Asparaginyl peptide ligase mediated single domain antibody radiolabeling

Abstract

The present invention relates generally to the field of radiochemistry. In particular, the present invention provides a method of producing a site-specific radiolabeled single domain antibody (sdAb) tracer mediated by an asparaginyl peptide ligase (PAL), optionally in the presence of a glutamyl cyclase (QC). The radiolabelled sdAb tracers prepared by the methods of the invention provide enhanced properties and are useful in diagnosis, surgery, therapy, therapy monitoring, and as imaging agents.

Description

Technical Field

[0001] This invention generally relates to the field of radiochemistry. Specifically, it provides a method for the production of site-specific radiolabeled single-domain antibody (sdAb) tracers mediated by asparagine acyl peptidase (PAL), an improvement of which is achieved in the presence of glutamine acyl cyclase (QC). The radiolabeled sdAb tracers prepared by the method of this invention offer enhanced properties and are suitable for diagnostics, surgery, treatment, therapy monitoring, and as imaging agents. Background Technology

[0002] Molecular imaging techniques, such as computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), ultrasound imaging (US), and optical imaging, provide non-invasive methods for characterizing physiological processes and linking molecular changes to clinical outcomes. Significant progress has been made in molecular imaging in recent years, including the development of new imaging devices, the discovery of new molecular targets, and the creation of multifunctional contrast agents. Furthermore, the imaging component can be replaced with therapeutic molecules (such as therapeutic radionuclides) or fluorescent molecules that can be used in photodynamic therapy.

[0003] For in vivo diagnostic purposes in molecular imaging, contrast agents or imaging tracers must ideally consist of a targeting molecule capable of specifically recognizing the target and a signal-emitting molecule that allows for in vivo detection. Bioconjugates between the two molecules are also crucial, especially for small protein candidates such as single-domain antibodies (sdAbs), which facilitate adequate tissue penetration, selective binding to the target, a high signal-to-noise ratio at the target site, and low overall in vivo retention or accumulation (due to elimination from the body, typically in the liver or kidneys).

[0004] Single-domain antibodies (sdAbs) are antibody fragments that constitute an interesting tool in the field of nuclear medicine for imaging purposes (e.g., via PET imaging) and for therapeutic purposes, with applications in various diseases (e.g., after site-specific conjugation of sdAbs with therapeutic radionuclides). As described in patent application number PCT / EP2019 / 055133 (WO2019 / 166622A1), a previously developed sdAb targeting human programmed death-ligand 1 (hPD-L1) exhibits favorable PET tracer properties for classifying potentially immunotherapy-suitable patients and predicting treatment outcomes (Chigoho, DM et al.). Pharmaceuticals14 , 550 (2021); Bridoux, J. et al., Biomolecules10 , 1-15 (2020)).

[0005] Fluorine-18 ( 18 F) is the most ideal PET radionuclide for clinical application. However, the harsh and complex conditions required for direct radioactive fluorination have hindered its development. 18 Development of F-labeled sdAbs. Enzymes have attracted much attention to facilitate the radiolabeling of sdAbs because they can perform radiolabeling under mild conditions in an aqueous medium while maintaining the integrity of the sdAb.

[0006] Novel asparagine acyl peptidase (PAL) families (e.g., disclosed in patent publications WO2020 / 226572A1, WO2018 / 056899A1, US2018 / 0274003A1, US2020 / 0172893A1 and WO2013 / 119184A1, the entire contents of which are incorporated herein by reference), such as the VyPAL2 enzyme in its active form, have kinetics that are unmatched by other ligases such as sorting enzyme A. Furthermore, they can accept a wide range of substrates consisting of short peptide linkers (stable or cleavable) or polyethylene glycol (PEG) and can be coupled with cytotoxic elements (e.g., monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), DM1, PDB dimer, SN38) or metal chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononanetriacetic acid (NOTA). The rapid reaction kinetics and substrate flexibility open new possibilities for the radiolabeling of sdAbs.

[0007] Therefore, there is a need to provide improved methods for enzyme-mediated production of labeled antibodies, their fragments, or mimics, which overcome or at least improve one or more of the aforementioned disadvantages. Summary of the Invention

[0008] This invention relates to a PAL-mediated method for preparing site-specific radiolabeled tracers with improved in vivo properties, wherein, in the presence of glutamine acyl cyclase (QC) and with optimized enzyme-to-substrate ratio, the yield is further improved to a conversion rate of over 90%.

[0009] In a first aspect, a method for preparing a site-specific radiolabeled tracer is provided, the method comprising providing i) Peptidyl asparaginyl ligase (PAL); ii) A first peptide comprising a P1-P1'-P2' tripeptide PAL motif at its C-terminus, wherein P1 is Asn or Asp, P1' is Gln or any nonproline amino acid, and P2' is a hydrophobic amino acid such as Val or Leu or Ile or a β-branched amino acid. iii) A second peptide comprising a P1''-P2'' motif at the N-terminus, wherein P1'' is Gly and P2'' is a hydrophobic amino acid such as Val or Leu or Ile, or a β-branched amino acid, wherein the second peptide is modified with a functional moiety to form a functionalized peptide. iv) Contact the peptidyl asparagine acyl ligase (PAL) with the first peptide and the functionalized peptide to form a conjugate of the first peptide and the functionalized peptide. The method further includes (a) Radiolabeled conjugate, or (b) Radiolabel the functionalized peptide prior to contact with the PAL.

[0010] In some embodiments, the contact step can be performed in the presence of glutamine acylcyclase (QC).

[0011] In some embodiments, the first peptide is an antibody or a functional fragment thereof. In other embodiments, the antibody or functional fragment thereof may be a single-domain antibody (sdAb). In some embodiments, the sdAb is a programmed death-ligand 1 (hPD-L1) sdAb or a carcinoembryonic antigen (CEA) sdAb. In some embodiments, the sdAb is selected from the group consisting of (hPD-L1)-NGL-His6, (hPD-L1)-NQL-His6, and (CEA)-NQL-His6 sdAb.

[0012] In some embodiments, the P1-P1'-P2' tripeptide PAL motif can be Asn-Gln-Leu (NQL) or Asn-Gly-Leu (NGL).

[0013] In some embodiments, the functionalized peptide may include a -GIGK-, -GIGG-, or -GIGGGK- motif. In some embodiments, the functionalized peptide may be selected from the group consisting of: GIGK-NOTA, GIGK-DOTA, GIGK-MeTz, GIGGGK-DOTA, GIGK-NOTA- 18 F]AlF, GIGK-Py-DABCO, Ac-GIGK-Py-DABCO, GIGK-Py-NMe3, Ac-GIGK-PyNMe3 and GIGK-FPy.

[0014] In some implementations, the PAL may be VyPAL2, butelase-1, butelase-2, VyPAL3, OaAEP1b-C247A, HeAEP3, AtLEGγ, VuPAL1, HaPAL1, OaAEP1b, or a functional fragment or variant thereof.

[0015] In some implementations, the PAL is VyPAL2 or a functional fragment or variant thereof.

[0016] In other embodiments, the QC may be human glutamine acylcyclase, mouse glutamine acylcyclase, fruit fly glutamine acylcyclase, Arabidopsis glutamine acylcyclase, cone snail glutamine acylcyclase, rattlesnake glutamine acylcyclase, bacterial glutamine acylcyclase, and their functional fragments or variants.

[0017] In some implementations, the QC is human QC or a functional segment or variation thereof.

[0018] In some implementations, the molar ratio of PAL:QC is 1:0.1 or 1:0.5.

[0019] In some embodiments, the functionalized peptide may include a chelating agent or a tetrazine group.

[0020] In some embodiments, the chelating agent is selected from the group consisting of: 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA).

[0021] In some embodiments, the conjugate or functionalized peptide may be selected from gallium-68 ( 68 Ga), gallium-67 ( 67 Ga), Lutetium-177 ( 177 Lu) and Fluorine-18 ( 18 Radioactive nuclides of group F are radiolabeled.

[0022] In some embodiments, the radionuclide is [ 18 Fluorine-18 in the form of F]AlF ( 18 F).

[0023] In a second aspect, a site-specific radiolabeled tracer prepared by the method described in the first aspect is provided.

[0024] In some embodiments, the site-specific radiolabeled tracer includes human programmed death-ligand 1 (hPD-L1) sdAb or carcinoembryonic antigen (CEA) sdAb.

[0025] In some implementations, the site-specific radiolabeled tracer may be selected from the group consisting of: [ 68 Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb and [ 177 Lu]Lu-DOTA-NHCS-KGIG-CEA sdAb.

[0026] In a third aspect, a site-specific radiolabeled tracer as described in the second aspect is provided for use as an imaging agent.

[0027] In a fourth aspect, the use of the site-specific radiolabeled tracer as an imaging agent according to the second aspect is provided.

[0028] In a fifth aspect, the use of the site-specific radiolabeled tracer described in the second aspect in the manufacture of diagnostic imaging agents for molecular imaging is provided.

[0029] In a sixth aspect, a method for treating a subject's disease is provided, the method comprising administering to the subject a site-specific radiolabeled tracer according to the second aspect.

[0030] In a seventh aspect, the use of the site-specific radiolabeled tracer described in the second aspect in the manufacture of a medicament for treating a subject’s disease is provided.

[0031] In some implementations, the disease is cancer.

[0032] Advantageously, the methods of this disclosure provide a rapid and efficient way to generate site-specific radiolabeled tracers based on sdAbs suitable for molecular imaging. More advantageously, the site-specific radiolabeled antibody tracers of this disclosure also exhibit improved in vivo properties compared to antibody tracers obtained using chemical site-specific strategies. These and other advantages of this disclosure will become apparent to those skilled in the art from the following detailed description. Attached Figure Description

[0033] The accompanying drawings illustrate the disclosed embodiments and serve to explain the principles of the disclosed embodiments. However, it should be understood that the drawings are for illustrative purposes only and are not intended to define limitations on the invention.

[0034] Figure 1 The synthetic steps of solid-phase peptide synthesis are shown. (A) Boc-protected GIGK-peptide backbone (on resin). (B) p-SCN-Bn-NOTA for functionalization of the free amine group on the lysine side chain. (C) GIGK-NOTA peptide cleaved from resin to form TFA salt, TFA = trifluoroacetic acid. (D) COOH-PEG4-MeTz for functionalization of the free amine group on the lysine side chain. (E) GIGK-MeTz peptide cleaved from resin to form TFA salt.

[0035] Figure 2 The structure of BCN-NODAGA is shown.

[0036] Figure 3 The purified and filtered [description] 68 Radioactive RP-HPLC of Ga]Ga-NOTA-KGIGN-(hPD-L1) sdAb. Rt( 68 Ga-labeled sdAb) = 7.42 min (99%), Rt (free) 68 Ga) = 3.30 min (1%).

[0037] Figure 4 Depicting [ 67 Radioactive SEC of Ga]Ga-DOTA-KGGGIGN-R3b23 sdAb (crude mixture). Rt( 68 Ga-labeled sdAb) = 4.85 min (30%), Rt (free) 68 Ga) = 5.70 min (70%).

[0038] Figure 5 The purification of tC18 is shown. 18 Radioactive RP-HPLC of F]AlF-NODAGA-BCN on an XBridge C18 column. Rt([ 18 F]]AlF-NODAGA-BCN) = Rt = 3.05 min (92%), Rt([ 18 F]AlF / NaF) = 1.25 min (4%), Rt (radioactive impurities due to degradation) = 1.77 min (4%).

[0039] Figure 6The chemically MeTz-functionalized hPD-L1 sdAb and [ 18 [F]AlF-NODAGA-BCN was clicked onto a Superdex 75 Increase 3.2 / 300GL column after purification and filtration for radioactive SEC. Rt([ 18 F]AlF-NODAGA-BCN-MeTz-mal-cys-(hPD-L1) sdAb) = 11.4 min (>95%).

[0040] Figure 7 The diagram shows NQL-His6hPD-L1 sdAb containing 1 eq. and [ 1 eq.] 18 The crude mixture of F]AlF-NOTA-KGIG was analyzed by radiometric SEC and UV spectra (280 nm) on a TSKgel column after 1 hour of reaction. Rt([ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb) = 9.87 min (41%), Rt([ 18 [F]AlF-NOTA-KGIG) = 11.97 min (59%). The sample was diluted in PBS / Tween 20 and a signal was generated under UV at Rt(Tween 20) = 6.8 min.

[0041] Figure 8 The purification and filtration on a Superdex 75 Increase 3.2 / 300GL column are shown. 18 Radioactive SEC and UV spectra (280 nm) of F]AlF-NOTA-KGIG)-(hPD-L1) sdAb. Rt([ 18 F]AlF-NOTA-KGIG)-(hPD-L1)sdAb) = 11.6 min (95%), Rt([ 18 [F]AlF-NOTA-KGIG) = 15.53 min (5%), sample diluted in PBS / Tween 20 0.1%. Rt(Tween 20) = 6.8 min.

[0042] Figure 9 Describes the [contains 1 eq.] 18 Radioactive SEC spectra of a crude mixture of F]AlF-NOTA-KGIG, 1 eq. of hPD-L1sdAb tagged with NQL-His6, and 0.01 eq. of an enzymatically catalyzed mixture incubated on a TSKgel column for 30 min. Rt([ 18F]AlF-NOTA-KGIG-(hPD-L1) sdAb) = 10.02 min (79%). R([ 18 F]AlF-NOTA-KGIG) = 11.25 min (9%). Rt (radioactive impurities) = 15.90 min (12%).

[0043] Figure 10 The QCO (quality control) for the radiolabeled sdAb after purification and filtration is shown. (A) Radioactive RP-HPLC analysis was performed on a PLRP-S column. Rt([ 18 F]AlF-NOTA-KGIG-sdAb = 6.42 min (93%). Rt (smaller radioactive impurity) = 3.85 min (7%). (B) Analyzing the same sample in UV spectrum at 280 nm. Rt(sdAb) = 6.25 min (100%). Since the radioactive detector is located after the UV detector of the HPLC system, a shift of ±20 seconds between the UV and radioactive signals is expected.

[0044] Figure 11 This study illustrates the in vitro cell binding of a 3 nM radiolabeled hPD-L1 sdAb analog to hPD-L1 positive (POS) cells. hPDL-1 negative (NEG) cells and POS cells blocked with a 100-fold overdose of non-radiolabeled sdAb served as controls to assess specificity. CPM = counts per minute. p<0.001; , p<0.01)(A)[ 68 The results of Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb. (B)[ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb. (C)[ 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb. (D)[ 18 F]F-NODAGA-BCN-MeTz-mal-cys-(hPD-L1) sdAb.

[0045] Figure 12 It shows 18 F-labeled sdAb analogs in hPD-L1 POS Affinity assay for cell saturation on 624-MEL cells (K) D (A)[ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb (K D= 2.5 nM), (B)[ 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb (K D = 4.34 nM) and (C) [ 18 F]F-NODAGA-BCN-MeTz-mal-cys-(hPD-L1) sdAb (K D The relationship between cell binding activity (expressed as counts per minute (CPM)) of sdAb (3.18 nM) and sdAb concentration (nM).

[0046] Figure 13 The graph depicts the injection of healthy mice (N = 3 mice per group) (left bar graph with diagonal stripes). 18 F]F-NOTA-KGIG-(hPD-L1) sdAb and (right-side bar chart with horizontal stripes)[ 18 Biodistribution profile of F]F-NOTA-KGIG-(R3b23) sdAb 80 min post-absorption. Nb = sdAb = single-domain antibody. Results are expressed as mean ± standard deviation, in %A / g = % of activity per gram of organ injection. (p<0.0001).

[0047] Figure 14 The graph depicts the injection of healthy mice (N = 3 mice per group) (left bar graph with checkerboard pattern). 68 Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb and (right-side bar chart with diagonal stripes)[ 18 Biodistribution profile of F]F-NOTA-KGIG-(hPD-L1)sdAb 80 min post-injection. Nb = sdAb = single-domain antibody. Results are expressed as mean ± standard deviation, in %A / g = % of activity per gram of organ injection. p < 0.001; (p<0.01).

[0048] Figure 15 The image shows the injection (left bar chart with dotted pattern) in healthy mice (N = 3 mice per group). 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb、(intermediate bar chart with diagonal stripes)[ 18 F]F-NOTA-KGIG-(hPD-L1) sdAb and (unpatterned right-side bar chart)[ 18Biodistribution profile of F]F-NODAGA-BCN-MeTz-mal-cys-(hPD-L1)sdAb 80 min after administration. Nb = sdAb = single-domain antibody. Results are expressed as mean ± standard deviation, in %A / g = % of activity per gram of organ injection. p<0.01; (p<0.05).

[0049] Figure 16 The structures of peptide substrates radiofluorinated via nucleophilic substitution are shown. The resulting product is a TFA salt (not shown in the figure). (Top) The yield of GIGK-Py-DABCO obtained was 37%. The yield of the aminoacetyl protected variant of the peptide (Ac-GIGK-Py-DABCO) obtained was 47%. (Bottom) The yield of GIGK-Py-NMe3 obtained was 50%. The yield of the aminoacetyl protected variant of the peptide (Ac-GIGK-Py-NMe3) obtained was 36%.

[0050] Figure 17 The study described the composition of (hPD-L1)-NQL-His6sdAb (1 eq. at 1 mg / mL) and 1 eq. (3.58 MBq). 18 A crude mixture of [F]AlF-NOTA-NHCS-KGIG and 0.01 eq. of VyPAL2 (v / vyPAL2, +GluC 0.5 eq.) reacted at 40 °C and pH 6.5 for 15 min, followed by a radioactive RP-HPLC chromatogram on a PLRP-S column. 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb) = 7.13 min (92%), Rt([ 18 F]AlF-NOTA-KGIG) = 6.32min (8%). (Internal reference SGD-2274-149).

[0051] Figure 18 The following is a list of reagents containing (hPD-L1)-NQL-His6sdAb (1 eq. at 1 mg / mL) and 1 eq. (8.8 MBq). 18 F]AlF-NOTA-NHCS-KGIG and 0.1 eq. of VyPAL2 (relative to VyPAL2, +GluC 0.5 eq.) (VyPAL2 has been used at 900 MBq) 18 A crude mixture pre-irradiated with NaF salt for 7 minutes was reacted at 40 °C and pH 6.5 for 7 minutes, and the resulting image was then subjected to radioactive RP-HPLC on a PLRP-S column. Rt([ 18F]AlF-NOTA-KGIG-(hPD-L1) sdAb) = 6.83min (85%), Rt([ 18 [F]AlF-NOTA-KGIG) = 6.03 min (15%). The injection peak at 2.5–4 min contains excess radioactive salt, therefore the signal in this region is saturated. Figure 17 In comparison, due to the use of a newer PLRP-S column with higher column pressure, the retention time shifted by 0.3 min, resulting in a slightly earlier retention time. (Internal reference SGD-2686-017).

[0052] Figure 19 The RP-HPLC chromatogram of the non-radioactive reference peptide at 215 nm UV on a PLRP-S column is shown, with Rt(FPy-KGIG) = 6.28 min. (Internal reference: JBR-2690-041). Compared with the radioactive peptide, Rt is consistent with the expected Rt at 6.43 min, with a typical offset of 0.15 min due to the delay between UV and the radioactive detector.

[0053] Figure 20 The UV spectrum of a crude mixture containing 1 eq. of R3b23-NQL-His6sdAb and 1 eq. of a non-radioactive reference FPy-KGIG peptide was plotted at 280 nm on a C4 column, reacted at 37 °C for 5 min in the presence of 0.1 eq. of VyPAL2 (relative to VyPAL2, +1 eq. of GluC). Rt(R3b23-NQL-His6), Rt(presumed R3b23-GIGK-PyF) = 7.53 min. (Internal reference: SGD-2686-117).

[0054] Figure 21 The rough [ 177 The radioactive RP-HPLC chromatogram of Lu]Lu-DOTA-mal-CEA sdAb after reaction at 60 °C for 2 h on a PLRP-S column. Rt([ 177 Lu]Lu-DOTA-mal-CEA) = 7.82 min, Rt(free[ 177 Lu]LuCl3) =3.20 min (Internal reference: SGD-2686-097).

[0055] Figure 22 The rough [ 177 The radioactive RP-HPLC chromatogram of Lu]Lu-DOTA-NHCS-KGIG-CEA sdAb reacted on a PLRP-S column at 40 °C for 45 min. Rt([177 Lu]Lu-DOTA-NHCS-KGIG-CEA) = 8.00 min, Rt(free[ 177 Lu]LuCl3) = 3.40 min (Internal reference: JBR-2690-044).

[0056] Figure 23 A table showing the parameter optimization affecting the radiofluorination of the NOTA-GIGK peptide is presented. The percentage of complexed AlF was measured on an XBridge C18 analytical column. Detailed Implementation

[0057] The following detailed description refers to specific details and embodiments in which the invention can be practiced by way of example. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

[0058] For convenience, the references mentioned in this specification are listed in the form of a bibliography and appended at the end of the embodiments. The full contents of such references are incorporated herein by reference, but their inclusion in the specification does not imply that they form part of common general knowledge.

[0059] definition For convenience, certain terms used in the specification, embodiments, and appended claims are collected herein.

[0060] Generally, the technical, scientific, and medical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. In addition, the following technical comments and definitions are provided. These definitions should not in any way limit the scope of the invention to these terms alone, but are set forth for a better understanding of the following description.

[0061] As used herein, “a” or “an” may mean one or more species, unless otherwise stated or the context otherwise requires.

[0062] As used herein, the term "amino acid" can refer to natural and / or non-natural or synthetic amino acids, including both D- and L-optical isomers, amino acid analogs (e.g., ortholeucine is an analog of leucine), and peptide mimics. As used in the context of this application, the term "amino acid" generally refers to 20 naturally occurring L-amino acids: Gly, Ala, Val, Leu, He, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr, and Trp.

[0063] As used herein, the term "comprising" or "including" should be interpreted as indicating the presence of the features, integers, steps, or components mentioned, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. However, in the context of this disclosure, the term "comprising" or "including" also includes "consisting of". Variations of the word "comprising" such as "comprise" and "comprises", and variations of "including" such as "include" and "includes", have correspondingly changing meanings.

[0064] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a polymer consisting of at least two amino acids covalently linked by an amide bond. A peptide is considered a short amino acid chain, while a polypeptide is a long amino acid chain, and proteins tend to have a stable structure and may include modifications (e.g., glycosylation or phosphorylation). The term “protein” may encompass both naturally occurring and artificially generated (e.g., engineered or variant) full-length proteins as well as functional fragments of proteins.

[0065] As used herein, the term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., biological activity or function, such as enzymatic activity or antigen-binding properties), such as the ability to catalyze a linkage reaction between two peptides. Functional fragments can be of any size as long as they retain the activity / function of the full-length protein / enzyme / antibody. For example, antibody fragments can retain the basic specificity and affinity of a full-length antibody. Examples of antibody fragments include, but are not limited to, antigen-binding fragments (Fab), F(ab')2, single-chain variable region fragments (scFv), single-domain antibodies (sdAb) (also known as nanobodies), variable domains (VhH) of heavy-chain-only antibodies, microantibodies, biantibodies, etc.

[0066] As used herein, the term "functionalized peptide" refers to a peptide sequence that has been modified and / or complexed with one or more functional groups, portions, compounds, and / or pharmaceutical agents to enhance and / or broaden the peptide's function, thereby achieving its intended purpose, such as for medical applications. For example, a peptide targeting cancer may be functionalized to include a connector carrying a therapeutic agent as part of a drug delivery system. As used in this invention, a peptide may be functionalized to preferably include a pharmaceutical agent suitable for use as a diagnostic agent and / or for therapeutic applications. For example, the functionalized peptides of this disclosure may be modified to include a radiolabeled chelating agent. Other functional groups, portions, compounds, and / or agents applicable to this invention include, but are not limited to, chelating agents such as 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), tetrazine portions such as MeTz, 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, and 1,2,4,5-tetrazine, probes / connectors such as bicyclic nonyne, functional groups such as DABCO and NMe3, and other detectable and / or optically labeled substances such as horseradish peroxidase (HRP) or green fluorescent protein (GFP).

[0067] As used herein, a “single-domain antibody” (sdAb) or nanobody refers to an antibody fragment comprising a single monomeric variable antibody domain or an engineered constant domain that promotes target binding exclusively. It is capable of selectively binding to a specific antigen, much like a full-length antibody. Methods for generating sdAbs are known in the art. It should be understood that any antibody or functional fragment thereof may be used in this invention for the purposes of this invention, provided that it is appropriately modified to include a P1-P1'-P2' tripeptide PAL recognition motif to facilitate ligation reactions using PAL, optionally in the presence of glutamine acyl cyclase (QC).

[0068] As used herein, the term "radiolabeling" refers to a method of labeling peptides / proteins using a radioisotope. Various radiolabeling strategies for incorporating radionuclides into peptides / proteins are known in the art. These include, for example, direct labeling, indirect labeling via a prosthetic group, and indirect labeling via complexation. It is understood that radioisotopes can be directly integrated into peptide molecules through electrophilic substitution or indirectly integrated into peptide molecules via conjugation. In other cases, radioactive metals can be labeled via complexation with a chelator / chelating agent. Examples of radionuclides / radioisotopes include, but are not limited to, gallium-68 (…). 68 Ga), gallium-67 ( 67Ga), indium-111 ( 111 In), Lutetium-177 ( 177 Lu), Fluorine-18 ( 18 F), Iodine-124 ( 124 I), iodine-131 ( 131 I), iodine-123 ( 123 I), iodine-125 ( 125 I), phosphorus-32( 32 P), Strontium-90 ( 90 Sr), Strontium-89 ( 89 Sr), Yttrium-90 ( 90 Y), Radium-223 ( 223 Ra), Radium-226 226 Ra), Cesium-137 137 Cs), Technetium-99m ( 99m Tc), Tin-117 ( 117m Sn), Samarium-153 153 Sm), Erbium-169 169 Er), Rhenium-188 188 Re), Rhenium-186 ( 186 Re), Thallium-201 201 Tl), carbon-11 ( 11 C), Oxygen-15 ( 15 O-), Copper-64 ( 64 Cu), Lead-212 ( 212 Pb), astatine-211 ( 211 At), Actinium-225 ( 225 Ac), Terbium-149 / 161 / 155 / 152 ( 149 / 161 / 155 / 152 Tb) and Iridium-192 192 Ir).

[0069] As used herein, the term "peptide asparagine acyl ligase (PAL)" refers to a specific group of enzymes that primarily catalyze peptide bond formation. In this respect, PAL typically performs site-specific ligation reactions and recognizes the P1-P1'-P2' tripeptide motif for ligation after P1, where P1 is typically Asn or Asp, and P1' and P2' can be any naturally occurring amino acid such as Gly, Ala, Val, Leu, Ile, Phe, Cys, Met, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr, and Trp. PAL cleaves the first peptide / protein following P1 in the PAL tripeptide motif and ligates the first peptide / protein to the P1''-P2'' motif of the second peptide / protein.

[0070] As used herein, the term "QC" refers to glutamine acyl cyclase (QC) and QC-like enzymes. QC and QC-like enzymes have the same or similar enzymatic activity, namely, catalyzing the intramolecular cyclization of N-terminal glutamine acyl and glutamine acyl residues in peptides and proteins to form pyroglutamine residues (pGlu). In this respect, the molecular structure of QC-like enzymes differs fundamentally from that of QC.

[0071] As used herein, the term "variant" refers to an amino acid sequence in which one or more amino acids of a non-variant reference sequence have been altered, but which retains the ability to recognize its targets and affect its function. Variants may have "conserved" changes, where the substituted amino acids have similar structural or chemical properties (e.g., replacing leucine with isoleucine). Less frequently, variants may have "non-conserved" changes (e.g., replacing glycine with tryptophan). Similar minor alterations may also include amino acid deletions or insertions, or both. Guidelines for determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological activity can be found using computer programs well known in the art, such as DNASTAR® software (DNASTAR, Inc. Madison, Wisconsin, USA).

[0072] The following is a description of exemplary, non-limiting embodiments of the present invention.

[0073] This invention provides a novel and improved method for the rapid and reagent-efficient generation of radiolabeled single-domain antibodies (sdAbs) with improved in vivo properties.

[0074] Therefore, one aspect of this disclosure provides a method for preparing a site-specific radiolabeled tracer, the method comprising providing: i) Peptidyl asparagine acyl ligase (PAL); ii) A first peptide comprising a P1-P1'-P2' tripeptide PAL motif at its C-terminus, wherein P1 is Asn or Asp, P1' is Gln or any nonproline amino acid, and P2' is a hydrophobic amino acid such as Val, Leu or Ile or a β-branched amino acid. iii) A second peptide comprising a P1''-P2'' motif at the N-terminus, wherein P1'' is Gly and P2'' is a hydrophobic amino acid such as Val or Leu or Ile, or a β-branched amino acid, wherein the second peptide is modified by a functional motif to form a functionalized peptide. iv) Contact the peptidyl asparagine acyl ligase (PAL) with the first peptide and the functionalized peptide to form a conjugate of the first peptide and the functionalized peptide. The method further includes (a) Radiolabeled conjugate, or (b) Radiolabel the functionalized peptide prior to contact with the PAL.

[0075] It should be understood that PAL can undergo site-specific ligation reactions and requires a minimal tripeptide recognition motif P1-P1'-P2' for ligation after P1, where P1 is typically Asn or Asp, and P1' and P2' can be any naturally occurring amino acid Gly, Ala, Val, Leu, Ile, Phe, Cys, Met, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr, and Trp.

[0076] For the purposes of this invention, P1 is preferably Asn or Asp, P1' is preferably Gln or any non-proline amino acid, and P2' is preferably a hydrophobic amino acid such as Val, Leu, or Ile, or a β-branched amino acid. In some embodiments, P1 is preferably Asn, and P1' is preferably Gln, Gly, or Ser. In some embodiments, the PAL motif of the P1-P1'-P2' tripeptide is an Asn-X-Leu motif, wherein X is Gln(Q), Gly(G), or Glu(E). In some preferred embodiments, the first peptide may comprise an Asn-Gln-Leu (NQL) or Asn-Gly-Leu (NGL) tripeptide PAL motif.

[0077] It should be understood that any peptide can be used as the first peptide in this invention, provided that it is appropriately modified to include the P1-P1'-P2' tripeptide PAL recognition motif as described herein to facilitate the linkage reaction.

[0078] It should also be understood that the first peptide may be further modified to include an affinity tag such as a His tag to facilitate its purification process. Therefore, in some embodiments, the first peptide may include a His6 tag at its C-terminus. In other embodiments, the first peptide may include a -NGL-His6 or -NQL-His6 tag at its C-terminus.

[0079] In some implementations, the first peptide may be an antibody, a scaffold protein, an antibody mimic, or a functional fragment thereof.

[0080] In some embodiments, the first peptide may be an antibody or a functional fragment thereof. Specifically, the antibody or functional fragment thereof may be selected from the group consisting of Fab, F(ab')2, scFv, and sdAb.

[0081] In some embodiments, the antibody or its functional fragment is an sdAb. In some embodiments, the sdAb may be selected from the group consisting of hPD-L1 sdAb, R3b23 sdAb, and CEA-targeting sdAb. In some embodiments, the sdAb is selected from the group consisting of (hPD-L1)-NGL-His6, (hPD-L1)-NQL-His6, R3b23-NGL-His6, R3b23-NQL-His6, and (CEA)-NQL-His6 sdAb.

[0082] In some embodiments, the first peptide may also be a scaffold protein and / or an antibody mimic, or a functional fragment thereof. Examples of antibody mimics may include, but are not limited to, affibody molecules, DARPin, affimer, anticalcin, alpha body, anticlonal protein, ankylosing smear repeat unit multimer, monomeric antibody, Kunitz domain peptide, and nanoclamp, etc. For the purposes of this invention, it should be understood that PAL cleaves the first peptide after P1 in the PAL motif of the tripeptide and links the first peptide to the P1''-P2'' motif of the functionalized peptide.

[0083] In some embodiments, P2'' may be a hydrophobic amino acid or a β-branched amino acid. Examples of hydrophobic amino acids may include Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, Tyr, and Trp. Examples of β-branched amino acids include Thr, Val, and Ile. In some embodiments, P2'' is a hydrophobic amino acid selected from Val, Leu, or Ile.

[0084] In some embodiments, the P1''-P2'' motif of the functionalized peptide may be a -GI motif. In various other embodiments, the functionalized peptide may include a -GIGK-, -GIGG-, or -GIGGGK- motif at its N-terminus.

[0085] In some embodiments, the functionalized peptides are selected from the group consisting of: GIGK-NOTA, GIGK-DOTA, GIGK-MeTz, GIGGGK-DOTA, GIGK-NOTA-[ 18 F]AlF, GIGK-Py-DABCO, Ac-GIGK-Py-DABCO, GIGK-Py-Nme3, Ac-GIGK-PyNMe3 and GIGK-FPy.

[0086] Those skilled in the art will understand that different PALs and their variants having the desired protein ligase activity may be suitable for the practice of this invention. Therefore, in some embodiments, the PAL may be VyPAL2, butelase-1, butelase-2, VyPAL3, OaAEP1b-C247A, HeAEP3, AtLEGγ, VuPAL1, HaPAL1, OaAEP1b, or a functional fragment or variant thereof. In other embodiments, the PAL is VyPAL2 or a functional fragment or variant thereof.

[0087] In some embodiments, the PAL may be selected from the group consisting of: VyPAL2 comprising the amino acid sequence shown in SEQ ID NO: 32, butelase-1 comprising the amino acid sequence shown in SEQ ID NO: 33, butelase-2 comprising the amino acid sequence shown in SEQ ID NO: 34 or SEQ ID NO: 35, VyPAL3 comprising the amino acid sequence described in SEQ ID NO: 36, OaAEP1b-C247A comprising the amino acid sequence shown in SEQ ID NO: 37, HeAEP3 comprising the amino acid sequence shown in SEQ ID NO: 38, AtLEGγ comprising the amino acid sequence shown in SEQ ID NO: 39, VuPAL1 comprising the amino acid sequence shown in SEQ ID NO: 440, HaPAL1 comprising the amino acid sequence shown in SEQ ID NO: 41, OaAEP1b comprising the amino acid sequence shown in SEQ ID NO: 42, and functional fragments or variants thereof.

[0088] In other preferred embodiments, PAL may be VyPAL2 comprising the amino acid sequence shown in SEQ ID NO: 32, or a functional fragment or variant thereof.

[0089] It should be understood that the presence of glutamine acyl cyclase (QC) improves the substrate-to-substrate linkage reaction catalyzed by PAL. Therefore, in some embodiments, the contact step is carried out in the presence of glutamine acyl cyclase (QC).

[0090] It is also envisioned that various QCs with the desired QC enzymatic activity may be suitable for use in the practice of this invention. Therefore, in some embodiments, the QC may be human glutamine acylcyclase, mouse glutamine acylcyclase, Drosophila glutamine acylcyclase, Arabidopsis glutamine acylcyclase, Conophytum glutamine acylcyclase, Rattlesnake glutamine acylcyclase, bacterial glutamine acylcyclase, or functional fragments or variants thereof.

[0091] In some embodiments, the QC is human glutamine acylcyclase or its functional fragments or variants.

[0092] In some embodiments, the QC may be selected from the group consisting of: human glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 43, mouse glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 44, Drosophila glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 45, Arabidopsis glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 46, Conophytum glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 47, Rattlesnake glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 48, bacterial glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 49, and functional fragments or variants thereof.

[0093] In some preferred embodiments, QC is a human glutamine acylcyclase comprising the amino acid sequence shown in SEQ ID NO: 43 or a functional fragment or variant thereof.

[0094] Those skilled in the art will understand that the function of a protein / enzyme is directly related to its structure and sequence, and that there is a positive correlation between sequence identity and functional similarity. Methods for determining protein sequence identity are known in the art. Therefore, the sequences of the enzymes of this disclosure can be sufficiently modified, as long as the enzyme retains its functionality and exhibits the desired activity.

[0095] In some embodiments, PAL may be VyPAL2 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 33; butelase-1 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 33; butelase-2 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 34 or 35; VyPAL3 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 36; or VyPAL3 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 33; or butelase-1 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 33; or butelase-2 comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 36 OaAEP1b-C247A, which has an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 37, includes HeAEP3, which has an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 38, includes AtLEGγ, which has an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 39, includes VuPAL1, which has an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 40, and includes amino acid sequences with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 37 ... including those with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 37, including those with at least 85%, at least 90%, at HaPAL1, which includes an amino acid sequence shown in NO:41 having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:42, or OaAEP1b, which includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:42.

[0096] In some embodiments, QC may be a human glutamine cyclase comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 43; a mouse glutamine cyclase comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 44; a Drosophila glutamine cyclase comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 45; an Arabidopsis glutamine cyclase comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 46; or a human glutamine cyclase comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 43 ... Cone snail glutamine cyclases having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 48, including rattlesnake glutamine cyclases having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 49, or bacterial glutamine cyclases having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 49.

[0097] Table 1. The single-domain antibodies (sdAbs), functionalized peptides, conjugates, radiolabeled sdAb tracers, peptide asparagine acyl ligases (PALs) and glutamine acyl cyclases (QCs) of the present invention and their amino acid sequences.

[0098]

[0099] The reaction rate of the methods disclosed herein can be controlled by varying the ratio of one or more enzymes to the associated substrate. In some embodiments, a small amount of enzyme (e.g., 0.01 eq of a PAL + QC mixture relative to the substrate) may be sufficient to implement the invention. The enzyme-to-substrate ratio used depends considerably on the substrate and the specific application.

[0100] In some embodiments, the ratio of the first peptide:functionalized peptide:PAL is in the range of 1-5:1-25:0.1 to 0.5. In some embodiments, the ratio of the first peptide:functionalized peptide:PAL is 1:1:0.25. In some embodiments, the ratio of the first peptide:functionalized peptide:PAL is selected from the group consisting of: 1:5:0.25, 1:25:0.25, 2:1:0.25, 1:5:0.25, and 1:1:0.25.

[0101] In some embodiments, the ratio of the first peptide:functionalized peptide:PAL+QC mixture is in the range of 1:1:0.01 to 1:1:0.1. In some embodiments, the ratio of the first peptide:functionalized peptide:PAL+QC mixture is 1:1:0.01.

[0102] It should be understood that changing the amount of QC may also affect the reaction rate. Therefore, in some embodiments, the molar ratio of PAL:QC is in the range of 1:0.1 to 1:1. In some embodiments, the molar ratio of PAL:QC is 1:0.1. In other embodiments, the molar ratio of PAL:QC is 1:0.5.

[0103] For the purposes of this invention, the functionalized peptides described herein may be suitably functionalized to include one or more functional groups, one or more portions, one or more compounds, and / or one or more pharmaceutical agents that can promote radiolabeling, their use as diagnostic agents, and / or their use as therapeutic agents. In this regard, it should be understood that the functionalized peptides used in this invention can be prepared using standard techniques known to those skilled in the art of synthetic organic chemistry, or can be derived by referring to relevant literature.

[0104] In some embodiments, the functionalized peptide is functionalized using a chelating agent or a tetrazine moiety. In some embodiments, the tetrazine moiety is -MeTz.

[0105] In some embodiments, the chelating agent is selected from the group consisting of: 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA).

[0106] In some embodiments, the functionalized peptide may further include bicyclic nonyne (BCN) or trans-cyclooctene (TCO).

[0107] In some embodiments, the tetrazine-functionalized peptide can form a conjugate of the first peptide-(tetrazine-functionalized peptide), and this conjugate is further complexed with a bicyclic nonyne / TCO-functionalized chelating agent / reactive moiety prior to radiolabeling. In some embodiments, the bicyclic nonyne-functionalized chelating agent comprises a chelating agent selected from the group consisting of: 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA).

[0108] In various embodiments, the conjugate or functionalized peptide may be radiolabeled with a radionuclide selected from the group consisting of gallium-68 (GaN-68). 68 Ga), gallium-67 ( 67 Ga), indium-111 ( 111 In), Lutetium-177 ( 177 Lu), Fluorine-18 ( 18 F), Iodine-131 ( 131 I), iodine-123 ( 123 I), iodine-125 ( 125 I), phosphorus-32 ( 32 P), Strontium-90 ( 90 Sr), Strontium-89 ( 89 Sr), Yttrium-90 ( 90 Y), Radium-223 ( 223 Ra), Radium-226 226 Ra), Cesium-137 137 Cs), Technetium-99m ( 99m Tc), Tin-117 ( 117m Sn), Samarium-153 153 Sm), Erbium-169 169 Er), Rhenium-188 ( 188 Re), Rhenium-186 ( 186 Re), Thallium-201 201Tl), carbon-11 ( 11 C), Oxygen-15 ( 15 O-), Copper-64 ( 64 Cu), Lead-212 ( 212 Pb), astatine-211 ( 211 At), Actinium-225 ( 225 Ac), Terbium-149 / 161 / 155 / 152 ( 149 / 161 / 155 / 152 Tb) and Iridium-192 192 Ir).

[0109] In some embodiments, the conjugate or functionalized peptide is selected from gallium-68 ( 68 Ga), gallium-67 ( 67 Ga), Lutetium-177( 177 Lu) and Fluorine-18 ( 18 Radioactive nuclides of group F are radiolabeled.

[0110] In some embodiments, the radioactive nuclide is gallium-68 (GaN-68). 68 Ga) or fluorine-18 ( 18 F). In other embodiments, fluorine-18 ( 18 F) is [ 18 The form of F]AlF.

[0111] On the other hand, site-specific radiolabeled tracers prepared by the methods described herein are provided.

[0112] In some embodiments, the site-specific radiolabeled tracers provided herein are selected from the group consisting of: [ 68 Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) and [ 177 Lu]Lu-DOTA-NHCS-KGIG-CEA sdAb.

[0113] On the other hand, this article provides site-specific radiolabeled tracers, as described herein, for use as nuclear imaging agents.

[0114] In another aspect, the use of the site-specific radiolabeled tracer disclosed herein as a nuclear imaging agent is provided.

[0115] In another aspect, the use of the site-specific radiolabeled tracers disclosed herein in the manufacture of diagnostic imaging agents for nuclear imaging is provided.

[0116] In another aspect, a method for treating a subject's disease is provided, the method comprising administering to the subject a site-specific radiolabeled tracer according to the second aspect.

[0117] In another aspect, the use of the site-specific radiolabeled tracer described in the second aspect in the manufacture of a medicament for treating a disease is provided.

[0118] In some implementations, sdAb targets diseased cells, such as cancer cells, and the site-specific radiolabeled antibody tracer described herein is used with therapeutic radionuclides (such as... 177 Lu、 211 At or 225 Ac) is used for radioactive labeling.

[0119] Overexpression of PD-L1 is associated with poor clinical outcomes in cancers such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, melanoma, breast cancer, non-small cell lung cancer, papillary thyroid cancer, testicular cancer, and bladder cancer, and the detection of its levels has significant clinical implications. Carcinoembryonic antigen (CEA) overexpression has been observed in patients with various cancers, including gallbladder cancer, urethral bladder cancer, endometrial cancer, colon cancer, thyroid cancer, lung cancer, uterine cancer, pancreatic cancer, gastric cancer, breast cancer, and ovarian cancer. CEA can be used clinically as a cancer biomarker; it can also be used as a prognostic biomarker for cancer after radiotherapy and chemotherapy, a predictor of cancer treatment, and a therapeutic target.

[0120] In some embodiments, the disease is a tumor or cancer. In other embodiments, the cancer can be pancreatic cancer, prostate cancer, ovarian cancer, melanoma, breast cancer, non-small cell lung cancer, papillary thyroid cancer, testicular cancer, bladder cancer, stomach cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer and / or gallbladder cancer, urethral bladder cancer, endometrial cancer, colon cancer, thyroid cancer, lung cancer, uterine cancer, pancreatic cancer, stomach cancer, breast cancer, and ovarian cancer. In some embodiments, the tumor is a neuroendocrine tumor or carcinoid tumor.

[0121] Unless otherwise stated or apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may assume any particular value or subrange within the range in various embodiments, accurate to one-tenth of the lower limit unit of the range, unless the context explicitly specifies otherwise. Unless otherwise stated or apparent from the context, “about” with respect to a numerical value generally refers to a range of values ​​falling within ±10%, ±5% in some embodiments, ±1% in some embodiments, and ±0.5% in some embodiments. In any embodiment of a numerical value beginning with “about,” an embodiment in which an exact value is described is provided. When an embodiment in which the numerical value does not begin with “about” is provided, an embodiment in which the value begins with “about” is also provided. When a range is preceded by “about,” unless the context explicitly specifies otherwise, “about” in the provided embodiments applies to the lower and upper limits of the range, or to either the lower or upper limit. When phrases such as “at least,” “at most,” “not exceeding,” or similar phrases appear before a series of numbers, unless the context explicitly specifies otherwise, it should be understood that the phrase applies to each number in the list in various embodiments (it should be understood that, depending on the context, 100% of the value, e.g., a value expressed as a percentage, may be the upper limit). For example, "at least 1, 2, or 3" should be understood to mean "at least 1, at least 2, or at least 3" in various implementations. It should also be understood that any and all reasonable lower and upper limits are explicitly considered.

[0122] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of illustration only and are not intended to limit the invention.

[0123] Example As described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, ColdSprings Harbor Laboratory, New York (2012), standard molecular biology techniques known in the art but not specifically described are generally followed.

[0124] Example 1: Materials and General Methods 1.1 Reagents All reagents and solvents were purchased from Sigma-Aldrich (Overijse, Belgium) or VWR (Oud-Heverlee, Belgium). Buffers used for coupling reactions and for radiolabeling were prepared using metal-free water (Honeywell, Fluka, Brussels, Belgium) and metal contaminants were purified using Chelex 100 resin (Sigma-Aldrich, Belgium). p-SCN-Bn-NOTA was purchased from Macrocyclics (Plano, United States of America). BCN-NODAGA was purchased from Macrocyclics (Dijon, France). VyPAL2 was prepared according to the methods described by Zhang et al. and Hemu et al. (prozymogen expression, low-pH prozymogen activation, and purification of the activated form). Human glutamine acylcyclase (QC) was purchased from Abcam (ab206806), aliquoted, and stored at -80°C until mixed with VyPAL2 at a stoichiometric ratio of 0.1:1.

[0125] 1.2 Analysis Quality control (QCO) was performed on Chromolith HR RP18 50–4.8 mm columns at a flow rate of 3 mL / min, with the following gradients: 3–97% ACN / H2O (with 0.1% TFA) over 4 min, detected at 300, 280, 254, and 214 nm. Preparative HPLC was also performed on Vydac C18 22 × 150 mm 10 µm columns at a flow rate of 20 mL / min, with the following gradients: 10–60% ACN / H2O with 0.1% TFA over 20 min.

[0126] Analytical and preparative HPLC SEC columns were purchased from Cytiva (Hoegaarden, Belgium). Site-specific functionalized (non-radioactive) sdAbs were SEC purified using PBS at pH 7 on a Superdex 75 Increase 10 / 300 GL column installed on an NGC medium-pressure chromatography system (BioRad, Tansel, Belgium) at a flow rate of 0.8 mL / min.

[0127] The modified sdAb was subjected to QCO analysis using 2× PBS (5.36 mM KCl, 273.8 mM NaCl, 2.94 mM KH2PO4, 16.2 mM Na2HPO4) via SEC at a flow rate of 0.150 mL / min on a Superdex 75 Increase 3.2 / 300 GL, with monitoring at 280 and 215 nm. The system was mounted on a Hitachi HPLC system equipped with a 5110 Hitachi pump, a 5210 Hitachi autosampler, and a 5430 Hitachi diode array detector.

[0128] [ 18 The radiochemical purity (RCP) of F]AlF-NODAGA-BCN was determined by radioactive RP-HPLC on a Hitachi system connected to a radioactive detector (GABI STAR, Elysia Raytest, Tillman, Belgium) and equipped with an analytical XBridge C18 5 μm 4.6×150 mm (Waters, Antwerp, Belgium) using the following gradients: A = H2O + 0.1% TFA and B = ACN + 0.1% TFA (HPLC grade); at 2.5 mL / min, 0–2 min 20–37% B; 2–6 min 37–50% B; 6–7 min 90% B.

[0129] The determination was performed by radioactive RP-HPLC on a Hitachi system equipped with an analytical XBridge column. 18 RCP of F]AlF-NOTA-KGIG was performed using the following gradient: A = H2O + 0.1% TFA and B = ACN + 0.1% TFA (HPLC grade); at 2.5 mL / min, 0–5 min 5–50% B; 5–6.5 min 90% B, or on a PLRP-S 300 A 5 µM 250×4.6MM column (Agilent) using the following gradient: A = H2O + 0.1% TFA and B = ACN + 0.1% TFA (HPLC grade); at 1 mL / min, 0–6 min 7–90% B; 6–10 min 90% B.

[0130] 18RCP of F-labeled sdAb was evaluated on the Hitachi system by the following: radioactive SEC on a Superdex 75 Increase 3.2 / 300 GL at 0.15 mL / min with 2×PBS, and / or on a TSKgelSUPER SW2000 (Tosoh Biosciences, Amsterdam, Netherlands) at 0.35 mL / min with 2×PBS; and by radioactive RP-HPLC on a PLRP-S column after diluting the sample with 2×PBS + 0.1% Tween 80, using the same method described above.

[0131] Using the same method as described above, the sample was diluted 2x with 0.1 M sodium citrate at pH 4.5 + 0.1% Tween 80 and evaluated on an RLRP-S column. 68 RCP of Ga-labeled sdAb.

[0132] The RCP of 67Ga-labeled sdAb was assessed by radio-SEC in 2×PBS on Superdex 75 Increase 5 / 150 GL at a flow rate of 0.45 mL / min for 12 min. Samples were diluted with 0.1 M sodium citrate pH 4.5 + 0.1% Tween 80.

[0133] The determination was performed using adhesive-free glass microfiber paper impregnated with silica gel (instant thin-layer chromatography, iTLC-SG) (Agilent Technologies, Degerm, Belgium). 68 / 67 RCP of Ga-labeled compounds was eluent using 0.1 M sodium citrate buffer at pH 4.5–5.

[0134] 1.3 Production of sdAb and extraction of periplasmic extract As previously described, nanobodies are produced (Vincke, C. et al., Methods mol. biol. 907 , 145-176 (2012)). In brief, the production and purification of hPD-L1 sdAb and control sdAb R3b23 involved: cloning the cDNA encoding the sdAb into the vector pHEN6 (which contains an N-terminal pelB signal for periplasmic transport), introducing a C-terminal NXL-His6 tag (X = G, Q) (for 2 L production), or a C-terminal adapter-His6-cys-tag (for 5 L production). The expression plasmid was then transformed into WK6 E. coli ( E. coliIn cells, under antibiotic selection conditions, growth was performed in shake-flasks at 37°C with baffles, followed by overnight expression induction with IPTG at 28°C. Overnight induced cultures were harvested by centrifugation, and periplasmic extracts were harvested by osmotic shock. Ni-NTA beads (Thermo Fisher, 250 µL per tube) were added to Falcon tubes (typically 8 x 50 mL) containing sdAb from the periplasmic extract, and the mixture was shaken for 1 h. The tubes were centrifuged and the supernatant was discarded. The tubes were then filled to 50 mL with metal-free PBS, shaken for 30 min, centrifuged, and the supernatant was discarded. The beads were added to a PD-10 column, and the tubes were washed twice with PBS (10 mL), followed by the addition of PBS to the column. The sdAbs were eluted from the column with 5 x 1 mL imidazole buffer (34 g / L in PBS; pH = 7.4). The recovered sdAbs were purified by SEC at a flow rate of 1 mL / min on a HiLoad 16 / 600 Superdex at 75 pg in PBS. Target fractions were collected, allowing for the recovery of the purified sdAbs: 20 mg of (hPD-L1)-NGL-His6 (production yield = 16.8 mg / mL), 25.7 mg of (hPD-L1)-NQL-His6 (production yield = 5.14 mg / mL), 35.1 mg of R3b23-NGL-His6 (production yield = 17.6 mg / mL), 22.2 mg of R3b23-NQL-His6 (production yield = 11.1 mg / mL), and 50 mg of (hPD-L1)-linker-his6-cys sdAb (production yield = 10 mg / L).

[0135] 1.4 Surface Plasmon Resonance All measurements were performed at 25°C on a Biacore T200 device (GE Healthcare) using Hepes buffer (0.01 M Na HEPES, pH 7.4; 0.15 M NaCl, 3 mM EDTA, 0.005% Tween 20) as the run buffer. All recombinant proteins were dissolved in 10 mM sodium acetate (pH 5.0) to a final concentration of 10 µg / mL and immobilized on the CM5 sensor chip using a linker chemistry of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Unreacted EDC-NHS linkers were blocked with 1 M ethanolamine-HCl. For all measurements, the SPR signal in the flow cell containing the immobilized protein was subtracted from the SPR signal in a flow cell that had undergone the same procedure but with the recombinant protein omitted to obtain the specific binding signal (response units, RU). The affinity of purified sdAb for human PD-L1 was evaluated on immobilized PD-L1 protein.

[0136] 1.5 Thermal stability (melting point) of sdAb The melting temperatures of the starting sdAb and the modified sdAb were determined using the protein melting program of a real-time PCR instrument. Samples were prepared by mixing 12.5 μg of sdAb with 7.5 μL of Cypro orange dye (Thermo Fisher, 300 × dilution) in PBS to a final volume of 25 μl. Blank samples contained NH4OAc. Samples were prepared in triplicate.

[0137] 1.6 SDS-PAGE and Western Blotting Samples were prepared as follows: sdAb was diluted in Leammli reducing buffer (Bio-Rad) and heated at 95°C for 4 min. 40 µL of protein sample was added to a Novex™ WedgeWell™ 8-16% Tris-glycine gel (LiveTechnologies Europe BV). A sample containing unmodified His6-tagged sdAb was prepared as a positive control. Prestained Protein Ladder (10 to 180 kDa, ThermoFisher) was added to the gel. The gel was placed in an XCell SureLock™ mini-cell electrophoresis system connected to a ZOOM® dual power supply (ThermoFischer) and run under the following conditions: in TGS buffer (BioRad), 80 V for 5–10 min to allow protein permeation, followed by 180 V for 45 min–1 h. The SDS-PAGE gel was stained with Instant Blue Coomassie protein stain (Abcam) for 20 min. The gel was scanned on an Amsersham imager (GE Healthcare) using colorimetric reverse transmission illumination (automatic) mode. For Western blotting, membrane transfer (Bio-Rad) was performed at 70–100 V for 45 min–1 h in TG transfer buffer (BioRad) pH 8.3 containing 20% ​​MeOH. The membrane was blocked with 1 g of milk powder (Nestlé) in 50 mL of PBS and incubated at RT with gentle shaking for 1 h with the primary antibody (mouse anti-His6, 4E3D10H2 / E3, dilution ThermoFisher Scientific), followed by incubation with the secondary antibody (goat anti-mouse IgG HRP, Sigma Aldrich) at the manufacturer's recommended dilution. Between each step, the membrane was washed twice with 10 mL of PBS + 0.1% Tween 80 and 30 mL of PBS. The membrane was incubated in the dark at RT for 20 min with freshly prepared HRP-developing solution (18 mg chloronaphthol (Sigma-Aldrich) in MeOH and 20 μL H2O2 (Sigma-Aldrich) in TPA buffer pH 7.5 (0.5 M NaCl, 23 mM Trisma-base)). The membrane was scanned on an Amersham imager using the following parameters: chemiluminescence, colorimetric markers, gradation = default (U) 8x, exposure = 6 sec (manual).

[0138] 1.7 Mice and cell lines Dr. SL Topalian (National Cancer Institute) provided HLA-A 0201+ 624- MEL cells. 624-MEL cells were stably transduced to express hPD-L1, and as previously described (Broos, K. et al., Cancers (Basel) 11 Characterized in 2019, 624-MEL cells were cultured in RPMI 1640 medium supplemented with 10% fetal clone I serum (Thermo Scientific, Belgium), 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, and non-essential amino acids. Five- to six-week-old female C57BL / 6 mice were purchased from Charles River. All experiments were conducted in accordance with European guidelines for animal experimentation, license number LA1230272. The experiments were approved by the Ethical Committee for the use of laboratory animals of the Vrije Universiteit Brussel (18-272-14 and 22-272-3). Intravenous injection was administered via the tail vein. Animals were anesthetized with 2.5% isoflurane in oxygen for injection and euthanasia.

[0139] 1.8 Peptide Synthesis The GIGK sequence was synthesized using solid-phase peptide synthesis. In a 5 mL sintered syringe reactor, rinkamide resin (244 mg, 0.41 mmol / g loading, 0.1 mmol graduation) was swollen in DMF (3 mL) for 10 min, then filtered under vacuum. Next, the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL) according to standards, and then deprotected with an alkaline solution of 4-methylpiperidine (4 MP) in DMF (20% vol., 2 mL) using Fmoc and shaking for 5 min. After filtration, the solution was removed, and the deprotection step was repeated with shaking for 15 min. The solution was then filtered off, and the resin was washed. HBTU (3 eq) and the first amino acid (Fmoc-L-Lys(Alloc)-OH) (3 eq) were weighed in a glass vial and dissolved in DMF (3 mL). After complete dissolution, DIEA (4 eq) was added to the solution, and the mixture was stirred for 5 min. The mixture was added to the resin and shaken for 60 min. The reagents were then filtered off, and the resin was washed with DMF and DCM. A Kaiser test was performed to ensure that all free primary amines had reacted. For each desired amino acid on the peptide, the steps from deprotection to the Kaiser test were repeated: Fmoc-Gly-OH, Fmoc-L-Ile-OH, and finally Boc-Gly-OH, to obtain the GIGK(Alloc) sequence.

[0140] For information on acetylation, please see the following paragraphs: "Ac-GIGK-Py-DABCO, GIGK-Py-DABCO, Ac-GIGK-Py-NME3 and GIGK-Py-NME3".

[0141] A solution of PhSiH3 (24 eq) and Pd(PPh3)4 (0.2 eq) in DCM (3 mL) was added to a syringe and shaken for 30 min to remove the Alloc protecting group on lysine. This deprotection step was repeated a second time with fresh solution to obtain the GIGK sequence. The resin was then washed standardly and rinsed six times with a mixture of 2 mg / mL diethyl dithiocarbamate solution (1 mL) and 0.5% (v) DIEA solution (1 mL) to remove residual palladium. The resin was washed standardly again. The desired reaction portion was then coupled (see the following paragraph).

[0142] GIGK-NOTA Synthesis Coupling was accomplished by adding a solution of p-NCS-Bn-NOTA (1.4 eq) and DIEA (9 eq) in DCM / DMF (2:1) (3 mL) to the resin and shaking at room temperature for 16 h. The solution was then filtered off and the resin was washed with DMF and DCM. The product was cleaved from the resin using an acidic TFA / TIS / H2O (9:0.5:0.5 (v)) solution for 1 h. The filtrate was collected and the product was cleaved from the resin again using a fresh acidic solution. The collected filtrate was concentrated under reduced pressure, and the peptide was cryogenically precipitated in diethyl ether and centrifuged. The precipitate was separated and dried. The resulting crude peptide was dissolved in ACN / H2O (1:1) and lyophilized.

[0143] The product was then purified by preparative HPLC on a Vydac C18 22x150 mm 10 µm column at a flow rate of 20 mL / min with the following gradient: 10-60% ACN / H2O + 0.1% TFA over 20 min. Fractions were combined according to purity and lyophilized. A white powder of GIGK (NHCS-Bn-NOTA) (12.9 mg, 14.7 µmol, yield 14%) was obtained. Purity (HPLC): >97%, t r = 1.44 min.

[0144] GIGK-MeTz Synthesis From this point onward, the product was protected from light. Coupling was performed by adding a commercially available solution of MeTz-PEG4-COOH (1.4 eq.), HBTU (1.5 eq.), and DIEA (9 eq.) to DCM / DMF (2:1) (3 mL). The reaction mixture was shaken for 16 h. The solution was filtered off, and the resin was washed with DMF and DCM. Peptide cleavage was completed within 1 h using an acidic TFA / DCM (1:1) solution. The filtrate was collected, and the product was cleaved again using a fresh acidic solution. The collected filtrate was concentrated under reduced pressure, and the peptide was cryogenically precipitated in diethyl ether and centrifuged. The precipitate was separated and dried. The resulting crude peptide was dissolved in ACN / H2O (1:1) and lyophilized.

[0145] The product was then purified by preparative HPLC on a Vydac C18 22x150 mm 10 µm column at a flow rate of 20 mL / min with the following gradient: 10-60% ACN / H2O + 0.1% TFA over 20 min. Fractions were combined according to purity and lyophilized. A bright pink powder, GIGK(NH-PEG4-Bn-MeTz) (20.2 mg, 22.4 µmol, yield 22%), was obtained. Purity (HPLC): >97%, t r= 2.60 min.

[0146] Ac-GIGK-Py-DABCO, GIGK-Py-DABCO, Ac-GIGK-Py-NME 3 and GIGK-Py-NME 3 For Ac-GIGK-Py-DABCO and Ac-GIGK-Py-NME3, the acetylation reaction was carried out using acetic anhydride (10 eq.) and DIPEA (5 eq.) in a solution of DCM (3 mL). The mixture was added to the resin and shaken for 1 h. The solution was filtered under vacuum, the resin was washed with standard solution, and a Kaizer test was performed. Alloc deprotection was then performed as described above.

[0147] The precursor DABCO-Py-TFP or NMe3-Py-TFP (2 eq.) was coupled to the lysine side chain in the presence of DIEA (5 eq.) and DMF (3 mL). The mixture was shaken for 2 h. Standard washing was performed. The resin was cleaved by adding a solution of TFA / triisopropylsilane (TIS) / H2O (90:5:5 (v / v / v)) to the resin and shaking for 2 h. The filtrate and 2 x 3 mL DCM wash buffer were collected in Falcon tubes. Most of the solvent was evaporated under reduced pressure. Diethyl ether (cold) was added, and the Falcon tubes were placed in a refrigerator overnight. The diethyl ether was removed under reduced pressure, and the product was lyophilized. The crude product was purified by preparative HPLC. All fractions were analyzed by analytical HPLC, combined according to their purity, and lyophilized. The resulting product was analyzed by HPLC and MS.

[0148] GIGK-Py-DABCO: LC-MS (m / z): C28H46N9O5+ calculated value 588.73, measured value 588.39. Purity (HPLC): 98%, Rt=2.09 min, 37% yield. GIGK-Py-NME3: LC-MS (m / z): C25H43N8O5+ calculated value 535.67, measured value 535.38. Purity (HPLC): ≥99%, Rt=2.12 min, 50% yield. Ac-GIGK-Py-DABCO: LC-MS (m / z): C30H48N9O6+ calculated value 630.77, measured value 630.37. Purity (HPLC): 90%, Rt=2.26 min, 36% yield. Ac-GIGK-Py-NME3: LC-MS (m / z): C27H45N8O6+ Calculated value 577.71, Measured value 577.39. Purity (HPLC): ≥99%, Rt=2.29 min, 36% yield. 1.9 Site-specific VyPAL2-mediated sdAb functionalization GIGK-MeTz peptide (25 eq., 4.85 mg, 5.19 µmol) was dissolved in reaction buffer (PBS pH = 6.5; 600 µL). (hPD-L1)-NGL-His6sdAb (1 eq., 3 mg, 0.215 µmol) and VyPAL2 enzyme (0.025 eq, 166 µg, 5.37 nmol) were added to the mixture. The reaction mixture was brought to a final volume of 3 mL with Fluka water (calculated based on the 71.5 µM sdAb concentration in the reaction mixture) and incubated at 37°C. After 2 hours, the reaction was terminated with 10x PBS stop buffer (pH = 7.4, 300 µL). Ni-NTA beads (100 µL / mg protein, Thermo Scientific) were added to an Eppendorf container, centrifuged, and the supernatant was discarded. Add 315 µL of termination buffer to the beads, centrifuge, and discard the supernatant. Repeat this process twice. Transfer the beads to a reaction mixture containing 200 µL of termination buffer and vortex the reaction mixture for 1 hour. Centrifuge the reaction mixture and filter the supernatant (Millex®-GV low protein binding Durapore® 13mm, 0.22 µm membrane). Wash the beads with 200 µL of termination buffer, vortex, and centrifuge. Recover the supernatant and use it to rinse the filter. Collect the filtrate and transfer it to a vivispin tube (5MWCO, Sartorius, Schalbeck, Belgium). Concentrate the reaction mixture to a volume of 300 µL by centrifuging at 3900 RPM. Recover the solution by centrifuging at 2100 RPM with the tube inverted. Rinse the membrane with 200 µL of metal-free PBS and combine the two solutions. The functionalized sdAb is purified by SEC. The highest concentration fractions collected were combined to recover purified (hPD-L1)-GIGK-MeTz sdAb (51 ± 12% recovery, N = 2). QCO was performed by SEC, SDS-PAGE, WB, and MS. The procedure was repeated on the control R3b23-NGL-His6 sdAb to obtain R3b23-GIGK-MeTz with a recovery of 59% (N = 1). The procedure was repeated on a 1.2 mg sdAb scale (keeping the ratio constant) to functionalize with GIGK-NOTA peptides to obtain (hPD-L1)-GIGK-NOTA (47% recovery, N = 1) and with GIGGGK-DOTA to obtain R3b23-GIGGGK-DOTA (39% recovery, N = 1).

[0149] 1.10 Site-specific sdAb functionalization via Michael addition Site-specific chemical Michael addition was performed according to existing procedures (Chigoho, DM et al., Pharmaceuticals14 , 550 (2021)). First, the cys-tagged sdAb was reduced. To this end, 2.5 mL (1.2 mg / mL, 37 µM, 3 mg) of the dimerized sdAb in PBS (pH 7.4) was reduced with 25 µL of a stock solution of 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 7) and a 90-fold molar excess of 2-MEA (Acros Organics, Fisher Scientific, Mellerbeck, Belgium). The mixture was incubated at 37 °C for 90 min to complete the reduction. The resulting solution was buffer-exchanged and purified on a PD-10 column pre-equilibrated with 0.2 M NH4OAc at pH 6. Next, mal-PEG4-MeTz (Conju-probes, San Diego, USA) was conjugated to the sdAb. For this purpose, the purified reduced sdAb (1 mg / mL) was incubated at 37 °C for 2 h in a 5-fold molar excess of mal-PEG4-MeTz and 30 µL of 0.5 M EDTA stock solution (pH 7). The reaction mixture was then concentrated and purified by SEC. The product was subjected to QCO analysis by SEC, SDS-PAGE, and ESI-Q-ToF-MS. The recovery of (hPD-L1)-linker-cys-mal-PEG4-MeTz was 52% (N=1).

[0150] 1.11 Direct of NOTA-KGIG-(hPD-L1) sdAb 68 Ga mark Add 1 mL (850 MBq) of [ 68 Ga]GaCl3 from germanium-68 / gallium-68 generator (Galli Eo) TMElute in IRE ELiT (Frerught, Belgium) and add to NOTA-KGIG-(hPD-L1) sdAb (7.97 nmol, final concentration 3.47 µM) diluted with 1 mL of 1 M NaOAc pH 5. After incubation at RT for 10 min, apply the reaction mixture to a PD10 column (Cytiva) pre-equilibrated with 25 mL of PBS. Allow the sample to permeate the column and add the applied sample to 2.5 mL. The product is the elution fraction of PBS (total elution volume 3.5 mL). Discard the first fraction of 0.5 mL and combine the subsequent 2 mL and filter through a 0.22 µm PVDF low protein binder filter (Merck Millipore). RCP was evaluated by radioactive RP-HPLC and radioactive iTLC on a PLRPS column. Based on [ 68 The initial activity of Ga]GaCl3 and the attenuation-corrected radiochemical yield (DC-RCY) of the filtered sample were calculated, with both values ​​being attenuated at the same time point.

[0151] 1.12 DOTA-KGGGIG-R3b23 sdAb direct 67 Ga test mark [ 67 Ga]Ga-citrate solution was purchased from Curium Pharmaceuticals (Amsterdam, Netherlands). As previously stated (Bridoux, J. et al., Biomolecules10 , 1-15 (2020); Scasnar, V.&van Lier,JE, Eur J Nucl Med20 , 273-273 (1993)) obtained [ 67 Ga]GaCl3. The DOTA-KGGGIG-R3b23sdAb solution (0.67 nmol, 14.2 µM in the total reaction mixture) was adjusted to pH 5 with 5 M NH4OAc pH 5 (calculated volume to achieve 0.5 M in the total reaction mixture), and then 10 µL (12.3 MBq) of [Ga]GaCl3 was added. 68 Ga]GaCl3. The reaction mixture was incubated at 65 °C for 10 min. RCP was evaluated on a Superdex 75 Increase 5 / 150GL column by radiometric (iTLC).

[0152] 1.13 [ 18 F]AlF production Using a cyclotron (Cyclone KIUBE, IBA, Autin-Leuven, Belgium), 2.55 mL of […] was irradiated with 18-MeV protons. 18 O]H2O was produced on-site for approximately 3 minutes (50 μA, 4.8 bar). 18 F. The enriched water was transferred to an AllInOne (Trasis) automated system and passed through a pretreated Sep-Pak Light QMA column (WAT023525, Waters, Belgium). The QMA was rinsed twice with 3 mL of water and eluted with 300 μL of 0.9% NaCl solution. 18 [F]NaF. Add 100 μL (1 — 2.5 GBq) of [ 18 Add 10 μL (20 nmol) of AlCl3 solution (2 mM AlCl3 trace metal in 0.1 M NaOAc buffer pH 4.5) to the F]NaF solution and place it under RT for 5 min.

[0153] 1.14 Enzyme-mediated radiofluorination of (hPD-L1)-NXL sdAb [ 18 Synthesis of F]AlF-NOTA-GIGK (reaction volume = 220 μL) In Eppendorf, a solution of 3 mM GIGK-NOTA in 0.2 M NaOAc pH 4.5 buffer (total reaction volume 0.2 mM) was diluted with 0.2 M NaOAc pH 4.5 buffer (total volume 0.05 M) and EtOH (25% of total volume). [The solution was then diluted with...] 18 F]AlF (50% of total volume, 0.300 — 1 GBq) was added, and Eppendorf was heated at 100 °C for 15 min. The reaction mixture was diluted with ice-cold Fluka water (900 µL). The reaction mixture was applied to a 1 cc (50 mg) tC18SEP-PAK Vac column (Waters, pre-activated with 2 mL of metal-free EtOH and 2 mL of Fluka water). The column was dried, washed with Fluka water (2 x 2.5 mL), and eluted with ACN (200 µL). The eluent fraction was aliquoted into 1 mL I-Erconic flasks and evaporated under a stream of N2 at 43 °C.

[0154] sdAb with NXL tag and [ 18 Enzyme-mediated coupling between F]AlF-NOTA-GIGK (reaction volume = 611 µL) When using NGL-tagged sdAbs in combination with VyPAL2 enzymes, the reaction was carried out at a 1:1:0.25 sdAb:peptide:one or more enzyme ratios; when using NQL-tagged sdAbs in combination with a mixture of VyPAL2+QC enzymes, the reaction was carried out at a 1:1:0.01 ratio. The concentration of sdAbs in the reaction mixture was kept constant at 71.5 µM.

[0155] To dry [ 18 Add PBS buffer (pH 6.5, 20% of total volume), an NXL-tagged sdAb, and one or more enzymes to F]AlF-NOTA-KGIG (50-150 MBq). Add water to reach the desired reaction volume. After 30 min, purify the solution via SEC using a 5 mL Pierce column (Thermo Scientific) or a PD10 desalting column, as recommended by the manufacturer. For Pierce column elution with PBS (0.5 mL), discard the first fraction and retain the next 1.5 mL, filtering through a 0.22 µm PVDF low-protein binder filter (Merck Millipore). For PD10 column elution with PBS (0.5 mL), discard the first 0.5 mL fraction and combine the next 1.5 mL, filtering through a 0.22 µm PVDF low-protein binder filter (Merck Millipore). RCP was evaluated by radioactive RP-HPLC on a PLRPS column and / or by radioactive SEC on a TSKgel or Superdex 75 Increase 3.2 / 300GL column. DC-RCY was based on the […]. 18 The initial activity and the activity after filtration of F]NaF were calculated.

[0156] 1.15 (hPD-L1) MeTz-functionalized sdAb analogue click radiofluorination [ 18 Synthesis of F]AlF-NODAGA-BCN A solution of 4 mM BCN-NODAGA in 0.2 M NaOAc buffer at pH 4.5 (10 µL) was diluted with 0.2 M NaOAc (40 µL) and EtOH (50 µL). 110 µL of […] was added to this solution. 18F]AlF (1 GBq) was added, and Eppendorf was heated at 95 °C for 7 min. After the reaction was complete, the solution was immediately rapidly diluted with ice-cold Fluka water (900 µL) and transferred to a fresh Eppendorf container. The reaction mixture was applied to a 1 cc (50 mg) tC18 SEP-PAK Vac column (Waters, pre-activated with 2 mL of metal-free EtOH and 2 mL of Fluka water). The column was dried, washed with Fluka water (2 x 2.5 mL), and eluted with EtOH (200 µL). RCP was evaluated by RP-HPLC before and after tC18 purification.

[0157] MeTz-functionalized sdAb analogues and [ 18 Click coupling between F]AlF-NODAGA-BCN Add 223.6 µL of Fluka water (calculated concentration of sdAb in the reaction mixture was 0.35 mg / mL) and 114.4 mL of EtOH (containing ±90 MBq of […] to an enzymatically or chemically MeTz-functionalized sdAb (200 µg, 232.5 µL). 18 [F]AlF-NODAGA-BCN (calculated volume of EtOH in the reaction mixture is 20%). After 30 min, the reaction mixture was applied to a PD10 column (Cytiva) pre-equilibrated with 25 mL of PBS. The sample was allowed to permeate the column, and the applied sample was added to 2.5 mL. The product was eluted with 3.5 mL of PBS, 0.5 mL per fraction. The first fraction was discarded, and the next 1.5 mL were combined and filtered through a 0.22 µm PVDF low-protein binder filter (Merck Millipore). RCP was assessed by radioactive SEC on a TSKgel column prior to purification. RCP was assessed on a Superdex 75 3.2 / 300 column after purification.

[0158] 1.16 Radiofluorination test of GIGK-NME3 peptide The lyophilized GIGK-NME3 peptide was dissolved in anhydrous DMSO (Sigma Aldrich). The automated AllInOne Trasis module was used to process the […]. 18 F]F - The enriched water was collected in vials. It was then applied to a QMA carbonate PlusLight column (Waters, P#186004540). NEt3•HCO3 solution (6 mg / mL, in 80% ACN) was used as a phase transfer catalyst (PTC) for elution. 18When using F, QMA requires pretreatment with 0.5 M NaHCO3. When using K... 222 When using K₂CO₃ solution (ABX, P#K-2620SYN) for elution, QMA must be pretreated with 8.4% NaHCO₃. The assay uses either 1.78 µmol or 0.178 µmol of peptide, calculated to have a radiolabeled concentration of 10 mg / mL or 1 mg / mL. Under NEt₃•HCO₃ conditions, 64 µL of elution solution (2 µmol PTC) is used. For K₂CO₃ / K₂CO₃... 222 Each test used 100 µL (equivalent to 1.44 µmol of base and 9.98 µmol of PTC). During radiolabeling, peptide concentration, solvent volume, and temperature all varied. RCP was evaluated by radioactive RP-HPLC on a PLRP-S column after 15 min of reaction.

[0159] 1.17 Cell Binding Studies In hPD-L1 positive (hPD-L1) POS The binding affinity of radiolabeled sdAbs to 624-MEL cells was tested. Two days prior to the experiment, 1 mL of a solution containing 5 x 10⁻⁶ sdAbs was added to each well of a 24-well plate. 4 Cells were cultured in RPMI (Thermo Fisher) medium and allowed to adhere at 37°C. One h prior to the experiment, the culture plates were cooled to 4°C. The supernatant was removed, and cells were incubated with 500 μL of 3 nM or 6 nM radiolabeled sdAb solution in unreplenished medium at 4°C for 1 h (N = 3 wells per condition). Unbound fractions were collected, and wells were washed 2x with ice-cold PBS. Cells were lysed with 0.75 mL of 1 M NaOH at RT for 5 min, 2x. All fractions were collected and counted using a gamma counter (Perckin-Elmer). Following the same procedure, cells were cultured in hPD-L1... NEG 624-MEL cells and hPD-L1 POS On cells, the specificity was determined in the presence of 100 moles of excess unlabeled competitor (unmodified sdAb). The percentage of binding activity was calculated as follows: the measured activity of the binding fraction divided by the total activity in the well.

[0160] 1.18 Cell saturation measurement In hPD-L1 POS The affinity of radiolabeled hPD-L1 sdAb was tested on 624-MEL cells. Two days before the experiment, 1 mL of 5 x 10⁻⁶ g of sdAb was added to each well of a 24-well plate. 4Cell culture medium was prepared and cells were allowed to adhere at 37°C. One hour before the experiment, the plates were cooled to 4°C. The supernatant was removed, and the cells were incubated for 1 hour at 4°C with 500 μL of radiolabeled sdAb solutions at different concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.4 nM, and 0.1 nM) in unreplenished medium (N = 3 wells per condition). Wells were treated in the same manner as in cell binding studies. To correct for specific binding, the same procedure was simultaneously applied to a second plate containing 100 moles of excess unlabeled competitor (unmodified sdAb) in each well. KD was calculated using the "One Site - Complete and Non-Specific Binding" analysis in Prism software.

[0161] 1.19 In vivo biodistribution study Healthy female C57BL / 6 mice (five to six weeks old, N = 3 per group) were injected with 22 µg of [ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb (2.75 GBq / µmol at injection), 22 µg of [ 18 F]AlF-NOTA-KGIG-R3b23 sdAb (0.98 GBq / µmol at injection), 5 µg of [ 68 Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb (30.87 GBq / µmol at injection), 22 µg of [ 18 F]AlF-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb (1.73 GBq / µmol at injection) or 24.7 μg of [ 18 F]AlF-NODAGA-BCN-MeTz-mal-cys-(hPD-L1) sdAb (1.78 GBq / μmol at injection). Animals were awakened after injection and euthanized 80 min later under isoflurane anesthesia via cervical dislocation. After euthanasia, major organs and tissues were dissected, weighed, and counted using a gamma counter according to known activity standards. The amount of radioactivity in organs and tissues is expressed as a percentage of injected activity per gram (%IA / g).

[0162] 1.20 Statistical Analysis Results are expressed as mean ± standard error of mean. A nonparametric Mann-Whitney U test was performed to compare the datasets. The legend indicates sample size and the number of replicates. The number of asterisks in the figures indicates statistical significance as follows: P<0.05; P<0.01; P<0.001; P<0.0001.

[0163] Example 2: Generation, quality control and characterization of SdAb The production of sdAb achieved a normal yield, and its standard quality control procedures and characterization were in line with expectations (Table 2).

[0164] Table 2. Summary of production yield, quality control, and characterization of the initial sdAb. SEC = Size Exclusion Chromatography. MS = Mass Spectrometry. SPR = Surface Plasmon Resonance. SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. MP = Melting Point. NM = Not Measured. NA = Not Applicable.

[0165]

[0166] Example 3: Peptide Synthesis The first peptide base, GIGK-(NHCS-Bn-NOTA) peptide ( Figure 1 C (called GIGK-NOTA) is a chelating agent-functionalized peptide that can be coupled to an sdAb engineered with an asparagine-glycine-leucine-hexahistine (NGL-His6) recognition tag. The VyPAL2 enzyme specifically recognizes and cleaves the NGL tag to form an sdAb-acyl-enzyme covalent intermediate. Following nucleophilic substitution of the intermediate with the peptide substrate, the reaction generates an sdAb specifically coupled to its C-terminus. The NOTA chelating agent can also bind to (radioactive) metals (such as...) 68 Ga or [ 18 F]AlF) complexation. Commercially available chelating agent p-NCS-Bn-NOTA ( Figure 1 B), in the presence of a base, reacts with the GIGK tetrapeptide sequence ( Figure 1 A) Coupling. Then, in the presence of a scavenger, the peptide is simultaneously cleaved from the resin using an acidic TFA solution, followed by Boc deprotection. After preparative HPLC purification, the GIGK-NOTA peptide ( Figure 1 C) The product was recovered as TFA salt in a yield of 14% and a purity of ≥97%, as determined by HPLC (214 nm).

[0167] Similarly, the second peptide substrate, GIGK(NH-PEG4-Bn-MeTz peptide (referred to as GIGK-MeTz), is a tetrazine-functionalized peptide that can be coupled to an NGL-His6-tagged sdAb via VyPAL2 enzyme catalysis. This tetrazine functionalization strategy allows for subsequent click radiolabeling using a commercially available bicyclic nonyne-functionalized chelating agent (BCN-NODAGA). Figure 2 This offers the following advantages: functionalized sdAbs can be properly purified and isolated, and more intermediate QCOs can be performed. Furthermore, these functionalized nanobodies can be click-conjugated to prosthetic groups (PGs) with other functions.

[0168] Commercially purchased MeTz-PEG4-COOH ( Figure 1 D) An amide bond is formed by coupling the GIGK tetrapeptide sequence with the carboxylic acid of MeTz-PEG4-COOH and the free amine on the peptide. The condensation is carried out in the presence of a base and an HBTU activator. Since the tetrazine moiety exhibits photosensitivity, the treatment of the tetrazine-functionalized product requires minimizing light exposure. The peptide is then simultaneously cleaved from the resin and deprotected by Boc. However, due to the sensitivity of the tetrazine moiety to TFA, a 1:1 TFA / DCM solution was used to obtain the GIGK-MeTz peptide (…). Figure 1 E) is a TFA salt with a yield of 22% and a purity ≥97%, as determined by HPLC (214 nm).

[0169] Example 4: VyPAL2-mediated site-specific functionalization, quality control, and characterization of sdAbs hPD-L1 sdAbs tagged with NGL-His6 must first be site-specifically functionalized with MeTz peptides in order to interact with [in subsequent steps]. 18 Click radiolabeling was performed on BCN-NODAGA PG labeled with F]AlF. The NODA peptide was also functionalized for non-radioactive analysis and... 68 A reference for Ga testing labeling. R3b23-NGL-His6sdAb was also coupled with MeTz-GIGK peptide as a control condition. The synthesized GIGGGK-DOTA peptide was coupled with R3b23 sdAb for testing with gallium-67 ( 67 The peptide was labeled with Ga (SPECT radionuclides) with the aim of using therapeutic radiometals for labeling in the future. The peptide reacted with sdAb in phosphate-buffered saline (PBS) at pH 6.5 at 37°C for 2 h in the presence of VyPAL2 enzyme.

[0170] Enzymes, unreacted sdAbs, and free His6-tags were removed from the mixture using immobilized metal affinity chromatography (IMAC) on Ni-NTA resin. Size exclusion chromatography (SEC) was used to separate the functionalized sdAbs from impurities not removed by IMAC, such as unreacted peptides. Table 3 shows the recoveries of the bioconjugation process, as well as quality control and characterization assays.

[0171] Table 3. Summary of VyPAL2-mediated bioconjugation with NGL-His6-tagged sdAbs and its quality control and characterization. SdAb = Single-domain antibody. QCO = Quality Control. SEC = Size Exclusion Chromatography. MS = Mass Spectrometry. SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. NM = Not Measured. NA = Not Applicable. N = Number of Repetitions when N>1.

[0172]

[0173] Western blot (WB) gel electrophoresis (data not shown) revealed that the functionalized sdAb contained a His6 tag, indicating that further optimization of IMAC purification is needed. Although the enzyme carried the His6 tag, it was not detected. Mass spectrometry analysis of the sample showed the expected molecular weight of the functionalized sdAb. However, unreacted sdAb was still visible in the spectrum, confirming the results of SDS-PAGE / WB analysis: a small amount of unreacted sdAb remained in solution, meaning that the process can be optimized.

[0174] Example 5: sdAb with cys tag and mal-PEG 4 - Site-specific Michael addition between MeTz, QCO and representation First, the (hPD-L1) linker-His6-cys-tag (dimeric form) was reduced in the presence of 2-mercaptoethylamine (2MEA). Following the reduction step, site-specific coupling with mal-PEG4-MeTz was performed on the free thiol group of the C-terminal cysteine ​​residue. The conjugated sdAb was purified by SEC with a recovery of 52%. QCO by SDS-PAGE showed the presence of the monomer, and SEC showed a purity >98%. No dimeric sdAb was observed. MS characterization showed a main peak at the expected Mw (15.752 kDa). No initiating dimeric sdAb or monomeric sdAb was observed.

[0175] Example 6: Direct conversion of NOTA-KGIG-(hPD-L1) sdAb 68 Ga mark In other applications 18 Prior to strategy F, NOTA-KGIG-(hPD-L1) sdAb had already been used. 68 Ga labeling is more direct due to its radiochemical properties. sdAb was successfully radiolabeled with a decay-corrected radiochemical yield (DC-RCY) of 45% and a radiochemical purity (RCP) >99%. Figure 3 (This can be assessed using radioactive RP-HPLC on a PLRP-S column.) The apparent molar activity at the end of production was 44.79 GBq / µmol.

[0176] Example 7: Direct comparison of DOTA-KGGGIG-R3b23 with sdAb67 Ga test mark Before proceeding with other treatment strategies involving therapeutic radiometals, the DOTA-KGGGIG-R3b23 control sdAb was used. 67 Ga labeling is used because radiochemistry is more direct. The labeling was tested at 65°C. Radiochemical SEC at 10 and 30 min showed 30% radiolabeled sdAb ( Figure 4 The reaction yield can be increased, however, due to radiochemical conditions and... 67 Unlike Ga, therapeutic radionuclide lutetium-177 ( 177 Lu) performs other optimization steps.

[0177] Example 8: Indirect click radiofluorination of MeTz-sdAb analogs The BCN-NODAGA chelating agent was radiolabeled to facilitate further click reactions with site-specific MeTz-functionalized sdAbs. Radiofluorination was achieved through […]. 18 The [F]AlF complexation is carried out, this [ 18 F]AlF is obtained from the automatic sequence of AllInOneTrasis. 18 The reaction between [F]NaF and AlCl3 occurs at room temperature. BCN-NODAGA and [ 18 The complexation reaction between F and AlF was carried out at 95 °C for 7 min, and the resulting product was immediately cooled to avoid degradation, as BCN-NODAGA is unstable at 100 °C for an extended period. The QCO of the unpurified mixture showed that it contained 20% radioactive fluorinated BCN-NODAGA and 80% free fluorinated BCN-NODAGA. 18 F(with[ 18 F]NaF and / or [ 18 F]AlF form). Then purified using a tC18 column [ 18 The intermediate [F]AlF-NODAGA-BCN was purified and eluted with EtOH. After purification, the RCP of the intermediate was 92%. Figure 5 Even if the product is not completely pure, it can still be used for the next step because radioactive impurities such as free [ 18 F]NaF / AlF does not affect click response.

[0178] The click coupling between the electron-rich dienophile BCN moiety and the electron-deficient diene MeTz of an enzymatically or chemically functionalized sdAb is termed the reverse electron-demanding Diels-Alder (IEDDA) reaction. This reaction is rapid and occurs under mild conditions, producing only nitrogen gas as a byproduct. The reaction was carried out at RT in an aqueous solution (PBS, pH 7) and 20% EtOH for 30 min. The mixture was then purified using a PD-10 column pre-equilibrated with a buffer compatible with in vivo injection (0.9% NaCl + 5 mg / mL vitamin C, or PBS). Based on the activity measured from the collected fractions, the estimated coupling yield was 25%. Radioactive SEC analysis of the filtered product showed an RCP of >95% (…). Figure 6 The attenuation-corrected radiochemical yield (DC-RCY) is 2%, based on the initial activity (in [ 18 The apparent molar activity was calculated from the activity measured in AlF / NaF solution and after filtration, with both values ​​corrected for decay at the same time point. The apparent molar activity was 3.87 GBq / μmol (N=2).

[0179] Example 9: With NXL-His 6 Enzyme-mediated radiofluorination of tagged sdAb First, optimize the production of GIGK-NOTA-[ 18 The conditions for [F]AlF were determined, and suitable analytical methods were developed. The Nota-functionalized peptides were prepared in 0.2 M NaOAc buffer at pH 4.5 and supplemented with [F]AlF. 18 The mixture was diluted in a solvent of [F]AlF / NaF. The reaction mixture was then heated at 100 °C for 15 min.

[0180] In order to determine [ 18 The optimal conditions for the chelation of AlCl3 with GIGK-NOTA were first explored using EtOH or ACN as solvents, followed by heating for 10 or 15 min. In these tests, the following parameters remained constant: 0.5 eq. AlCl3 with 1 eq. GIGK-NOTA. 在反应小瓶中 = 0.2 mM, [AlCl3] 在反应小瓶中 = 0.020 mM, final volume solvent content 25%, pH = 4.5. Various parameters such as the concentrations of AlCl3 and GIGK-NOTA were evaluated, as well as [ 18 F]AlF equivalent number. After the reaction, each sample was diluted with cold water to stop the reaction. Samples were analyzed by radioactive RP-HPLC on an XBridge C18 analytical column, and observed at Rt = 0.83 min. 18 F]AlF, observed at Rt = 3 min [18 F]AlF-GIGK-NOTA. Marking conditions and [ 18 F]AlF complexation yield report in Figure 23 middle.

[0181] When using 0.1 eq. of AlCl3 ( Figure 23 The first line) resulted in a complexation rate of 16%; however, under the same conditions, a better complexation rate of 35% was obtained when 0.5 eq. of AlCl3 was used for the reaction. Figure 23 (Line 2). Higher equivalence numbers result in lower yields; 1.5 eq. of AlCl3 ( Figure 23 (Line 5) resulted in a 9% complexation rate, while under the same conditions, the complexation rate was 15% at a reaction of 0.5 eq. ( Figure 23 (Line 4). Therefore, it is assumed that non-radioactive Al material is related to [ 18 F]AlF competes for NOTA chelation. This reduces the concentration of GIGK-NOTA by half. Figure 23 The third and second lines) result in similar complexation yields (30% vs. 35%). However, when the concentration is reduced to one-third of its original value ( Figure 23 (Line 4), the yield was negatively affected, at only 15%, while at higher concentrations ( Figure 23 The yield of the second row is 35%.

[0182] Before performing enzyme-mediated labeling of sdAb, first isolate [ 18 F]AlF-NOTA-KGIG substance. First, dilute the reaction mixture with cold water to a total volume of 1.1 mL. Then, apply the solution to a C18 column and rinse the C18 column twice with water to remove salt and [ 18 [18F]AlF / NaF. Next, the product was eluted with ACN. The QCO analysis method for [18F]AlF-NOTA-KGIG was transferred to the PLRP-S method, which allows for the separation of free 18F, peptides, and radiolabeled sdAb final products using a single gradient.

[0183] Successfully formed [ 18 Following F]AlF-NOTA-KGIG, the optimal conditions for coupling with (hPD-L1)-NGL-His6sdAb were determined. First, the effects of the peptide / sdAb ratio and reaction time were evaluated. [The following text appears to be incomplete and requires further context: "Containing [...] 18The ACN eluent of F]AlF-NOTA-KGIG was evaporated at 43 °C under a nitrogen stream. Solutions of sdAb (at different eq.) and VyPAL2 (0.025 eq.) diluted in 20% (v) PBS pH = 6.5 and 30% (v) water (to maintain a constant sdAb concentration) were added to the dried peptide, and the mixture was heated at 40 °C for the duration of the reaction. The sample was diluted in 2 × PBS + Tween 80 0.1% and analyzed by radioactive SEC on a TSKgel column to determine the percentage of radioactivity coupled to the sdAb.

[0184] Table 4. Optimization using VyPAL2 enzyme 18 The coupling reaction between F-labeled Nota peptides and sdAbs tagged with NGL-His6. The coupling activity was measured by radioactive SEC.

[0185]

[0186] The results listed in Table 4 show that when the sdAb / peptide ratios were 0.04 and 0.20 (reactions 1 and 2, respectively), almost all the expected radioactivity was coupled to the sdAb within one hour (4% and 18%, respectively). When the sdAb / peptide ratio was 1:1 (reaction 6), the percentage of activity coupled to the sdAb appeared to remain stable (around 50%) after heating for 30, 45, or 60 min, indicating that the reaction was completed in just 30 min. This further demonstrates the high efficiency of the VyPAL2 enzyme, as coupling with other enzymes (such as sorting enzyme A) must be carried out within 16 h. Higher ratios were also tried, using 2 and 5 times the amount of sdAb as PG, respectively (rows 4 and 5, respectively), which increased the coupling percentage to 60% and 72%, respectively. However, these reactions required a large amount of sdAb protein, which may not be cost-effective for future translational applications. The reaction mixture from row 3 (…) Figure 7 Further purification was performed on a PD10 desalting column. The PD10 eluent was fractionated to investigate whether the peptides eluted with the eluent.

[0187] After PD10 purification, the initial elution volume of 0.5 mL was discarded, and the remaining 1.5 mL was collected and analyzed by radioactive SEC analysis. Figure 8 The figure shows that the RCP of the recovered product was 95.2%. The remaining 4.8% was peptides that could have been eluted with fraction 4. Therefore, adding more fractions to the pool would certainly elute more free peptides and lower the final RCP.

[0188] The purified radioactive products were attenuated and then analyzed by SDS-PAGE / WB. SDS-PAGE analysis showed a band near the expected Mw of the sdAb. However, WB analysis showed some unlabeled sdAbs still present in solution, which was expected given the incomplete labeling and the presence of a few unreacted sdAbs still carrying the His6-tag. Another band corresponding to the His-tagged enzyme was observed on WB, indicating that some enzymes were not eliminated by the PD10 column desalting and remained in the purified solution.

[0189] Overall, the recovered product was suitable for in vivo studies due to its RCP > 95%. The obtained product had a DC-RCY of 7% (measured in the diluted reaction mixture prior to purification on tC18) and was active after filtration. Both values ​​were corrected for decay at the same time point. The apparent molar activity after production was 2.94 GBq / µmol.

[0190] 9.2 Cascade Labeling Reactions Using VyPAL2 + QC Mixture To improve radiochemical yield, an enzymatic mixture (VyPAL2 + QC enzyme) was used to mediate fluorination between hPD-L1 sdAb tagged with NQL and radiofluorinated GIGK-NOTA. First, the GIGK-(NHCS-Bn-NOTA) peptide was radiolabeled and isolated as described above. Using 1 eq. of hPD-L1-NQL-His6 and 1 eq of the labeled peptide in PBS buffer (pH 6.5), […]. 18 Enzyme-mediated radiofluorination was performed using an enzymatic mixture of [F]AlF-GIGK-NOTA and 0.01 eq. The reaction was carried out at 43 °C for 30 min. After 30 min incubation, radioactive SEC showed 11% of the remaining peptide (F). Figure 9 Small radioactive impurities were detected at 15.90 min, which may represent free radioactive contaminants. 18 F or degraded peptides. These are more likely due to impurities attached to that particular column, as no unexpected radioactive impurities were detected by radioactive RP-HPLC (data not shown). It is estimated that 72% of the radioactivity is related to sdAb.

[0191] The mixture was then purified using a PD-10 column. After purification and filtration, QCO, as determined by radioactive RP-HPLC, showed an RCP of 93% ( Figure 10 A). A small amount of radioactive impurities (7%) were detected. The UV spectrum at 280 nm shows a chemical purity of 100%. Figure 10 B). The obtained product has good purity and is suitable for in vivo studies.

[0192] The DC-RCP was 11%, which is higher than the value obtained previously when coupled with the VyPAL2 enzyme (DC-RCP=7%).

[0193] Example 10: In vitro binding study To evaluate the binding specificity of radiolabeled sdAb to its target hPD-L1 expressed on cells, radiolabeled sdAb was added to hPD-L1 at two different concentrations (3 nM and 6 nM). POS or hPD-L1 NEG MEL624 hPD-L1 cells were blocked using a 100-fold molar excess of non-radioactive sdAb. POS Cells were used as a control to assess binding specificity. After incubation, unbound fractions were removed, and cell-related activities were measured. Figure 11 A, 11B, 11C, and 11D show the results for the following tested compounds; 68 Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb and [ 18 F]F-NODAGA-BCN-MeTz-mal-cys-(hPD-L1) sdAb. For all sdAb analogues, both radiolabeled cell-related activities showed activity against hPD-L1. POS Cell-specific binding, which is achieved through control conditions (hPD-L1) NEG The absence of cell-related activity was confirmed in cells and in excess of unlabeled sdAb. This assay confirmed the specificity of hPD-L1 sdAb to its target, but could not assess affinity.

[0194] Example 11: In vitro cell saturation determination In hPD-L1 POS The affinity (dissociation constant K) of radiolabeled sdAb analogues was tested on 624-MEL cells. D Cells were incubated in plates containing radiolabeled sdAb at concentrations ranging from 300 nM to 0.1 nM, and wells were treated in the same manner as in cell binding studies. Figure 12 A, 12B, and 12C show the results for the following tested compounds; 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb,[ 18F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) sdAb and [ 18 F]F-NODAGA-BCN-MeTz-mal-cys-(hPD-L1) sdAb. All obtained values ​​were within the expected range for hPD-L1 sdAb, indicating that hPD-L1 sdAb did not have a negative impact on affinity after its functionalization and radiolabeling.

[0195] Example 12: Biodistribution study in healthy mice To investigate its biodistribution in vivo, radiolabeled sdAb was injected into three healthy mice. The mice were euthanized 80 minutes post-injection (pi). Organs were collected, and the viability of each organ was measured.

[0196] First, it is administered via injection of an enzyme mixture. 18 F]AlF-NOTA-KGIG radiofluorinated hPD-L1 and control R3b23sdAb ( Figure 13 Aside from retention in the kidneys due to the excretion pathway of sdAb, no expected background signal was observed. As expected, hPD-L1 sdAb showed significantly lower renal retention compared to control sdAb.

[0197] Then, we studied and separated the [prefunctionalized versions] that had been pre-functionalized with VyPAL2. 68 Biological distribution of Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb, and then using 68 Ga direct labeling. Compare this biological distribution with previous data from [ 18 Data obtained from F]AlF-tagged analogues were compared. Figure 14 Since the two final products are identical except for the radionuclide, an enzyme-mediated strategy was used to observe the radiofluorination of sdAb instead of the usual prefunctionalization strategy followed by direct radiofluorination. 68 The effects of Ga markers are quite interesting.

[0198] Finally, the obtained [ 18 The biodistribution of F]AlF-NOTA-KGIG-(hPD-L1) sdAb was compared with that of enzymatically or chemically prefunctionalized MeTz sdAbs. This comparison is interesting because chemical strategies are one of the commonly used site-specific strategies performed on radiolabeled sdAbs.

[0199] In summary, compared with chemical prefunctionalization, enzymatic prefunctionalization of sdAb reduced renal retention by half. When an enzyme-mediated radioactive fluorination strategy was used to obtain […] 18When F]F-NOTA-KGIG-(hPD-L1) was used, renal retention was further reduced by 3.2x compared to the chemical strategy.

[0200] In summary, these data indicate that radiofluorination of hPD-L1 sdAb using the VyPAL2 enzyme (in combination with the QC enzyme) not only promotes and enhances the overall efficiency of the radiochemical process but also provides advantages in terms of in vivo properties.

[0201] Example 13: Novel peptides for radiofluorination via nucleophilic substitution To obtain the data previously shown, [ 18 F]AlF is used as a metalloid radionuclide because it is compatible with the use of radioactive halogens [ 18 F]F - Compared to nucleophilic substitution, complexation chemistry is more direct. However, using [ 18 F]F - This will increase the yield of radiofluorination of peptides, and by combining non-radiolabeled peptide precursors with... 18 F-labeled peptides are isolated to increase specific activity. Furthermore, this would benefit overall enzymatic coupling yield. Additionally, radioactive metals are known to increase renal retention compared to radioactive halogens. For these reasons, one objective is to develop peptides with leaving groups, such as DABCO and NMe3, for radiofluorination via nucleophilic substitution.

[0202] The precursors DABCO-Py-TFP and NMe3-Py-TFP were obtained in good yield from 6-chloronicotinic acid in two steps. Esterification was performed using N,N'-dicyclohexylcarbodiimide (DCC) and 2,3,5,6-tetrafluorophenol (TFP-OH) as starting materials to produce the ester Cl-Py-TFP. The byproduct dicyclohexylurea (DCU) was removed by filtration, while unreacted reagents were easily removed by liquid extraction. Since Cl-Py-TFP is highly activated by the electron-withdrawing TFP ester moiety, nucleophilic aromatic substitution of chlorine with DABCO and NMe3 is advantageous. The DABCO-Py-TFP precursor was obtained by reacting Cl-Py-TFP with DABCO under the same conditions described by Zlatopolskiy et al. (2019). The product was obtained after a filtration step in 72% yield. Using the conditions described by Bouvet et al. (2016), NMe3-Py-TFP precursors with a yield of 72% and a purity of ≥99% were also obtained by reacting a solution of NMe3 in THF with Cl-Py-TFP.

[0203] The coupling between the precursor and the peptide backbone (composed of a GIGK sequence) was performed similarly to that described for the NOTA and MeTz peptides in Example 3. The coupling step was carried out over 2 h using 2 eq. of the precursor in the presence of 5 eq. of base. Since the precursor was already activated by the existing TFP group, no additional activator was required. To prevent nucleophilic substitution of the amine group of the glycine residue on the leaving group of the peptide during radiofluorination, acetyl-protected peptide variants were generated. The products were purified by RP-HPLC, yielding products with good purity and varying yields. Figure 16 ).

[0204] Next, the GIGK-NME3 peptide was subjected to radiofluorination testing by changing several parameters described in Table 5.

[0205] Table 5. Radiofluorination test of GIGK-NME3 peptide (pep) with variations in base, phase transfer catalyst (PTC), solvent, temperature, and activity. Radiochemical purity (RCP) was assessed by radioactive RP-HPLC after 15 min of reaction.

[0206]

[0207] In the following steps, we will confirm whether the measured peaks are the expected products by testing various columns or by preparative RP-HPLC separation. 18 F-labeled peptides. We will also attempt radiofluorination of DABCO-functionalized peptides.

[0208] Example 14: Radiofluorination of hPD-L1 sdAb – Optimization of coupling with GIGK-NHCS-NOTA peptide.

[0209] The synthesis of GIGK-NHCS-NOTA peptide and its relationship with [[ were described earlier.] 18 Radiolabeling of F]AlF. It has been reported that 1 eq. of hPD-L1-NQL-His6 single-domain antibody (sdAb) and 1 eq. of labeled peptide [ were used in PBS buffer (pH 6.5). 18 Enzyme-mediated radiofluorination was performed using an enzymatic mixture of [F]AlF-GIGK-NOTA and 0.01 eq. of VyPAL2 + 0.1 eq. of GluC enzyme, relative to VyPAL2. The reaction was carried out at 43 °C for 30 min. Under these conditions, approximately 75% of the radiopeptide was conjugated to sdAb.

[0210] The optimization of reaction time and efficiency was studied.

[0211] For this purpose, non-radioactive optimization was performed on hPD-L1 sdAb using 0.1 eq. of VyPAL2 (relative to VyPAL2, + 0.5 eq. of GluC); and the conversion rate of the Nota peptide was >90% within 5 minutes.

[0212] For coupling with radiolabeled peptides, the equivalent of GluC enzyme was increased to 0.5 eq. (relative to VyPAL2), as follows: Figure 17 As shown, 92% of the conjugated peptide was obtained within 15 minutes of reaction.

[0213] for[ 18 F]AlF peptide, due to the presence of metal contaminants in the cyclotron (unrelated to enzymatic coupling), we are currently unable to increase its content. 18 The amount of F. However, we are interested in investigating the enzyme's resistance when exposed to higher levels of activity.

[0214] In this experiment, 900 MBq of [ 18 The F]NaF salt was dried and redissolved using an enzyme mixture. The mixture was heated at 43°C for 7 minutes for irradiation, followed by the addition of (hPD-L1)-NQL sdAb (1 eq., at 1 mg / mL) and 18 The F-labeled peptide substrate (1 eq.) was incubated for 7 minutes in the presence of 0.1 eq. of VyPAL2 (relative to VyPAL2, + 0.5 eq. of GluC). Under these conditions, 85% of the radiolabeled peptide was conjugated with sdAb, as shown in the figure. Figure 18 As shown.

[0215] This data appears to suggest that more active radiation (suitable for preclinical studies) does not affect enzyme efficiency. The next steps will involve increasing the activity of the peptide itself, investigating the reproducibility of the process, and developing robust purification methods to replicate preclinical studies in tumor-bearing mice.

[0216] Example 15: Radiolabeling using FPy-KGIG peptide via nucleophilic substitution.

[0217] In order to enable higher levels of radioactive manipulation of peptides, we are also investigating the use of novel peptides that include leaving groups that can be radioactively fluorinated through nucleophilic substitution, as described in Example 3.

[0218] The synthesis of two novel peptides, GIGK-Py-DABCO and GIGK-Py-NMe3, has been described previously. The labeling assay using the NMe3 peptide produced 65% radioactivity at 6.43 minutes, indicating that the phase transfer was achieved within 15 minutes at 95°C using kryptofix as a phase transfer catalyst and K₂CO₃ as a base. 18F was incorporated into the peptide in DMSO at a concentration of 1 mg / mL. A non-radioactive reference has now been synthesized, which can confirm the presence of the radiolabeled peptide at the expected Rt (see [link to relevant documentation]). Figure 19 ).

[0219] Using a non-radioactive reference, attempt to couple a non-radioactive element to (hPD-L1)-NQL-His6 and the control R3b23-NQL-His6sdAb. For example... Figure 20 As shown, peptide coupling can be observed by tracking the UV signal on the C4 column; however, this needs to be confirmed by mass spectrometry.

[0220] 1:1 sdAb (tested with hPD-L1 and control R3b23): peptide ratio, using 0.1 eq. of VyPAL2 (relative to VyPAL2, +1 eq. of GluC), 5 min reaction time.

[0221] The test showed Figure 20 The results shown are obtained on control sdAb R3b23: Rt = 7.15 min = unmodified sdAb, Rt = 7.53 = putative sdAb coupled with FPy-KGIG peptide.

[0222] Example 16: Enzymatic coupling to produce sdAb for labeling therapeutic radionuclides.

[0223] The sdAb targeting carcinoembryonic antigen (CEA) is synthetic and has an NQL-His6 or cysteine ​​tag at its C-terminus.

[0224] The goal is to associate the DOTA component with sdAb so that it can be used 177 Radiolabeled with Lu (a radionuclide used for therapeutic applications). For this purpose, the GIGK-NHCS-DOTA peptide was synthesized according to the protocol described in Example 3 above.

[0225] The GIGK-NHCS-DOTA peptide was conjugated with CEA sdAb according to the scheme described in Example 4 above.

[0226] Meanwhile, maleimide-DOTA (mal-DOTA) was purchased from CheMatech (Dijon, France) and conjugated with a cysteine-tagged CEA sdAb analogue according to the procedure described in Example 5 above.

[0227] By 177 Lu was added to DOTA-sdAb (19 µM in the mixture) at pH 4.5 and left for 2 hours. 177 Lu was used for testing and labeling. Under these conditions, CEA chemically coupled with mal-DOTA resulted in only [a certain effect] at 50°C.177 A 10% Lu incorporation rate is required, and the temperature needs to be raised to 60°C to achieve a 94% incorporation rate, such as... Figure 21 What I saw.

[0228] Enzyme-coupled CEA-GIGK-NHCS-DOTA was prepared using 16.6 μM... 177 Lu was labeled at 40°C for 45 min, yielding a satisfactory 80% concentration. 177 Lu is incorporated, such as Figure 22 As shown.

[0229] Summarize This disclosure describes a novel method for the rapid and reagent-efficient generation of radiolabeled single-domain antibodies (sdAbs) using asparagine acylpeptide ligase (PAL) alone or in tandem with glutamine acyl cyclase (QC), which improves their in vivo properties. In particular, the cascade reaction applied to sdAbs (PAL + QC) introduces an unparalleled tool: it allows for the efficient generation of pure homogeneous radiolabeled sdAbs within a timeframe (e.g., <1 hour) compatible with radioactive elements with short (<12 hours) half-lives, yielding near 100% and generating minimal radioactive waste.

[0230] Various strategies were employed to generate radiolabeled sdAb tracers, either using VyPAL2 enzyme alone or in combination with a QC enzyme (typically at a molar ratio of 1:0.1 or 1:0.5). These bioconjugation strategies were compared with a site-specific strategy based on chemical reactions rather than enzymatic reactions. Notably, the use of an enzymatic mixture enabled the sdAb with an NQL-His6 tag at its C-terminus to bind with a radiolabeled fluorinated peptide. 18 Nearly complete bioconjugation (>70%) was achieved between F]AlF-NOTA-KGIG. For the latter reaction, an equimolar ratio of sdAb:peptide was used. The resulting product [ 18 The F]AlF-NOTA-KGIG-(hPD-L1) sdAb exhibited superior in vivo activity compared to products obtained using chemical site-specific strategies. Characterization of the sdAb conjugates obtained using the enzymatic mixture indicates that this method is well-suited for the rapid and efficient generation of site-specific radiofluorination tracers based on sdAbs, with improved in vivo molecular imaging features.

[0231] Furthermore, peptide substrates can include other radiolabeling agents and / or reactive moieties for radiolabeling. This can be combined with specific amino acid sequences that promote radiochemical processes and further improve in vivo behavior, depending on the desired application, such as for diagnostic, surgical, therapeutic, or therapeutic monitoring purposes, and more specifically, as an imaging agent. This will be of great interest to hospitals and medical centers that frequently use radiation medicine.

[0232] References Bouvet, V., Wuest, M., Jans, HS, Janzen, N., Genady, AR, Valliant, JF, ...&Wuest, F. (2016). Automated synthesis of [18 F] DCFPyLvia direct radiofluorination and validation in preclinical prostate cancer models. EJNMMI research , 6 , 1-15. Bridoux, J., Broos, K., Lecocq, Q., Debie, P., Martin, C., Ballet, S., ...&Xavier, C. (2020). Anti-human PD-L1 nanobody for immuno-PET imaging: validation of a conjugation strategy for clinical translation. Biomolecules , 10 (10), 1388. Broos, K., Lecocq, Q., Xavier, C., Bridoux, J., Nguyen, TT, Corthals, J., ...&Breckpot, K. (2019). Evaluating a single domain antibody targeting human PD-L1 as a nuclear imaging and therapeutic agent. Cancers , 11 (6), 872. Chigoho, D. M., Lecocq, Q., Awad, R. M., Breckpot, K., Devoogdt, N.,Keyaerts, M., ...&Bridoux, J. (2021). Site-Specific radiolabeling of a humanPD-L1 nanobody via maleimide-cysteine chemistry. Pharmaceuticals , 14 (6), 550. Hemu, X., El Sahili, A., Hu, S., Wong, K., Chen, Y., Wong, Y. H.,...&Tam, J. P. (2019). Structural determinants for peptide-bond formation byasparaginyl ligases. Proceedings of the National Academy of Sciences , 116 (24),11737-11746. Š asnár, V.,&van Lier, J. E. (1993). The use of SEP-PAK Sl cartridgesfor the preparation of gallium chloride from the citrate solution. European Journal of nuclear medicine , 20 , 273-273. Vincke, C., Gutiérrez, C., Wernery, U., Devoogdt, N., Hassanzadeh-Ghassabeh, G.,&Muyldermans, S. (2012). Generation of single domain antibodyfragments derived from camelids and generation of manifoldconstructs. Antibody Engineering: Methods and Protocols, Second Edition , 145-176. Zhang, D., Wang, Z., Hu, S., Lescar, J., Tam, J. P.,&Liu, C. F.(2021). Vypal2: A Versatile Peptide Ligase for Precision Tailoring ofProteins. International Journal of Molecular Sciences , 23 (1), 458. Zlatopolskiy, B. D., Zischler, J., Krapf, P., Richarz, R., Lauchner,K.,&Neumaier, B. (2019). Minimalist approach meets green chemistry: synthesisof 18F‐labeled (hetero) aromatics in pure ethanol. Journal of labelled compounds and radiopharmaceuticals , 62 (8), 404-410。

Claims

1. A method for preparing a site-specific radiolabeled tracer, the method comprising providing i) Peptidyl asparagine acyl ligase (PAL); ii) A first peptide, wherein the first peptide comprises a P1-P1'-P2' tripeptide PAL motif at its C-terminus, wherein, P1 is Asn or Asp, P1' is Gln or any non-proline amino acid, and P2' is a hydrophobic amino acid such as Val or Leu or Ile or a β-branched amino acid. iii) A second peptide comprising a P1''-P2'' motif at the N-terminus, wherein P1'' is Gly and P2'' is a hydrophobic amino acid such as Val or Leu or Ile, or a β-branched amino acid, wherein the second peptide is modified by a functional motif to form a functionalized peptide. iv) Contact the peptidyl asparagine acyl ligase (PAL) with the first peptide and the functionalized peptide to form a conjugate of the first peptide and the functionalized peptide. The method further includes (a) Radiolabeled conjugate, or (b) Radiolabel the functionalized peptide prior to contact with the PAL.

2. The method according to claim 1, wherein, The first peptide is an antibody, a scaffold protein, an antibody mimic, or a functional fragment thereof.

3. The method according to claim 2, wherein, The first peptide is an antibody or a functional fragment thereof.

4. The method according to claim 3, wherein, The antibody or its functional fragment is a single-domain antibody (sdAb).

5. The method according to claim 4, wherein, The sdAb is an sdAb targeting hPD-L1 or an sdAb targeting CEA.

6. The method according to any one of the preceding claims, wherein, P2' and / or P2'' are hydrophobic amino acids selected from the group consisting of Val, Leu, and Ile.

7. The method according to any one of the preceding claims, wherein, The P1-P1'-P2' tripeptide PAL motif is an Asn-X-Leu motif, where X is Gln(Q), Gly(G), or Glu(E).

8. The method according to any one of the preceding claims, wherein, The P1-P1'-P2' tripeptide PAL motif is Asn-Gln-Leu (NQL) or Asn-Gly-Leu (NGL).

9. The method according to any one of the preceding claims, wherein, The first peptide includes a His6 tag at its C-terminus.

10. The method according to any one of the preceding claims, wherein, The first peptide includes a -NGL-His6 or -NQL-His6 tag at its C-terminus.

11. The method according to claim 10, wherein, The first peptide is selected from the group consisting of (hPD-L1)-NGL-His6, (hPD-L1)-NQL-His6 and (CEA)-NQL-His6 sdAb.

12. The method according to any one of the preceding claims, wherein, The P1''-P2'' motif is a -GI motif.

13. The method according to any one of the preceding claims, wherein, The functionalized peptide includes a -GIGK-, -GIGG-, or -GIGGGK- motif at its N-terminus.

14. The method according to claim 13, wherein, The functionalized peptide is selected from the group consisting of: GIGK-NOTA, GIGK-DOTA, GIGK-MeTz, GIGGGK-DOTA, GIGK-NOTA-[ 18 F]AlF, GIGK-Py-DABCO, Ac-GIGK-Py-DABCO, GIGK-Py-NMe3, Ac-GIGK-PyNMe3 and GIGK-FPy.

15. The method according to any one of the preceding claims, wherein, The PAL is selected from the group consisting of: VyPAL2 including the amino acid sequence shown in SEQ ID NO: 32, butelase1 including the amino acid sequence shown in SEQ ID NO: 33, butelase-2 including the amino acid sequence shown in SEQ ID NO: 34 or 35, VyPAL3 including the amino acid sequence shown in SEQ ID NO: 36, OaAEP1b-C247A including the amino acid sequence shown in SEQ ID NO: 37, HeAEP3 including the amino acid sequence shown in SEQ ID NO: 38, AtLEGγ including the amino acid sequence shown in SEQ ID NO: 39, VuPAL1 including the amino acid sequence shown in SEQ ID NO: 40, HaPAL1 including the amino acid sequence shown in SEQ ID NO: 41, OaAEP1b including the amino acid sequence shown in SEQ ID NO: 42, and functional fragments or variants thereof.

16. The method according to any one of the preceding claims, wherein, The PAL is VyPAL2, which includes the amino acid sequence shown in SEQ ID NO: 32, or a functional fragment or variant thereof.

17. The method according to any one of the preceding claims, wherein, The contact step was carried out in the presence of glutamine acylcyclase (QC).

18. The method according to claim 17, wherein, The stoichiometry of PAL:QC is 1:0.1 to 1:

1.

19. The method according to claim 18, wherein, The PAL:QC stoichiometry is 1:0.1 or 1:0.

5.

20. The method according to any one of claims 17 to 19, wherein, The QC is selected from the group consisting of: human glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 43, mouse glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 44, Drosophila glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 45, Arabidopsis glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 46, Conophytum glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 47, Rattlesnake glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 48, bacterial glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 49, and functional fragments or variants thereof.

21. The method according to any one of claims 17 to 20, wherein, The QC is a human glutamine acylcyclase comprising the amino acid sequence shown in SEQ ID NO: 43, or a functional fragment or variant thereof.

22. The method according to any one of the preceding claims, wherein, The functionalized peptides are functionalized using chelating agents or tetrazines.

23. The method according to claim 22, wherein, The chelating agent is selected from the group consisting of: 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA).

24. The method according to claim 22 or 23, wherein, The chelating agent was further functionalized using bicyclononyne.

25. The method according to claim 22, wherein, The tetrazine moiety is -MeTz.

26. The method according to claim 22 or 25, wherein, Tetraazine-functionalized peptides form conjugates of the first peptide-(tetraazine-functionalized peptide), and the conjugates are further complexed with a bicyclic nonyne functionalized chelating agent prior to radiolabeling.

27. The method according to claim 26, wherein, The bicyclic nonyne functionalized chelating agents include chelating agents selected from the group consisting of: 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA).

28. The method according to any one of the preceding claims, wherein, The conjugate or the functionalized peptide is selected from gallium-68 ( 68 Ga), gallium-67 ( 67 Ga), Lutetium-177( 177 Lu) and Fluorine-18 ( 18 Radioactive nuclides of group F are radiolabeled.

29. The method according to claim 28, wherein, The radionuclide is gallium-68 (GaN-68) 68 Ga) or with [ 18 Fluorine-18 in the form of F]AlF ( 18 F).

30. A site-specific radiolabeled tracer prepared by the method according to any one of claims 1 to 29.

31. The site-specific radiolabeled tracer according to claim 30, selected from the group consisting of: [ 68 Ga]Ga-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]AlF-NOTA-KGIG-(hPD-L1) sdAb,[ 18 F]F-NODAGA-BCN-MeTz-KGIG-(hPD-L1) and [ 177 Lu]Lu-DOTA-NHCS-KGIG-CEA sdAb.

32. The site-specific radiolabeled tracer according to claim 30 or 31, used as a nuclear imaging agent.

33. Use of the site-specific radiolabeled tracer according to claim 30 or 31 as a nuclear imaging agent.

34. Use of the site-specific radiolabeled tracer according to claim 30 or 31 in the manufacture of diagnostic imaging agents for nuclear imaging.

35. A method of treating a subject's disease, the method comprising administering to the subject a site-specific radiolabeled tracer according to claim 30 or 31.

36. Use of the site-specific radiolabeled tracer according to claim 30 or 31 in the manufacture of a medicament for treating a subject’s disease.

37. The method of claim 35, or the use of claim 36, wherein, The disease in question is a tumor or cancer.

38. The method of claim 35, or the use of claim 36, wherein, The sdAb targets diseased cells, and the radiolabeled tracer is labeled with a therapeutic radionuclide, such as... 177 Lu、 211 At or 225 Ac.

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