bone marrow (Larson 2015). Direct application of Affibody® molecules for radionuclide therapy is challenging due to high renal reabsorption and long-term retention of activity in the case of radiometal labels (Fortin 2008). Common methods applied for reducing renal uptake of radiolabeled proteins and peptides have proven inefficient for Affibody® molecules (Altai, 2013; Garousi 2020).

[0004] A solution to the problem of high renal reabsorption of radiolabeled Affibody® molecules is to apply pretargeting, a methodology that separates the effects of molecular recognition of cancer-associated abnormalities and radionuclide delivery (Frampas 2013, Altai 2017 JNM). In pretargeting, a target-specific primary drug coupled to a recognition tag is injected to localize it in the tumor. After clearance of the primary drug from the blood, a radiolabeled secondary probe with high affinity for the recognition tag is injected. Low renal uptake of the secondary probe is critical for the success of affibody-based pretargeting therapy. Affibody® molecules are attractive candidates for primary probes because they are rapidly cleared from the blood and slowly internalized by cancer cells (Wallberg 2008).

[0005] After evaluating different approaches (Altai 2016; Honarvar 2016), hybridization of complementary peptide nucleic acid (PNA) probes was selected for affibody-based pretargeting because it provided the best retention of activity in tumors. PNAs are a class of synthetic DNA analogs capable of Watson-Crick base pairing (Egholm 1993; Nielsen 1994). The PNA backbone is constructed of N-(2-aminoethyl)-glycine repeating units linked by amide bonds, and purine and pyrimidine nucleobases are linked to this scaffold via carboxymethyl linkers. PNAs are resistant to degradation by nucleases and proteases and have excellent stability in human serum (Demidov 1994). They are non-immunogenic and have low general toxicity. First-generation primary drug Z HER2:342 The molecular design of -SR-HP1 and secondary probe HP2 was successful because it provided high affinity and specificity of PNA hybridization, specific accumulation of the primary probe in the tumor, and efficient specific delivery of the radiometal (Westerlund 2015, Honarvar 2016). 177 Lu(Altai 2017 NMB, Westerlund 2018), 111 In (Westerlund 2015, Honarvar 2016) and 68 Labeling of HP2 with Ga (Vorobyeva 2018) resulted in significantly higher uptake in tumors than in kidneys, although kidney uptake was the highest among normal tissues.

[0006] Z HER2:342 -SR-HP1 / [ 177 Experimental treatment using the [Lu]Lu-HP2 pretargeting system significantly increased the median survival of mice bearing HER2-expressing xenografts ([ 177There was no observable bone marrow or kidney toxicity during the 66 days of treatment (compared to 32 days for [Lu]Lu-HP2 alone) (Westerlund 2018). However, further increases in the ratio of absorbed dose to tumor compared to normal tissues, primarily the kidney, are necessary to achieve a curative effect with such treatments.

[0007] A possible optimization parameter is the length of the secondary probe. Reducing the length can reduce the hydrodynamic radius of the probe, promoting both its extravasation and diffusion in the tumor interstitium, and improving both localization in the tumor and the uniformity of distribution within the tumor. However, there are obvious risks associated with reducing the size of the secondary probe. First, reducing the number of nucleic acid bases can reduce the strength of hybridization with the primary probe. Second, modifying the base composition can affect off-target interactions, resulting in increased uptake in normal tissues. For example, 177 Lu 111 In or 68 Small structural changes associated with substituting Ga resulted in significant differences in kidney uptake (Vorobyeva 2018) or blood, liver, and bone uptake (Altai 2017 NMB). Biodistribution further depends not only on the number and nature of the nucleobases, but also on their order in the PNA sequence. Binding to the mRNA encoding the MYC protein. 99m Scrambling the nucleobases of Tc-labeled antisense PNAs resulted in a greater than two-fold decrease in uptake in normal tissues (Rao 2003, Mather 2004). Therefore, experimental in vivo studies are needed to assess whether second-generation secondary probes would result in better biodistribution and dosimetry profiles. [Brief explanation of the drawings]

[0008] [Figure 1]SPR sensorgrams of single-cycle kinetic titrations of HP16, HP17, HP18, and HP19 binding to immobilized ZHER2:342-SR-HP15. Each PNA probe was injected at concentrations of 22.6, 45.3, 90.6, 181.25, and 362.5 nM. [Figure 2a] Normalized melting temperature curves shown for three PNA hybridization complexes HP15:HP16; HP15:HP17; and HP15:HP18. [Figure 2b] Normalized melting temperature curve for the HP15:HP19 PNA hybridization complex. [Figure 3A] In vitro binding specificity of the primary agent (A) [177Lu]Lu-ZHER2:342-SR-HP15 to SKOV3 and BT474. Data are shown as mean values ​​± SD from triplicate samples. [Figure 3B] In vitro binding specificity of the primary agent (B) [177Lu]Lu-HP16 to SKOV3 and BT474. Data are shown as mean values ​​± SD from triplicate samples. [Figure 3C] In vitro binding specificity of the primary agent (C) [177Lu]Lu-HP17 to SKOV3 and BT474. Data are shown as mean values ​​± SD from triplicate samples. [Figure 3D] In vitro binding specificity of the primary agent (D) [177Lu]Lu-HP18 to SKOV3 and BT474. Data are shown as mean values ​​± SD from triplicate samples. [Figure 4A] (A) Biodistribution of [177Lu]Lu-HP16 4 hours after injection in female Balb / c nu / nu mice bearing SKOV3 xenografts with and without prior injection of ZHER2:342-SR-HP15. Uptake is expressed as %ID / g and is presented as the mean ± SD (n = 5). [Figure 4B](B) Biodistribution of [177Lu]Lu-HP17 4 hours after injection in female Balb / c nu / nu mice bearing SKOV3 xenografts with and without prior injection of ZHER2:342-SR-HP15. Uptake is expressed as %ID / g and is presented as the mean ± SD (n = 5). [Figure 4C] (C) Biodistribution of [177Lu]Lu-HP18 4 hours after injection in female Balb / c nu / nu mice bearing SKOV3 xenografts with and without prior injection of ZHER2:342-SR-HP15. Uptake is expressed as %ID / g and is presented as mean ± SD (n=5). [Figure 5A] SPECT / CT image 4 hours post-injection of a HER2-expressing SKOV3 xenograft using (A) [177Lu]Lu-HP16 for pretargeting. [Figure 5B] SPECT / CT image 4 hours post-injection of a HER2-expressing SKOV3 xenograft using (B) [177Lu]Lu-HP17 for pretargeting. [Figure 5C] SPECT / CT image 4 hours post-injection of a HER2-expressing SKOV3 xenograft using (C) [177Lu]Lu-HP18 for pretargeting. [Figure 5D] SPECT / CT image 4 hours post-injection of a HER2-expressing SKOV3 xenograft using (D) [177Lu]Lu-HP2 for pretargeting. [Figure 6A] (A) Time-activity plot of [177Lu]Lu-HP16. Non-decay-corrected data were used for both kidney and tumor. [Figure 6B] (B) Time-activity plot of [177Lu]Lu-HP17. Non-decay-corrected data were used for both kidney and tumor. [Figure 6C] (C) Time-activity plot of [177Lu]Lu-HP18. Non-decay-corrected data were used for both kidney and tumor. [Figure 6D](D) Time-activity plot of [177Lu]Lu-HP2. Non-decay-corrected data were used for both kidney and tumor. Summary of the Invention

[0009] DISCLOSURE OF THE INVENTION A second-generation hybridization probe, primary HP15, and a set of secondary probes: HP16 (9-mer PNA), HP17 (12-mer PNA), HP18 (15-mer PNA), and HP19 (6-mer PNA) are disclosed herein (Table 1). As shown in the examples, probes bearing a DOTA chelator were designed, synthesized, and characterized in vitro. 177 The probes were labeled with Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution profiles of these novel probes were evaluated in BALB / C nu / nu mice bearing SKOV3 xenografts and were consistent with previously studied [ 177 Lu]Lu-HP2.

[0010] Characterization by SPR and UV spectroscopy confirmed the formation of high-affinity duplexes between HP15 and secondary probes HP16, HP17, HP18, and HP19, with affinity correlated with the length of the complementary PNA sequence. The three tested PNA-based probes (HP16, HP17, and HP18) bound specifically to HER2-expressing cells with high affinity (11–12 pM) in vitro. In vivo studies demonstrated that all [ 177 We demonstrated HER2-specific uptake of [Lu]Lu-labeled probes. The ratio of cumulative radioactivity in the tumor to that in the kidney depended on the size of the secondary probe and decreased with increasing number of nucleobases. This is the shortest PNA probe tested in vivo. 177 Lu]Lu-HP16 exhibited the highest tumor-to-kidney ratio and is the most promising secondary probe for affibody-mediated tumor pretargeting.

[0011] In a first aspect, the present invention provides a kit for targeting a diagnostic or therapeutic agent to a target site, comprising: (a) a first conjugate comprising: (i) a targeting moiety capable of selectively binding to a target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; and (b) a second conjugate comprising: (i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic agent portion, wherein the second PNA oligomer of the second hybridization probe portion is 14 bases or less in length.

[0012] In the present context, the term "kit" should be understood to mean or include a composition of chemical and / or biological compounds, such as a pharmaceutical composition.

[0013] According to the present invention, the use of a second PNA oligomer having a length of 14 bases or less will result in an improved (increased) tumor-to-non-tumor tissue ratio for the diagnostic or therapeutic agent when the first and second conjugates are administered via a pretargeting protocol to a mammal bearing a tumor, compared to an otherwise equivalent situation in which the diagnostic or therapeutic agent is administered without a pretargeting protocol, i.e., the targeting moiety is coupled to the therapeutic / diagnostic moiety in the same entity.

[0014] The relevant non-tumor tissue may vary depending on the specific application area.For example, in therapeutic applications, there is generally a non-tumor tissue (called dose-limiting tissue) that limits the toxicity of therapeutic agents to the dose that can be administered to patients without unacceptable side effects.The present invention can achieve a tumor-to-dose-limiting tissue ratio that is at least 2-fold (preferably at least 2.5-fold, more preferably at least 3-fold, most preferably at least 3.5-fold) higher than that of otherwise equivalent situations in which pre-targeting protocols are not used, i.e., when the targeting moiety is coupled to the therapeutic / diagnostic moiety in the same entity.In addition, the specific dose-limiting non-tumor tissue may depend on factors such as the nature of the targeting moiety and the therapeutic moiety.Non-tumor tissue may be, for example, kidney, bone, liver or stomach, preferably kidney.In diagnostic applications, it is desirable to minimize the interfering background signal of diagnostic markers from the tissues surrounding the tumor being investigated, especially blood.Therefore, non-tumor tissue may be blood.

[0015] In the present context, the term "pretargeting protocol" means that a first conjugate is administered to a mammal prior to administration of a second conjugate, such that association between the first and second conjugates occurs in vivo.

[0016] With respect to the first and second PNA oligomers, the term "complementary" PNA oligomer refers to a PNA oligomer that can form a double-stranded structure by matching base pairs. Preferably, there is complete complementarity between the two PNA strands, i.e., each base faces its opposite. However, the degree of complementarity can be less than perfect (100%), as long as the two probes can hybridize to form a structured duplex.

[0017] The degree of complementarity between two nucleic acid strands can vary from perfect complementarity (each nucleotide faces its opposite) to no complementarity (each nucleotide does not face its opposite).

[0018] Preferably, the target site is present in a mammalian protein, including human, expressed on the surface of tumor cells. Preferably, the target protein is not expressed or barely expressed in normal healthy tissues and is overexpressed on the surface of tumor cells. For example, the protein may be selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), carbonic anhydrase IX (CAIX), platelet-derived growth factor receptor β (PDGFR-β), nectin-4, cluster of differentiation 38 (CD38), cluster of differentiation 33 (CD33), cluster of differentiation 30 (CD30), cluster of differentiation 22 (CD22), and cluster of differentiation 79b (CD79b) (see Table 7). Preferably, the target protein is a human protein. Preferably, the target site is present in human epidermal growth factor receptor 2 (HER2).

[0019] The targeting moiety preferably comprises: Antibodies, e.g. monoclonal antibodies; · antibody fragments, such as single domain antibodies (sdAb; nanobodies), single chain variable fragments (scFv), antigen-binding fragments (Fab), diabodies (see Holliger et al., 1993), or minibodies (see Hu et al., 1996); engineered scaffold proteins, such as Affibody® molecules; and Peptides, e.g., tumor-targeting peptides selected from combinatorial libraries (see Liu et al., 2017), or peptide analogs based on endogenous peptides that bind to G protein-coupled receptors (GPCRs) (see Hauser, 2017; and Moody, 2018) is selected from the group consisting of:

[0020] In a preferred embodiment of the invention, the targeting moiety is an Affibody® molecule, such as an Affibody® molecule that targets HER2, such as Z HER2:342 or other similar molecules disclosed in Orlova et al., 2006 and WO2005 / 003156.

[0021] The term "Affibody® molecule" refers to a small, engineered scaffold protein that can be selected to bind a wide range of biomolecules with high affinity. Affibody molecules are small (58 amino acid residues) protein domains derived from one of the IgG-binding domains (Z domains) of staphylococcal protein A. Affibody molecules have a three-helix bundle structure that can be used as a scaffold for constructing combinatorial libraries from which affibody molecule variants targeting desired molecules can be selected. For a review, see, e.g., Tolmachev 2020.

[0022] Preferably, the first hybridization probe moiety is covalently linked to the targeting moiety. Preferably, the first hybridization probe moiety is conjugated to the targeting moiety by sortase A-mediated ligation, as described, for example, in Westerlund, 2015. Staphylococcus aureus sortase A is a transpeptidase that attaches surface proteins to cell walls; it cleaves between Gly and Thr in the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of the cell wall peptidoglycan. A recognition motif (LPXTG) is added to the C-terminus of the protein of interest, while a single glycine or oligoglycine peptide with a free N-terminus is added to the second molecule to be ligated. Upon addition of sortase to the molecule, the two molecules are covalently linked via a native peptide bond.

[0023] Optionally, one or both of the hybridization probe moieties include a moiety that improves solubility. The moiety can be a PEG-based linker containing a solubilizing moiety, such as (PEG)2, (PEG)4, (PEG)6, or the like. Preferably, the PEG-based linker is a linker containing 2-[2-(2-aminoethoxy)ethoxy]acetic acid (AEEA). Alternatively, or in addition, the linker contains a charged or polar amino acid, such as Glu, Asp, Ser, Thr, Lys, or Arg. Preferably, the solubilizing moiety includes AEEA.

[0024] According to the present invention, the length of the first PNA oligomer in the first hybridization probe portion is preferably at least the length of the second PNA oligomer in the second hybridization probe portion, and is 15 bases or less. More preferably, the length of said PNA oligomer in the first hybridization probe portion is 15 bases. A preferred 15-base sequence is the sequence cctggtgttgatgat (SEQ ID NO: 3). In a preferred embodiment of the present invention, said first hybridization probe portion has the structure GSScctggtgttgatgatEK-[linker]-E-NH2 or GSScctggtgttgatgatEK([chelator])-[linker]-E-NH2 have wherein G, S, E, and K represent the amino acids Gly, Ser, Glu, and Lys, respectively. In one embodiment, the first hybridization probe portion has the structure GSScctggtgttgatgatEK(DOTA)-AEEA-E-NH2 have (where DOTA is the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetic acid).

[0025] According to the present invention, the length of the second PNA oligomer in the second hybridization probe portion is 5 to 14 bases, for example, preferably 8 to 14 or 9 to 14 bases, or more preferably 9 to 12 bases (including the endpoints). The second PNA oligomer has a sequence that is complementary to, and therefore hybridizes with, the sequence of the first PNA oligomer in the first hybridization probe portion.

[0026] In a preferred embodiment, the second PNA oligomer is 9 bases in length. A preferred 9-base sequence is the sequence aacaccagg (SEQ ID NO: 4). In a preferred embodiment, the second hybridization probe portion has the structure [Chelator]-[Linker]-SSaacaccaggEEY-NH2 have wherein S, E, and Y represent the amino acids Ser, Glu, and Tyr, respectively. In one embodiment, the second hybridization probe portion has the structure DOTA-AEEA-SSaacaccaggEEY-NH2 have (where DOTA is the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetic acid).

[0027] In another preferred embodiment, the second PNA oligomer is 12 bases in length. A preferred 12-base sequence is the sequence atcaacaccagg (SEQ ID NO: 5). In a preferred embodiment, the second hybridization probe portion has the structure [Chelator]-[Linker]-SSatcaacaccaggEEY-NH2 have wherein S, E, and Y represent the amino acids Ser, Glu, and Tyr, respectively. In one embodiment, the second hybridization probe portion has the structure DOTA-AEEA-SSatcaacaccaggEEY-NH2 have (where DOTA is the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetic acid).

[0028] According to the present invention, the first and second hybridization probe portions may comprise PNA sequences having modifications such as substitutions, small deletions, insertions or inversions, while still maintaining the biological activity of the PNA sequences set forth as SEQ ID NOS: 1 to 3. Preferably, such modifications do not involve more than 1, 2 or 3 bases in any one of SEQ ID NOS: 1 to 3.

[0029] For example, the 9 base sequence shown as SEQ ID NO:4 can be modified with one or two substitutions or deletions, resulting in a sequence having at least 75% or at least 85% identity to SEQ ID NO:4.

[0030] Furthermore, the 12-base sequence shown as SEQ ID NO:5 can be modified with one, two, or three substitutions or deletions, resulting in a sequence having at least 75%, at least 83%, or at least 91% identity to SEQ ID NO:5.

[0031] The 15 base sequence shown as SEQ ID NO:3 may be modified with one, two or three substitutions or deletions, resulting in a sequence having at least 80%, at least 86%, or at least 93% identity to SEQ ID NO:3.

[0032] It will be understood that the modified PNA oligomer has a sequence that is complementary to, and therefore hybridizes with, the sequence of the PNA oligomer of the other hybridization probe portion. As mentioned above, the degree of complementarity may be less than perfect (100%). As a result, one or two mismatches may exist in such a duplex, but the hybridized conjugate will still be useful according to the present invention.

[0033] When the second conjugate contains a therapeutic moiety, the therapeutic agent can be a radionuclide or a cytotoxic drug suitable for use in antibody-drug conjugates (ADCs) or other drug-containing conjugates (for a review, see, e.g., Shim, 2020). For example, the cytotoxic drug can be selected from auristatins such as calicheamicin, monomethyl auristatin E / F (MMAE / F), and maytansinoids such as maytansine derivatives DM0-DM4. Preferably, the therapeutic agent is a radionuclide.

[0034] When the therapeutic agent is a radionuclide, the radionuclide is preferably lutetium-177 ( 177 Lu), Yttrium-90( 90 Y), Bismuth-212( 212 Bi), Bismuth-213( 213 Bi), astatine-211( 211 At), Actinium-255 ( 255 Ac), Copper-67( 67 Cu), Gallium-67( 67 Ga), and rhenium-186( 186 More preferably, the radionuclide is lutetium-177 ( 177 Lu).

[0035] When the second conjugate comprises a diagnostic agent moiety, said diagnostic agent preferably produces a signal detectable by a method selected from the group consisting of positron emission tomography (PET), single photon emission computed tomography (SPECT), and optical imaging.

[0036] In a preferred embodiment, the diagnostic agent is a radionuclide. When the method of signal detection is SPECT, the radionuclide is preferably technetium-99m ( 99m Tc), Indium-111( 111 In), lutetium-177( 177 Lu), iodine-123( 123 I), iodine-125( 125 I), gallium-67( 67 Ga), and copper-67( 67 More preferably, the radionuclide is selected from the group consisting of Indium-111 ( 111 In).

[0037] When the method is PET, the radionuclide is preferably gallium-68 ( 68 Ga), Fluorine-18( 18 F), iodine-122( 122 I), iodine-124( 124 I), and copper-64( 64 More preferably, the radionuclide is selected from the group consisting of gallium-68 ( 68 Ga).

[0038] When the method of signal detection is optical imaging, the diagnostic agent is preferably a fluorescent dye selected from the group consisting of cyanine dyes, porphyrin derivatives, phthalocyanines, squaraine derivatives, xanthenes, Alexa analogs, and BODIPY analogs.

[0039] When the diagnostic or therapeutic agent is a radionuclide, the second hybridization probe portion preferably comprises a chelating agent for radiometal complexation. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), and Diethylenetriaminepentaacetic acid (DTPA) In one preferred embodiment of the invention, the chelating agent is DOTA.

[0040] However, fluorine-18( 18 F), iodine-122( 122 I), iodine-123( 123 I), iodine-124( 124 I), and iodine-125( 125 It will be appreciated that some radionuclides, such as I), can be conjugated to hybridization probes without the aid of a chelating agent, for example, by fluorination or iodination.

[0041] When the diagnostic or therapeutic agent is other than a radionuclide, such as a cytotoxic drug, the diagnostic or therapeutic agent is preferably covalently linked to the second hybridization probe moiety. For example, the second hybridization probe moiety can be conjugated to the diagnostic or therapeutic agent moiety by sortase A-mediated ligation, e.g., as described in Westerlund, 2015.

[0042] Optionally, the kit according to the invention can remove circulating primary conjugates that are not bound to the target site. Detergent For example, as disclosed in International Publication WO96 / 40245, Detergent may be an anti-idiotypic antibody or an antigen-binding antibody fragment.

[0043] In a further aspect, the present invention provides a pharmaceutical composition comprising the kit defined above. The present invention further provides the use of the kit or pharmaceutical composition defined above (a) for targeting a diagnostic or therapeutic agent to a target site, and / or (b) for diagnosing, prognosing or treating a condition in a mammal, including a human.

[0044] In yet another aspect, the present invention provides a method for delivering a diagnostic or therapeutic agent to a target site in a mammal, including a human, said method comprising: (a)(i) a targeting moiety that selectively binds to a target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; administering to said mammal a first conjugate comprising: (b) optionally, administering to said mammal Detergent and administering the Detergent The delocalized first conjugate is released from the blood circulation. Removal enabling the (c)(i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety administering to said mammal a second conjugate comprising Including, The second PNA oligomer of the second hybridization probe portion is 14 bases or less in length, and said second PNA oligomer binds to the first PNA oligomer of the first conjugate, thereby targeting the diagnostic or therapeutic agent moiety to the target site.

[0045] In a preferred embodiment, the method as defined above refers to a method for the diagnosis, prognosis or treatment of a pathology in a mammal, including a human.

[0046] According to the above method, the first and second conjugates and any Detergent is preferably as defined above in the context of the kit disclosed by the present invention.

[0047] In yet another aspect, the present invention provides a diagnostic or therapeutic conjugate for use in a method of delivering a diagnostic or therapeutic agent to a target site in a mammal, including a human, said method comprising: (a)(i) a targeting moiety that selectively binds to a target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; administering to said mammal a targeted conjugate comprising: (b) optionally, administering to said mammal Detergent and administering the Detergent Delocalized targeted conjugates are released from the blood circulation Removal enabling the (c) administering said diagnostic or therapeutic conjugate to said mammal. Including, The diagnostic or therapeutic conjugate comprises: (i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety Including, The second PNA oligomer of the second hybridization probe portion is 14 bases or less in length, and said second PNA oligomer binds to the first PNA oligomer of the first conjugate, thereby targeting the diagnostic or therapeutic agent moiety to the target site.

[0048] In a preferred embodiment, the method as defined above refers to a method for the diagnosis, prognosis or treatment of a pathology in a mammal, including a human.

[0049] According to the above method, said targeting conjugate is preferably as the first conjugate defined above in the context of the kit disclosed by the present invention, and said diagnostic or therapeutic conjugate is preferably as the second conjugate defined above in the context of the kit disclosed by the present invention.

[0050] According to the above method, any Detergent is preferably as defined above in the context of the kit disclosed by the present invention.

[0051] In the methods and uses according to the present invention, the first and second conjugates may be administered intravenously, intraarterially, intrapleurally, intraperitoneally, intrathecally, subcutaneously or by perfusion.

[0052] In the methods and uses according to the present invention, the condition may be selected from the group consisting of cancer, infectious diseases, inflammatory diseases, and autoimmune diseases. Preferably, the condition is cancer.

[0053] In a preferred embodiment, the pathology is a cancer capable of forming solid tumors, and the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, head and neck cancer, gastric cancer, and colon cancer. In such cases, the target site is present in a preferably mammalian, more preferably human, protein selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), carbonic anhydrase IX (CAIX), platelet-derived growth factor receptor β (PDGFR-β), and nectin-4 (see Table 7). In a preferred embodiment, the method of the present invention is a method for in vivo detection or treatment of HER2-expressing cancer, particularly cancer associated with breast cancer or gastroesophageal cancer.

[0054] In another preferred embodiment of the invention, the condition is a blood cancer selected from the group consisting of melanoma, leukemia, and myeloma, in which case the target site is located on a mammalian, more preferably human, protein selected from the group consisting of Cluster of Differentiation 38 (CD38), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 30 (CD30), Cluster of Differentiation 22 (CD22), and Cluster of Differentiation 79b (CD79b) (see Table 7). DETAILED DESCRIPTION OF THE INVENTION

[0055] Numbered Items of the Invention Embodiments of the present invention include the items summarized in the following non-exclusive list:

[0056] 1. A kit for targeting a diagnostic or therapeutic agent to a target site, comprising: (a)(i) a targeting moiety capable of selectively binding to a target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; a first conjugate comprising: (b)(i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety a second conjugate comprising Including, The kit, wherein the second PNA oligomer of the second hybridization probe portion is 14 bases or less in length.

[0057] 2. The kit according to item 1, wherein the target site is present in a mammalian, including human, protein expressed on the surface of a cell, preferably a tumor cell.

[0058] 3. The kit of item 2, wherein the target site is present in a mammalian, including human, protein selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), carbonic anhydrase IX (CAIX), platelet-derived growth factor receptor β (PDGFR-β), nectin-4, cluster of differentiation 38 (CD38), cluster of differentiation 33 (CD33), cluster of differentiation 30 (CD30), cluster of differentiation 22 (CD22), and cluster of differentiation 79b (CD79b).

[0059] 4. The kit of item 3, wherein the target site is present in human epidermal growth factor receptor 2 (HER2).

[0060] 5. The targeting moiety is ·antibody; antibody fragments, such as single domain antibodies (sdAbs; nanobodies), single chain variable fragments (scFvs), antigen-binding fragments (Fabs), diabodies, or minibodies; engineered scaffold proteins, such as Affibody® molecules; and ·peptide 5. The kit according to any one of items 1 to 4, selected from the group consisting of:

[0061] 6. The kit of item 5, wherein the targeting moiety is an Affibody® molecule.

[0062] 7. The kit of any one of items 1 to 6, wherein one or both of the hybridization probes comprises a linker.

[0063] 8. The kit according to item 7, wherein the linker is a solubilizing moiety, preferably (a) a PEG-based linker, such as a linker comprising 2-[2-(2-aminoethoxy)ethoxy]acetic acid (AEEA); or (b) a peptide-based linker comprising a charged or polar amino acid.

[0064] 9. The kit of any one of items 1 to 8, wherein the length of the first PNA oligomer of the first hybridization probe portion is at least the length of the second PNA oligomer of the second hybridization probe portion, and is 15 bases or less.

[0065] 10. The kit according to item 9, wherein the PNA oligomer of the first hybridization probe portion is 15 bases in length.

[0066] 11. The kit according to item 10, wherein the 15 bases have the sequence cctggtgttgatgat (SEQ ID NO: 3).

[0067] 12. The kit of item 11, wherein the first hybridization probe portion has the following structure: GSScctggtgttgatgatEK(DOTA)-AEEA-E-NH2.

[0068] 13. The kit according to any one of items 1 to 12, wherein the complementary PNA oligomer of the second hybridization probe portion is 5 to 14 bases in length.

[0069] 14. The kit according to item 13, wherein the complementary PNA oligomer is 9 to 12 bases in length.

[0070] 15. The kit according to item 14, wherein the complementary PNA oligomer is 9 bases in length.

[0071] 16. The kit according to item 15, wherein the 9 bases have the sequence aacaccagg (SEQ ID NO: 4).

[0072] 17. The kit of item 16, wherein the second hybridization probe portion has the following structure: DOTA-AEEA-SSaacaccaggEEY-NH2.

[0073] 18. The kit according to item 14, wherein the complementary PNA oligomer is 12 bases in length.

[0074] 19. The kit according to item 18, wherein the 12 bases have the sequence atcaacaccagg (SEQ ID NO: 5).

[0075] 20. The kit of item 19, wherein the second hybridization probe portion has the following structure: DOTA-AEEA-SSatcaacaccaggEEY-NH2.

[0076] 21. The kit of any one of items 1 to 20, wherein the therapeutic agent is selected from the group consisting of a radionuclide, a calicheamicin, an auristatin such as monomethylauristatin E / F (MMAE / F), and a maytansinoid such as maytansine derivatives DM0 to DM4.

[0077] 22. The therapeutic agent is lutetium-177 ( 177 Lu), Yttrium-90( 90 Y), Bismuth-212( 212 Bi), Bismuth-213( 213 Bi), astatine-211( 211 At), Actinium-255 ( 255 Ac), Copper-67( 67 Cu), Gallium-67( 67 Ga), and rhenium-186( 186 22. The kit according to item 21, wherein the radionuclide is selected from the group consisting of:

[0078] 23. The kit of any one of items 1 to 22, wherein the diagnostic agent produces a signal detectable by a method selected from the group consisting of positron emission tomography (PET), single photon emission computed tomography (SPECT), and optical imaging.

[0079] 24. The kit according to item 23, wherein the diagnostic agent is a radionuclide.

[0080] 25. The method is SPECT and the radionuclide is technetium-99m ( 99m Tc), Indium-111( 111 In), lutetium-177( 177 Lu), iodine-123( 123 I), iodine-125( 125 I), gallium-67( 67 Ga), and copper-67( 67 Item 25. The kit according to item 24, wherein the compound is selected from the group consisting of: Cu).

[0081] 26. The method is PET and the radionuclide is gallium-68(68 Ga), Fluorine-18( 18 F), iodine-122( 122 I), iodine-124( 124 I), and copper-64( 64 Item 25. The kit according to item 24, wherein the compound is selected from the group consisting of: Cu).

[0082] 27. The kit of items 21 or 24, wherein the diagnostic or therapeutic agent is a radionuclide and at least the second hybridization probe portion comprises a chelator.

[0083] 28. The kit according to item 27, wherein the chelating agent is selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), and Diethylenetriaminepentaacetic acid (DTPA).

[0084] 29. The kit according to item 28, wherein the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0085] 30. A method for delivering a diagnostic or therapeutic agent to a target site in a mammal, including a human, said method comprising: (a)(i) a targeting moiety that selectively binds to a target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; administering to said mammal a first conjugate comprising: (b) optionally, administering to said mammal Detergent and administering the Detergentallowing the delocalized first conjugate to clear from the blood circulation; and (c)(i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety administering to said mammal a second conjugate comprising Including, The method, wherein the second PNA oligomer of the second hybridization probe portion is 14 bases or less in length, and said second PNA oligomer binds to the first PNA oligomer of the first conjugate, thereby targeting the diagnostic or therapeutic agent moiety to the target site.

[0086] 31. A method for the diagnosis, prognosis or treatment of a medical condition in a mammal, including a human, said method comprising the method of item 30 for delivering a diagnostic or therapeutic moiety to a target site in a mammal.

[0087] 32. A method for diagnosing a pathology, comprising administering the first and second conjugates of the kit according to any one of items 1 to 29 to a mammal, including a human.

[0088] 33. A method for treating a medical condition, comprising administering the first and second conjugates of the kit according to any one of items 1 to 29 to a mammal, including a human, in need of such treatment.

[0089] 34. The method according to any one of items 31 to 33, wherein the pathology is selected from the group consisting of cancer, infectious disease, inflammatory disease, and autoimmune disease; preferably cancer.

[0090] 35. The method of item 34, wherein the pathology is a cancer capable of forming solid tumors, and the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, head and neck cancer, gastric cancer, and colon cancer.

[0091] 36. The method of claim 35, wherein the target site is present in a human protein selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), carbonic anhydrase IX (CAIX), platelet-derived growth factor receptor β (PDGFR-β), and nectin-4.

[0092] 37. The method of item 34, wherein the condition is a blood cancer selected from the group consisting of melanoma, leukemia, and myeloma.

[0093] 38. The method of claim 37, wherein the target site is present in a human protein selected from the group consisting of cluster of differentiation 38 (CD38), cluster of differentiation 33 (CD33), cluster of differentiation 30 (CD30), cluster of differentiation 22 (CD22), and cluster of differentiation 79b (CD79b).

[0094] 39. A pharmaceutical composition comprising the first and second conjugates of the kit according to any one of items 1 to 29.

[0095] 40. The pharmaceutical composition according to item 39, for use in the diagnosis or treatment of a condition selected from the group consisting of cancer, infectious diseases, inflammatory diseases, and autoimmune diseases.

[0096] Experimental Method All buffers were prepared using high-quality Milli-Q® water and purified from metal contaminants using Chelex® 100 resin (Bio-Rad Laboratories, USA). 177 LuCl3 was purchased from PerkinElmer (Waltham, MA, USA). Radioactivity was measured using an automated gamma spectrometer equipped with a NaI(TI) detector (1480 Wizard, Wallac, Finland).

[0097] In vitro cell studies were performed using HER2-expressing ovarian cancer SKOV3 and breast cancer BT474 cells (both obtained from the American Type Culture Collection (ATCC)). Cells were cultured in RPMI medium (Flow Irvine, UK) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and PEST, which consisted of 100 IU / mL penicillin and 100 mg / mL streptomycin.

[0098] Data on in vitro studies and biodistribution were analyzed by unpaired two-tailed t test (for comparison of two sets of data) and ANOVA (for comparison of several sets of data) using GraphPad Prism (version 4.00 for Windows; GraphPad Software) to determine significant differences.

[0099] Synthesis and purification of PNA pretargeting probes Peptide nucleic acid monomers Fmoc-PNA-A(Bhoc)-OH, Fmoc-PNA-G(Bhoc)-OH, Fmoc-PNA-C(Bhoc)-OH, and Fmoc-PNA-T-OH were purchased from PolyOrg, Inc., Leominster, USA, or PNA Bio, Inc., Thousand Oaks, USA. Rink Amide resin (ChemMatrix, 0.50 mmol / g) was purchased from Biotage (Uppsala, Sweden). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was purchased from CheMatech, Dijon, France. Fmoc-NH-(PEG)2-CH2COOH (AEEA) was purchased from Merck KGaA, Darmstadt, Germany. Solvents and reagents for solid phase synthesis were obtained from commercial suppliers and used without further purification.

[0100] HP15 was synthesized on a 50 μmol scale in a 10 ml reaction vial using Rink Amide resin (ChemMatrix, 0.50 mmol / g) on ​​a Biotage® Initiator+Alstra microwave peptide synthesizer. Fmoc deprotection was performed in two steps at room temperature by treating the resin with piperidine-DMF (1:4) for 3 minutes, followed by piperidine-DMF (1:4) for 10 minutes. Coupling was performed using 5 equivalents of PNA or amino acid monomer, 5 equivalents of Oxyma, and 5 equivalents of DIC in DMF. A 10-minute coupling time at 75°C was used throughout the sequence, followed by a 2-minute capping step using NMP-lutidine-acetic anhydride (89:6:5).

[0101] To allow for site-specific introduction of DOTA, an orthogonally protected Lys(Mtt) was introduced. After four coupling steps (Fmoc-EK(Mtt)-[AEEA]-E-resin), automated synthesis was interrupted for selective side-chain deprotection of Lys(Mtt) by adding 5–10 fresh TFA:TIS:DCM (1:2:97) solutions, followed by vortexing for 1 min. DOTA coupling was carried out at room temperature for 1 h using 5 equivalents of DOTA, 5 equivalents of Oxyma, and 5 equivalents of DIC in DMF. After automated synthesis was resumed and all cycles were completed, the resin was washed with DMF, DCM, and finally MeOH, then dried overnight. The PNA-peptide hybrid was cleaved from the solid support using a mixture of TFA:HO:TIS (95:2.5:2.5) at room temperature for 4 h. Finally, the PNA product was extracted between diethyl ether and water and lyophilized from the aqueous phase.

[0102] The shortest complementary PNA probe, HP19, was synthesized on the same microwave peptide synthesizer as HP15. Fmoc deprotection, coupling, and capping were performed as in the synthesis of HP15. The DOTA chelator was manually coupled to the PNA probe at the end of the synthesis using the same protocol as for DOTA coupled to HP15. The final product was cleaved from the resin and extracted in the same manner as HP15. The synthesis was continuously monitored by Kaiser test. The molecular weight of the final product, HP19, was verified using MALDI-TOF analysis.

[0103] The other complementary PNA probes (HP16, HP17, and HP18) were synthesized manually using the same monomers, resin, and solvents as those used for the synthesis of HP15. Each coupling was performed using 5 equivalents of PNA monomer. Before addition to the resin, the PNA monomer was preactivated with 5 equivalents of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; Sigma-Aldrich) in the presence of 10 equivalents of DIEA in NMP and DMF for 1 minute. Each coupling was performed at room temperature for 30 minutes to 1 hour with gentle shaking. Capping of unreacted PNA was performed in the same manner as for HP15. Fmoc deprotection was performed using 20% ​​piperidine in NMP for 20 minutes at room temperature with gentle shaking. The synthesis was monitored by Kaiser test, followed by microcleavage of a few beads of resin followed by MALDI-TOF analysis. After synthesis was complete, the resin was washed extensively with NMP and then DCM and allowed to dry overnight. Subsequent cleavage of the complementary PNA probe and ether extraction was performed as for HP15.

[0104] RP-HPLC purification was performed using a semi-preparative Zorbax 300 SB-C18 column (9.4 × 250 mm, 5 μm particle size; Agilent, Santa Clara, USA) with a linear gradient of 5–50% B (where A = 0.1% TFA-HO and B = 0.1% TFA-CH3CN) over 25 min at a flow rate of 3 ml / min, a column temperature of 70 °C, and UV detection at 220 and 260 nm. Collected fractions were analyzed by MALDI-TOF (4800 MALDI-TOF / TOF, ABSCIEX) using an α-cyano-4-hydroxycinnamic acid matrix. Fractions determined to contain the correct product were pooled and lyophilized.

[0105] The purity of HP16, HP17, and HP18 was confirmed using analytical RP-HPLC on a Zorbax 300SB-C18 column (4.6 × 150 mm, 3.5 μm particle size; Agilent) followed by MALDI-TOF analysis. The identity and purity of HP19 were confirmed using MALDI-TOF analysis. The extinction coefficient at 260 nm (ε ) of each PNA probe was 260 ) is the PNA composition and the extinction coefficient of each PNA monomer (A: 13700 M -1 cm -1 , C:6600M -1 cm -1 , G:11700M -1 cm -1 , and T:8600M -1 cm -1 ) were estimated based on the extinction coefficients used for each probe throughout all experiments: HP16: 98000M -1 cm -1 ,HP17:126900M -1 cm -1 ,HP18:155800M -1 cm -1 ,HP19:64000M -1 cm -1 and HP15:150700M -1 cm -1 .

[0106] Creation of affibody-PNA conjugates pAY430-Z HER2:342 The -SR-H6 plasmid (Westerlund 2015) was transformed into BL21(DE3) chemically competent Escherichia coli (E. coli) cells (Life Technologies), and the cells were cultured in complex medium (tryptic soy broth with yeast extract) supplemented with kanamycin. Protein expression was induced by the addition of 1 mM IPTG (final concentration), and the culture was maintained overnight at room temperature and 150 rpm. Cells were harvested by centrifugation (4000 rcf, 10 min, 4°C), resuspended in IMAC binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.5), and then lysed by sonication. After centrifugation, the clarified supernatant was used for the Z HER2:342 The eluate was passed through an IMAC matrix (HisPur™ cobalt resin, Thermo Scientific) that captures SR-H6. The resin was washed with wash buffer (20 mM Tris-HCl, 300 mM NaCl, 30 mM imidazole, pH 7.5), and the protein was eluted using elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 7.5). The eluted Z HER2:342 -H6 was buffer exchanged into sortase A conjugation buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl pH 7.5) using a PD-10 desalting column (GE Healthcare). HER2:342 The purity and molecular weight of -SR-H6 were confirmed using SDS-PAGE and MALDI-TOF.

[0107] Z HER2:342 -SR-H6 was site-specifically conjugated to HP15 using sortase A-mediated ligation (SML). The SML method described below is based on a previously published protocol for affibody-PNA conjugation (Altai 2017). Srt A3 *A variant of sortase A with P94S / D160N / K196T (Chen et al., 2011) mutations, designated as , was used for conjugation. The glycine-modified HP15 probe was dissolved in 10% DMSO and heated at 80 °C for 5 min, after which the concentration was estimated based on the absorbance at 260 nm. 500 nmol of HP15, 1.25 μmol of Z HER2:342 -SR-H6 and 1.25 μmol NiCl2 were mixed in a final volume of 5 ml of sortase A conjugation buffer. * HP15 was added to the reaction mixture to a final concentration of 5 μM, and the reaction was allowed to proceed for 30 min before undergoing a reverse IMAC step using a pre-equilibrated IMAC matrix. The conjugation product, hydrolyzed protein by-products, and unconjugated HP15 could be collected in the flow-through after a 30-min incubation step with the given matrix. The collected flow-through was then buffer-exchanged to 10 mM NaOAc pH 3.6 on a PD-10 column and subsequently lyophilized. HER2:342 The Z-SR-HP15 conjugate was purified by RP-HPLC using the same column and solvent as for the purification of the PNA probe, but with a gradient of 5% to 50% B in 25 min and absorbance monitored at 220, 260, and 280 nm. HER2:342 The SR-HP15 conjugate fraction was collected, lyophilized, and analyzed by MALDI-TOF. To confirm the purity of the purified conjugate, it was analyzed by analytical HPLC (Zorbax 300SB-C18, particle size 3.5 μm, 4.6 × 150 mm, Agilent) and electrospray ionization mass spectrometry (ESI-MS, Impact II, Bruker). Unconjugated HP15 was analyzed by Z HER2:342 -could be used in a new conjugation reaction together with a new batch of SR-H6.

[0108] PNA probe and Z HER2:342The final concentration of -SR-HP15 was determined by measuring the absorbance at 260 nm. Since the contribution from the protein moiety to the total absorbance at 260 nm is negligible, the same extinction coefficient was used for HP15 and Z HER2:342 -Used for both SR-HP15.

[0109] Z to the HER2 receptor HER2:342 The binding of -SR-HP15 was confirmed by surface plasmon resonance (SPR).

[0110] Characterization of PNA pretargeting probes The kinetic parameters of PNA probe hybridization were analyzed using surface plasmon resonance (SPR) on a Biacore™ 8K instrument (GE Healthcare). Dextran Chip Series S Sensor CM5 (GE Healthcare) was coupled to four surfaces using standard amine coupling procedures to 385, 194, 185, and 353 resonance units (RU). HER2:342 The PNA probes were functionalized with -SR-HP15. The reference surface of each chip was subjected to activation followed by deactivation. Complementary PNA probes HP16, HP17, HP18, and HP19 were injected using single-cycle injections at five concentrations: 22.6, 45.3, 90.6, 181.25, and 362.5 nM. Each concentration was allowed to associate for 300 seconds, followed by the next injection and association step. After the injection of the final concentration, dissociation was allowed for 10,000 seconds (2 hours and 47 minutes), followed by regeneration with 10 mM HCl for 30 seconds, followed by 15 mM NaOH for 30 seconds. All runs were performed at 25°C in PBST (0.05% Tween-20) pH 7.4 with a flow rate of 50 μl / min. Kinetic parameters were calculated using a 1:1 binding model in Biacore Insight Evaluation software.

[0111] The melting temperatures of HP15:HP16, HP15:HP17, and HP15:HP18 were determined by monitoring UV absorbance at 260 nm (Chirascan™, Applied Photophysics) over a temperature range of 20 to 95 °C using a 1 °C / min temperature change. The PNA complexes were heated to 80 °C for 5 min, then hybridized at room temperature for 5 min, followed by UV monitoring at room temperature for 5 min. CD spectra were collected before and after determining thermal denaturation. The melting temperature of the HP15:HP19 complex was determined using a Varian Cary 50 Bio UV-visible spectrophotometer equipped with a single-cell Peltier thermostatically controlled cuvette holder. The temperature in the cell was adjusted between 20 and 80°C at a rate of 0.5°C / min, and measurements were taken at 260 nm after 60 seconds of equilibration at each temperature point.

[0112] Radiolabeling and in vitro stability 177 Radiolabeling of primary and secondary PNA probes with Lu was performed using a previously described method (Westerlund 2018). Briefly, 30 μg of peptide was dissolved in 100 μL of ascorbic acid (1 M, pH 5.5) by heating at 95 °C for 10 min, followed by sonication for 5 min to ensure complete dissolution. 3 μL (60–120 MBq) of Lu was added. 177 LuCl was added followed by vortexing. The mixture was incubated at 95° C. for 60 minutes. The reaction mixture was analyzed by radio-ITLC eluting with 0.2 M citric acid, pH 2.0.

[0113] loosely bound 177 To remove Lu, treatment with excess ethylenediaminetetraacetic acid tetrasodium salt (EDTA.Na4) was performed. A freshly prepared solution of EDTA.Na4 (10 mg / mL in Milli-Q water) was added to the reaction mixture in a 1000-fold molar excess and incubated at 95°C for 10 min. Due to the high radiochemical yield and purity, no further purification was required for the new secondary probe. 177 Lu]Lu-HP2 and [ 177Lu]Lu-Z HER2:342 -SR-HP15 was purified by size exclusion chromatography after EDTA treatment using a disposable NAP-5 column pre-equilibrated and eluted with 1% BSA / PBS.

[0114] To assess stability, a fraction of the freshly radiolabeled conjugate (0.4 μg) was incubated with a 500-fold molar excess of EDTA for 60 min at 37°C. Incubations in PBS were also performed as controls. Tests were performed in triplicate.

[0115] To verify the radio-ITLC results, reversed-phase HPLC was performed using a LaChrom Elite® system (Hitachi, VWR, Darmstadt, Germany) consisting of a serially connected L-2130 pump, UV detector (L-2400), and radioactive flow detector (Bioscan, Washington, DC, USA). An analytical column (Phenemenex, Aschaffenburg, Germany; Luna® 5 μm C18, 100 Å; 150 × 4.6 mm column) was used to measure the radioactivity. 177 Purity analysis of the [Lu]Lu-labeled compound was performed. HPLC conditions were as follows: A = 10 mM TFA / HO; B = 10 mM TFA / acetonitrile; UV detection at 220 nm; elution gradient: 5–70% B for 0–15 min, 70–95% B for 15–18 min, 5% B for 19–20 min; flow rate: 1.0 mL / min.

[0116] In vitro studies Cells, 10 6 Cell culture dishes were seeded at a density of cells / dish. A set of three dishes was used for each data point.

[0117] to HER2-expressing cells 177 Lu]Lu-Z HER2:342The specificity of the binding of Z-SR-HP15 was tested by incubating cells with 1 nM of the labeled conjugate for 1 h at 37 °C. To saturate the receptor, unlabeled Z-SR-HP15 was added for 5 min before the addition of the radiolabeled probe. HER2:342 (1000 nM) was added to the control set.

[0118] New 177 Lu]Lu-HP16, [ 177 Lu]Lu-HP17 and [ 177 Pretargeting specificity assays for [Lu]Lu-HP18 were performed using four sets of cell dishes as previously described (Honarvar 2016). To demonstrate pretargeting, one set of dishes was placed in Z HER2:342 The cells were incubated with SR-HP15 (1 nM) at 37°C for 1 hour and then washed. 177 Lu-labeled secondary probe (10 nM) was added, and the cells were incubated for 1 hour at 37°C. To demonstrate that pretargeting was HER2-mediated, a second set of dishes was incubated with excess affibody molecule Z. HER2:342 (1000 nM) for 5 min, followed by Z HER2:342 -SR-HP15 was added. 177 Lu-labeled secondary probe (10 nM) was added, and the cells were incubated for 1 hour at 37°C. To demonstrate that pretargeting was PNA-mediated, a third set of dishes was cultured in Z HER2:342 -SR-HP15, followed by incubation with excess unlabeled secondary probe (300 nM) for 30 min, then 177 In the fourth set, cells were incubated for 1 hour with a Lu-labeled secondary probe. 177 Nonspecific binding was assessed by incubation with Lu-labeled secondary probe alone. At the end of the incubation, cells were washed, detached with trypsin, and the radioactivity in the cells was measured to calculate the percentage of cell-bound radioactivity.

[0119] To assess the binding affinity of the radiolabeled conjugates to the HER2 receptor, a LigandTracer® yellow instrument (Ridgeview Instruments AB, Vange, Sweden) was used to measure the binding affinity of the conjugates to SKOV3 cells. 177 The kinetics of binding of [Lu]Lu-labeled probes and their dissociation from SKOV3 cells were measured. SKOV3 cells were seeded in a local area on cell culture dishes (Nunclon™, size 100620, NUNC A / S, Roskilde, Denmark). SKOV3 cells were incubated in duplicate dishes for 2 h at 2 °C. HER2:342 The cells were pre-saturated with 1 nM SR-HP15 and then washed three times to remove unbound primary drug. Increasing concentrations of the two radiolabeled molecules ([ 177 Lu]Lu-Z HER2:342 - 180 and 540 pM for SR-HP:15, [ 177 For Lu]Lu-labeled secondary probes, 1 and 5 nM) were added. Data were analyzed using Interaction Map™ software (Ridgeview Diagnostics AB, Uppsala, Sweden) to determine the association rate constant (k a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D ) was calculated. The analysis was performed in duplicate.

[0120] Cellular processing and retention in SKOV3 and BT474 cells was studied during interrupted incubation by the acid wash method (Wallberg 2008).

[0121] In vivo studies Animal experiments were performed in accordance with national legislation regarding work with laboratory animals. Approval was granted by the Ethics Committee for Animal Research in Uppsala. For tumor implantation, 10 7SKOV3 cells were subcutaneously injected into the right hind leg of female BALB / c nu / nu mice. Biodistribution experiments were performed 2 weeks after cell implantation. The average animal weight was 18±1 g. The average tumor weight was 0.23±0.11 g. For biodistribution measurements, mice were euthanized at the designated time points by an overdose of anesthesia (Rompun® / Ketalar®) followed by cardiac puncture. Organs of interest and tumors were harvested, weighed, and their radioactivity was measured. The percentage of the total injected dose per gram of sample (%ID / g) was calculated.

[0122] The pretargeting protocol used in this study was described by Westerlund et al. (Westerlund 2018) [ 177 It has been previously optimized for affibody-based PNA-mediated therapy using [Lu]Lu-HP2. 177 When both injection masses of [Lu]Lu-HP2 were doubled, the upscaling experiment showed 177 Based on this study, 50 μg of the primary drug and an equimolar amount of the secondary drug (0.69, 0.89, 1, and 1 μg for HP16, HP17, HP18, and HP2, respectively) were injected per mouse.

[0123] For biodistribution studies, mice were randomized into five groups: 30 mice, Z HER2:342 -SR-HP15 (4 nmol in 100 μL of PBS per mouse) was intravenously injected. After 16 hours, all mice 177 Lu]-HP16, [ 177 Lu]-HP17 or [ 177 Lu]-HP18 (194 pmol, 120 kBq in 100 μL of 2% BSA in PBS) was injected. Biodistribution was measured 4 and 144 hours after injection of the secondary probe. For comparison, first-generation [ 177 The biodistribution of [Lu]Lu-HP2 was measured by Z HER2:342 -SR-HP:1 was measured in the same way as when it was used as the primary drug.

[0124] To assess in vivo specificity, 177 The biodistribution of the [Lu]Lu-secondary probe was measured 4 hours after injection without prior injection of the primary agent.

[0125] After gamma counter measurements were completed, tumors were embedded in cryomedium (Neg-50™, Thermo Scientific, USA) and frozen at -80°C. Frozen tumors were cut into serial sections (30 μm thick) using a cryomicrotome (CryoStar™ NX70, Thermo Scientific, USA) and thaw-mounted onto glass slides. For digital autoradiography, slides containing sections were placed in cassettes and exposed to a phosphor screen overnight. The phosphor screen was scanned using a Cyclone® storage phosphor system at a resolution of 600 dpi and analyzed using OptiQuant software (PerkinElmer, USA).

[0126] Cumulative activity in the kidney and tumor was assessed to estimate the ratio of absorbed dose in the tumor and kidney. Estimation was based on a clinically validated two-time point approach (Freedman 2020). Biodistribution data were undecay-corrected, and the area under the time-activity plot was calculated using GraphPad Prism software. It was assumed that the primary absorbed dose was due to beta particles, since crossover doses were negligible and the absorption ratio would be equal to 1.

[0127] SPECT / CT imaging Secondary SKOV3 xenograft-bearing mice 177Seven nmol of primary drug was injected intravenously 16 hours before injection of the Lu-labeled probe (680 pmol, 9–13 MBq). Immediately prior to imaging (4 hours after injection), animals were sacrificed by CO2 asphyxiation. SPECT imaging was performed using a nanoScan SC (Mediso Medical Imaging Systems, Hungary). CT acquisition was performed using an X-ray energy of 50 keV. A 20-minute SPECT helical scan was acquired using energy windows 50–62, 103–124, and 188–230 keV. Data were reconstructed using Tera-Tomo™ 3D SPECT software.

[0128] Examples of inventions Example 1: Generation and characterization of affibody-PNA conjugates and complementary PNA probes PNA pretargeting probes HP15, HP16, HP17, HP18, and HP19 (Table 1) were prepared as described above. The probes were designed to avoid self-complementary sequences and extended stretches of purines (A and G), which are known to promote aggregation and therefore can make PNA-based probes difficult to synthesize and purify (Zhao 2020).

[0129] Affibody® Molecule Z HER2:342 -SR-H6 and HP15, Srt A3 * The ligation efficiency of the reaction was estimated to be 40% based on the integrated area under the peak at 260 nm in RP-HPLC. The 40% ligation efficiency was obtained by using the Z HER2:342This is lower than the 70% ligation efficiency previously reported for conjugation to SR-H6 (Altai 2017). However, HP15 has one glycine residue at its N-terminus, compared to three glycine residues at the N-terminus of HP1, which may affect ligation efficiency. The 40% ligation efficiency is within the same range as the 45% reported for conjugation of HP1 using wild-type sortase A (Westerlund, 2015).

[0130] Immobilized Z HER2:342 Hybridization of four complementary PNA probes to -SR-HP15 was analyzed by SPR. Representative sensorgrams of interactions analyzed using single-cycle injections are shown in Figure 1. The association rate constant, k a is 4.6 × 10 for HP16, HP17, and HP18, respectively. 4 , 4.3 × 10 4 and 5.7 × 10 4 M -1 s -1 Therefore, the on-rates of all three PNA probes interacting with HP15 are in the same range. a is 2.1 x 10 for HP19 7 M -1 s -1 The dissociation rate constant k d For HP16 it is 1.2 x 10 -5 s -1 , 7.2×10 for HP19 -2 s -1 For the other two complementary PNA probes (HP17 and HP18), k d The equilibrium dissociation constant of HP16 was calculated to be approximately 280 pM (Table 2), but Z HER2:342 It was estimated that HP17 and HP18 have higher affinity for -SR-HP15.

[0131] K determined for the interaction between HP15 and HP16 (nonamer) DThe K determined for the interaction between HP15 and HP19 (hexamer) (280 pM) appears to be sufficiently high affinity for the intended pretargeting application. D Although the affinity (3.4 nM) is higher, this lower affinity is expected to be sufficient for applications. Successful pretargeting using bispecific antibody constructs that bind both tumor-associated antigens and radiolabeled haptens has been previously demonstrated, and bifunctional antibodies and 111 The interaction between In-labeled benzyl EDTA derivatives was only 10 -9 ~10 -10 Estimated K of M D (Stickney 1991).

[0132] Hybridization between HP15 and the secondary probes HP16, HP17, or HP18 produced CD spectra with minima at approximately 215 nm and 260 nm. The induced signal is the result of PNA:PNA duplex formation upon hybridization between complementary PNA probes with C-terminal L-amino acids (Corradini 2012). The melting temperatures of each hybridization probe after duplex formation with HP15 were monitored at 260 nm and estimated to be 73°C, 75°C, and 87°C for HP16, HP17, and HP18, respectively (Figure 2a), and 55°C for HP19 (Figure 2b). Spectroscopic characterization indicates that HP16, HP17, and HP18 all form highly thermally stable duplexes with the primary PNA probe HP15. The melting temperatures of HP16, HP17, and HP19 are expected to be lower due to their shorter oligomer lengths compared to HP18. Although significantly lower than the melting temperatures of HP16, HP17, and HP18, the melting temperature of HP19 is still well above human body temperature.

[0133] Example 2: Radiolabeling and in vitro stability Table 3 shows 177The results of radiolabeling all probes with Lu are shown. After EDTA treatment, the radiochemical yield of the new secondary agent exceeded 98%. Therefore, further purification using NAP-5 was not performed for in vitro and in vivo studies. A NAP-5 column was used. 177 Lu]Lu-Z HER2:342 -SR-HP15 and [ 177 Purification of [Lu]Lu-HP2 yielded 100±0% radiochemical purity for both labels. 177 All Lu-labeled probes were stable in PBS and in the presence of EDTA for up to 1 h of incubation.

[0134] To verify the radioactive ITLC results, radioactive HPLC was performed to demonstrate that no fragmentation occurred after labeling and purification. The radioactive HPLC retention time for all probes was approximately 5.8 min. The retention time for the unlabeled probe was the same as that of the labeled probe.

[0135] Example 3: In vitro studies First-line drugs [ 177 Lu]Lu-Z HER2:342 The HER2-binding specificity of -SR-HP15 was tested using saturation experiments. When cells were pre-saturated with anti-HER2 affibody molecules, binding was significantly (p<0.0005) reduced (Figure 3A), demonstrating that binding was HER2-mediated. 177 Lu]Lu-Z HER2:342 The slow internalization of -SR-HP15 is advantageous for pretargeting applications, as this allows for long persistence of the primary hybridization probe on the surface of the target cell.

[0136] Z HER2:342 -SR-HP15 pretreated HER2-expressing cells 177 Lu]Lu-HP16, [ 177 Lu]Lu-HP17 and [ 177 The in vitro binding specificity of [Lu]Lu-HP18 was evaluated (Figures 3B, 3C, and 3D). HER2 receptors were saturated with anti-HER2 affibody molecules or ZHER2:342 - When SR-HP15-treated cells were preincubated with a large excess of unlabeled secondary drugs, binding of all secondary drugs was significantly (p<0.0005) reduced. Binding of all secondary drugs to cells without preincubation with primary drugs was significantly (p<0.0005) lower compared to pretargeting. 177 The specificity of [Lu]Lu-HP2 has been previously demonstrated (Altai 2017 NMB).

[0137] Z HER2:342 LigandTracer® measurements of the kinetics of binding to live SKOV3 cells pretreated with -SR-HP15 demonstrated extremely strong binding of all secondary probes. Interaction Map™ calculations showed that 177 Lu]Lu-Z HER2:342 -SR-HP15 and pre-targeted 177 The [Lu]Lu- secondary probe exhibited rapid association followed by very slow dissociation, with picomolar dissociation constants (K D ) resulted in K D The values ​​were 11–12 pM. There was no difference in the apparent dissociation constants between the secondary probes. Thus, decreasing the length of the PNAs from 15 to 9 nitrogenous bases was not associated with an observable decrease in their binding to the primary probes in the cellular assay.

[0138] After interrupted incubation, cellular processing and retention of total radiolabel by SKOV3 and BT474 cells was determined. 177 Lu]Lu-Z HER2:342 In the case of -SR-HP15, internalization was slow, which is typical for HER2-binding affibody molecules and their derivatives. 177 Lu]Lu-Z HER2:342 The internalization rate of -SR-HP15 was slightly higher in SKOV3 cells than in BT474 cells (18±2% for SKOV3 vs. 12±2% for BT474 at 24 hours). The internalization pattern of the labeled secondary probe was [ 177 Lu]Lu-Z HER2:342The pattern was similar to that of SR-HP15. The retention of radioactivity over time was [ 177 Lu]Lu-Z HER2:342 -SR-HP15 was the most expensive, but [ 177 The [Lu]Lu- secondary probe showed a faster release of bound radioactivity over time.

[0139] Example 4: In vivo studies Without prior injection of the primary probe [ 177 Data on the biodistribution of the Lu]Lu-labeled secondary probes are provided in Table 4. 177 Lu]-HP16 and [ 177 The biodistribution of [Lu]-HP17 was very similar, except for a small but significant difference in bone uptake. 177 Compared with [Lu]-HP18, the shorter variants had significantly lower uptake in blood, liver, and kidney. 177 Lu]-HP17 also has significantly lower uptake in lung and bone.

[0140] Z HER2:342 -Pre-injection of SR-HP15 (4 nmol) 177 The in vivo specificity results of the [Lu]Lu-secondary probe (4 hours after injection) are shown in Figure 4. HER2:342 -If SR-HP15 is pre-injected, all [ 177 Tumor uptake of the [Lu]Lu-secondary probe was significantly (p<0.00005) higher than without pre-injection. Interestingly, uptake in normal tissues was also significantly higher with pre-injection of the primary probe.

[0141] The dramatic increase in uptake of the secondary probe in tumors after pre-injection of the primary agent convincingly demonstrates the specificity of pretargeting. HER2:342 Increased uptake of the secondary probe after injection of -SR-HP15 was also observed in the blood, kidney, spleen, and muscle. This may be explained by the association of the secondary probe with the primary agent, which was not completely cleared from the circulation or re-entered the bloodstream after dissociating from receptors in the tumor. This effect was [177 Lu]-HP16 was less pronounced.

[0142] After injection of the primary probe in SKOV3-bearing mice [ 177 Lu]Lu-HP16, [ 177 Lu]Lu-HP17, [ 177 Lu]Lu-HP18 and [ 177 The results of the comparative biodistribution with [Lu]Lu-HP2 are shown in Table 5.

[0143] Biodistribution measurements indicate rapid clearance from blood and normal organs and tissues for all PNA-based probes studied. Some differences in biodistribution were observed between conjugates. Blood concentrations were significantly higher for PNA-based probes than for other secondary probes 4 hours after injection. 177 Lu]Lu-HP18 was significantly (p<0.0001) higher. 177 Lu]Lu-HP16, [ 177 Lu]Lu-HP17 and [ 177 The liver uptake for [Lu]Lu-HP2 (0.1 ± 0.0% ID / g) was comparable, whereas [ 177 The uptake was significantly (p<0.05) lower than that of [Lu]Lu-HP18 (0.2±0.1%ID / g). The only tissues with significant uptake were the kidney and tumor. 177 The renal uptake of Lu]Lu-HP16 (6±1%ID / g) was 177 Lu]Lu-HP18 (12±2%ID / g) and [ 177 [Lu] was significantly (p<0.05) lower than that for Lu-HP2 (10±2%ID / g). 177 Lu]Lu-HP16 showed the highest mean tumor uptake (24±6% ID / g), but the difference relative to the uptake of the other probes was not significant. The combination of lower kidney uptake and high tumor uptake was due to the [ 177 Lu]Lu-HP16 resulted in a higher tumor-to-kidney ratio (4-fold higher than kidney uptake). 177 Lu]Lu-HP16 and [ 177 Renal uptake of Lu]Lu-HP17 was 177The tumor uptake was significantly (p<0.005) lower than that of [Lu]Lu-HP18. 177 Lu]Lu-HP17 and [ 177 Lu]Lu-HP18 (both 5±1%ID / g)>[ 177 Lu]Lu-HP2(4±1%ID / g)>[ 177 Lu]Lu-HP16 (3±1% ID / g). 177 Lu]Lu-HP17 showed better retention of radioactivity in the tumor and faster clearance in the kidney (tumor uptake was 7-fold higher than kidney uptake), resulting in a higher tumor-to-kidney ratio 144 hours after injection.

[0144] Example 5: SPECT / CT imaging SPECT / CT imaging results (Figure 5) confirmed efficient affibody-based PNA-mediated pretargeting for all variants. The tumor was the site of highest uptake. The kidney and tumor were the only tissues with significant uptake. Radioactivity uptake in the tumor was significantly higher than in the kidney in each animal.

[0145] The distribution of radioactivity in the tumor was assessed using autoradiography. Four hours after injection, the distribution of activity was very uniform, reflecting the clustered nature of the xenografts. At later time points, the gradient of radioactivity concentration from the tumor core to the rim became more pronounced.

[0146] The estimated dosimetry results for the kidney and tumor are shown in Table 6 and Figure 6. The ratios of the areas under the time-activity plots for the tumor and kidney were 3.8, 2.8, 2.0, and 2.0 for HP16, HP17, HP18, and HP2, respectively. 177 These results indicate that [Lu]Lu-HP16 may provide the most favorable dosimetry for therapeutic use. JPEG0007795810000001.jpg243155 JPEG0007795810000002.jpg231155 JPEG0007795810000003.jpg230155 JPEG0007795810000004.jpg60155

[0147] [Table 1A]

[0148] [Table 1B]

[0149] [Table 2]

[0150] [Table 3]

[0151] [Table 4] a [ 177 Lu]-HP16 and [ 177 Significant difference between [Lu]-HP17 b [ 177 Lu]-HP16 and [ 177 Significant difference between [Lu]-HP18 c [ 177 Lu]-HP17 and [ 177 Significant difference between [Lu]-HP18 * Data for the GI tract and carcass including contents are presented as % of injected dose per total sample.

[0152] [Table 5] a [ 177Lu]Lu-HP16 and [ 177 Significant difference between Lu]Lu-HP18 (p<0.05) b [ 177 Lu]Lu-HP17 and [ 177 Significant difference between Lu]Lu-HP18 (p<0.05) C [ 177 Lu]Lu-HP18 and [ 177 Significant difference between Lu]Lu-HP2 (p<0.05) d [ 177 Lu]Lu-HP16 and [ 177 Significant difference between Lu]Lu-HP2 (p<0.05)

[0153] [Table 6]

[0154] [Table 7] [Sequence List Free Text]

[0155] Sequence Listings 1-7 <223> Synthetic PNA sequence

[0156] [ka] [ka]

Claims

1. 1. A kit for targeting a diagnostic or therapeutic agent to a target site present in a mammalian protein, including a human protein, expressed on the surface of a cell, preferably a tumor cell, comprising: (a) (i) a targeting moiety capable of selectively binding to said target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; a first conjugate comprising: (b) (i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety and a second conjugate comprising Including, the second PNA oligomer of the second hybridization probe portion is 8 to 12 bases in length; A kit wherein the length of the first PNA oligomer in the first hybridization probe portion is at least the length of the second PNA oligomer in the second hybridization probe portion, and is 15 bases or less.

2. 2. The kit of claim 1, wherein the target site is present in a mammalian, including human, protein selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), carbonic anhydrase IX (CAIX), platelet-derived growth factor receptor β (PDGFR-β), nectin-4, cluster of differentiation 38 (CD38), cluster of differentiation 33 (CD33), cluster of differentiation 30 (CD30), cluster of differentiation 22 (CD22), and cluster of differentiation 79b (CD79b).

3. 3. The kit of claim 2, wherein the target site is present in human epidermal growth factor receptor 2 (HER2).

4. the targeting moiety, ·antibody; - antibody fragments, such as single domain antibodies (sdAb; nanobodies), single chain variable fragments (scFv), antigen-binding fragments (Fab), diabodies, or minibodies; engineered scaffold proteins, such as Affibody® molecules; and ·peptide 4. The kit of claim 1, wherein the kit is selected from the group consisting of:

5. 5. The kit of claim 4, wherein the targeting moiety is an Affibody® molecule.

6. One or both of the hybridization probe moieties may be a solubilizing moiety, preferably (a) a PEG-based linker, such as a linker comprising 2-[2-(2-aminoethoxy)ethoxy]acetic acid (AEEA); or (b) Peptide-based linkers containing charged or polar amino acids 6. The kit of claim 1, comprising:

7. The kit of any one of claims 1 to 6, wherein the second PNA oligomer of the second hybridization probe portion is 9 to 12 bases in length.

8. 8. The kit of any one of claims 1 to 7, wherein the therapeutic agent is selected from the group consisting of a radionuclide, a calicheamicin, an auristatin such as monomethyl auristatin E / F (MMAE / F), and a maytansinoid such as maytansine derivatives DM0 to DM4.

9. 9. The kit of any one of claims 1 to 8, wherein the diagnostic agent produces a signal detectable by a method selected from the group consisting of magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), X-ray imaging, ultrasound, and optical imaging.

10. 1. A pharmaceutical composition for targeting a diagnostic or therapeutic agent to a target site present in a mammalian protein, including a human protein, expressed on the surface of a cell, preferably a tumor cell, comprising: (a) (i) a targeting moiety capable of selectively binding to said target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; a first conjugate comprising: (b) (i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety and a second conjugate comprising Including, the second PNA oligomer of the second hybridization probe portion is 8 to 12 bases in length; A pharmaceutical composition, wherein the length of said first PNA oligomer in said first hybridization probe portion is at least the length of said second PNA oligomer in said second hybridization probe portion, and is 15 bases or less.

11. 11. The pharmaceutical composition of claim 10 for use in the diagnosis or treatment of a condition selected from the group consisting of cancer, infectious diseases, inflammatory diseases, and autoimmune diseases, preferably cancer.

12. 1. A pharmaceutical composition comprising a diagnostic or therapeutic conjugate for use in a method for delivering a diagnostic or therapeutic agent to a target site in a mammal, including a human, said method comprising: (a) (i) a targeting moiety that selectively binds to the target site; and (ii) a first hybridization probe portion comprising a first PNA oligomer; administering to said mammal a targeted conjugate comprising: (b) optionally administering a clearing agent to the mammal to allow the clearing agent to remove the delocalized targeted conjugate from the circulation; (c) administering the pharmaceutical composition to the mammal. Including, the diagnostic or therapeutic conjugate (i) a second hybridization probe portion comprising a second PNA oligomer complementary to the first PNA oligomer; and (ii) a diagnostic or therapeutic moiety Including, the second PNA oligomer of the second hybridization probe portion is 8-12 bases in length, and the second PNA oligomer binds to the first PNA oligomer of the targeting conjugate, thereby targeting the diagnostic or therapeutic agent moiety to the target site; A pharmaceutical composition, wherein the length of said first PNA oligomer in said first hybridization probe portion is at least the length of said second PNA oligomer in said second hybridization probe portion, and is 15 bases or less.

13. 13. The pharmaceutical composition of claim 12, wherein the method is for the diagnosis, prognosis or treatment of a condition in a mammal, including a human, and the condition is selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an autoimmune disease, preferably cancer.

14. the condition is a cancer capable of forming solid tumors, and the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, head and neck cancer, gastric cancer, and colon cancer; 14. The pharmaceutical composition of claim 13, wherein the target site is present in a human protein selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), carbonic anhydrase IX (CAIX), platelet-derived growth factor receptor β (PDGFR-β), and nectin-4.

15. 14. The pharmaceutical composition of claim 13, wherein the condition is a blood cancer selected from the group consisting of melanoma, leukemia, and myeloma.

16. 16. The pharmaceutical composition of claim 15, wherein the target site is present on a human protein selected from the group consisting of cluster of differentiation 38 (CD38), cluster of differentiation 33 (CD33), cluster of differentiation 30 (CD30), cluster of differentiation 22 (CD22), and cluster of differentiation 79b (CD79b).

17. 17. The pharmaceutical composition of any one of claims 12 to 16, wherein the second PNA oligomer of the second hybridization probe portion is 9 to 12 bases in length.

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