An immune checkpoint PD-L2 targeted single-chain antibody and radionuclide marker and application thereof

Abstract

The present invention belongs to the fields of medical biological detection technology, radiochemistry and nuclear medicine, and relates to an immune checkpoint PD-L2 targeted single-chain antibody and radionuclide marker and application. The single-chain antibody includes a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO: 2. The radionuclide-labeled antibody fragment (scFv-hFc) PET tracer targeting PD-L2 provided by the present invention has stable properties in vitro and in vivo, good imaging effect, and high affinity and specificity for PD-L2. Preclinical evaluation shows that this series of tracers is expected to become a clinical imaging agent for evaluating tumor PD-L2 expression.

Description

Technical Field

[0001] The present invention belongs to the fields of medical biological detection technology, radioactive chemistry and nuclear medicine, and specifically relates to the screening of PD-L2 specific antibody fragments and applications thereof, and in particular to a screening method, radionuclide labeling and applications thereof of the antibody fragments. Background Art

[0002] Immune checkpoint inhibitor therapy (ICI) is an important type of treatment in tumor immunotherapy. Its mechanism is that immune checkpoints are a type of immunosuppressive molecules that can regulate the breadth and depth of immunity and avoid damage and destruction of normal cells or tissues. However, in the tumor microenvironment, in order to meet their own growth needs, tumor cells suppress the function of the immune system through certain means, so that immune cells cannot normally identify and kill tumor cells, resulting in so-called immune escape. In the process of immune escape, programmed cell death protein-1 (PD-1) and its ligands play an important role. PD-1 is an immunosuppressive protein expressed on the surface of T cells and is a member of the CD28 superfamily. Its ligands are divided into two categories, namely PD-L1 and PD-L2, both of which can be expressed on the surface of tumor cells. Tumor cells bind to PD-1 through PD-L1 or PD-L2, ultimately inhibiting the ability of immune cells to recognize and eliminate tumors. ICI treatment is based on immune checkpoints. By blocking the binding of receptors and ligands, immune escape is overcome, immune function is upregulated, and then the purpose of tumor inhibition or killing is achieved. With the approval and application of monoclonal antibody drugs targeting PD-1 / PD-L1, multiple prospective studies have confirmed that ICI treatment can significantly improve the survival rate of patients with advanced non-small cell carcinoma and prolong the survival time of patients. However, how to screen potential beneficiaries before treatment, monitor the efficacy of ICI treatment during treatment, and grasp the indications for discontinuation in time to effectively reduce the side effects of ICI treatment has become the core issue of this treatment. At present, immunohistochemistry is used clinically to quantify the expression of PD-L1 as one of the means to screen patients.

[0003] However, the above screening method has the problem of possible trauma caused by puncture sampling, and the site taken may lead to false negative results due to the heterogeneity of the tumor; on the other hand, the clinical benefits of PD-1 monoclonal antibody treatment are also seen in patients with PD-L1-negative tumors, which suggests that only evaluating PD-L1 is not enough for patient screening. Therefore, the present invention determines the research object as another ligand of PD-1, PD-L2. PD-L2 is one of the important ligands in the PD-1 signaling pathway. It is the second important ligand found to bind to PD-1 after PD-L1. It is located on chromosome 9 and the encoded protein can bind to PD-1, thereby inhibiting the function of immune cells. This is the main biological characteristic of PD-L2. Compared with PD-L1, the affinity of PD-L2 binding to PD-1 is about 2-6 times higher, which means that PD-1 may be more inclined to bind to PD-L2. At the same time, in multiple cancers such as colorectal cancer, esophageal cancer, pancreatic cancer and renal cell carcinoma, the expression of PD-L2 has been reported as an independent marker for poor prognosis. Combined with ICI treatment, it was found that the response rate of patients with both PD-L1 and PD-L2 positive (27.5%) was greater than that of patients with only PD-L1 positive (11.4%), and immunohistochemistry results showed that there was a certain correlation between the expression of PD-L1 and PD-L2 (R 2 =0.3930, P<0001). In addition, studies have confirmed that the therapeutic effect of PD-L1 monoclonal antibody is related to PD-L2 expression. The possible reason is that after blocking the binding of PD-L1 and PD-1, PD-1 can bind to its ligand PD-L2, and immune escape still exists. Therefore, PD-L2 is expected to be used as a biomarker for screening potential beneficiaries of ICI treatment and evaluating the efficacy.

[0004] Single-chain antibody (scFv) is a small molecule composed of the variable region of the heavy chain and the variable region of the light chain of an antibody connected by a peptide chain. It is the smallest functional structural unit with antibody activity. The molecular weight of scFv is about 28kDa. The antibody heavy chain variable region (VH) and the light chain variable region (VL) are connected into a recombinant gene through an artificially synthesized linker gene. The antibody expressed by the recombinant gene is called scFv. Structurally, the N-terminus of the heavy chain can be connected to the C-terminus of the light chain, or the N-terminus of the light chain can be connected to the C-terminus of the heavy chain. The advantages of scFv as a tracer are small molecular weight, strong penetration, short half-life, and low immunogenicity. Summary of the invention

[0005] The purpose of the present invention is to provide a single-chain antibody E6scFv targeting PD-L2 and an engineered antibody fragment E6scFv-hFc prepared by screening with phage library display technology, and to provide a radioactive tracer labeled with a radionuclide and a preparation method and application thereof. The radionuclide-labeled antibody fragment-type PET tracer targeting PD-L2 provided by the present invention has stable in vitro and in vivo properties, good imaging effect, and high affinity and specificity for PD-L2. Preclinical evaluation shows that this series of tracers is expected to become a clinical imaging agent for evaluating tumor PD-L2 expression.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides an immune checkpoint PD-L2 targeting single-chain antibody, wherein the single-chain antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO: 2.

[0007] According to a preferred embodiment, a linker is further provided between the heavy chain variable region (VH) and the light chain variable region (VL). Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 3, and the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO: 4.

[0008] In order to further increase its in vivo stability as a tracer and its ability to bind to the target, the present invention further incorporates a crystallizable fragment (Fc) of human IgG1 and modifies it into a bivalent scFv-hFc antibody fragment. Specifically, the second aspect of the present invention provides an immune checkpoint PD-L2 targeted engineered antibody fragment E6scFv-hFc, wherein the engineered antibody fragment is a fusion protein of the above-mentioned single-chain antibody and the hFc fragment, and preferably, the amino acid sequence of the hFc fragment is shown in SEQ ID NO: 5.

[0009] According to a more specific embodiment of the present invention, the engineered antibody fragment scFv-hFc is a bivalent antibody, and the sequence of each chain is shown in SEQ ID NO:6.

[0010] The present invention provides a PD-L2 targeted engineered antibody fragment E6scFv-hFc, which is based on a human single-chain antibody (scFv) library, an E6scFv sequence screened by phage library display technology, integrated with the hFc fragment sequence of IgG1, expressed in HEK293 cells by transfection, and then purified by Protein A.

[0011] The third aspect of the present invention provides a DNA fragment encoding the above-mentioned single-chain antibody or the above-mentioned engineered antibody fragment.

[0012] Specifically, the DNA sequence encoding the heavy chain variable region (VH) is shown in SEQ ID NO:7, and the DNA sequence encoding the light chain variable region (VL) is shown in SEQ ID NO:8.

[0013] According to a specific embodiment of the present invention, the DNA sequence encoding the linker is shown in SEQ ID NO: 9, and the sequence of the DNA fragment encoding the single-chain antibody is shown in SEQ ID NO: 10.

[0014] According to a specific embodiment of the present invention, the DNA sequence encoding the hFc fragment is shown in SEQ ID NO: 11, and the sequence encoding the engineered antibody fragment is shown in SEQ ID NO: 12.

[0015] The fourth aspect of the present invention provides a radionuclide-labeled engineered antibody fragment, wherein the engineered antibody fragment is the engineered antibody fragment described above.

[0016] The present invention provides a radionuclide-labeled antibody fragment E6scFv-hFc. The radionuclide-labeled E6scFv-hFc is obtained by subjecting the antibody fragment E6scFv-hFc to N-bromosuccinimide-mediated or bifunctional chelating agent coupling and then radionuclide labeling.

[0017] In the present invention, the radioactive nuclide is preferably a radioactive isotope of iodine, 68 Ga, 64 Cu, 89 Zr, 18 F. 177 Lu, 225 Ac, 86 Y. 90 Y. 111 In, 11 C. 211 At 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 213 Bi, 212 Bihe 99m At least one of Tc, the radioactive isotope of iodine is preferably 124 I. 123 I. 125 I or 131 I.

[0018] The bifunctional chelating agent includes, but is not limited to, at least one of NOTA, DOTA, DFO, RESCA, DTPA, THP, HYNIC and SHNH.

[0019] The fifth aspect of the present invention provides the use of the above-mentioned radionuclide-labeled engineered antibody fragment in the preparation of a PET / CT molecular diagnostic imaging agent targeting PD-L2.

[0020] The sixth aspect of the present invention provides a diagnostic or therapeutic radiopharmaceutical targeting PD-L2, the drug comprising the engineered antibody fragment labeled with the above-mentioned radionuclide. When the labeled nuclide is a radionuclide for imaging, the drug is a diagnostic radiopharmaceutical, and when the labeled nuclide is a therapeutic radionuclide, the drug is a therapeutic radiopharmaceutical.

[0021] The specific labeling method can adopt various radionuclide labeling methods known in the art.

[0022] The present invention provides a method for preparing the radionuclide-labeled antibody fragment E6scFv-hFc. The method can be divided into two parallel technical schemes according to the different nuclides used.

[0023] for 64 Cu or 89 The Zr-labeled antibody fragment targeting PD-L2 can be prepared according to the following steps:

[0024] (1) mixing E6scFv-hFc and a bifunctional chelating agent to obtain a labeled precursor, wherein the molar ratio of E6scFv-hFc to the bifunctional chelating agent is 1:(8-10);

[0025] (2) Adoption 64 Cu or 89 Zr performs a labeling reaction on the labeling precursor, and the labeling precursor 64 Cu or 89 The dosage ratio of Zr is (0.1-0.3 mg): (6×10 7 -3×10 8 Bq).

[0026] When radioactive isotope of iodine is used for labeling, the preparation method comprises: mixing E6scFv-hFc with a salt of a radioactive isotope of iodine, N-bromosuccinimide and a reaction buffer for labeling reaction; the usage ratio of E6scFv-hFc, the salt of a radioactive isotope of iodine and N-bromosuccinimide is (0.1-0.3 mg):(60-90 KBq / μL):(20-100 μg).

[0027] According to a more specific embodiment, the preparation method comprises:

[0028] 0.5-1.0 mL of 60-90 KBq / μL Na 124 I solution was added with an appropriate amount of 0.1M pH=7.2 PB buffer and 0.1-0.3mg antibody fragment E6scFv-hFc, and then 20-100μg N-bromosuccinimide was added to react at room temperature for 1min. After the reaction was completed, 0.1-0.3mL 10% human serum albumin was added to the reaction system to terminate the reaction; the obtained reaction solution was purified by PD-10 column to obtain 124 I-labeled antibody fragment 124 I-E6scFv-hFc.

[0029] For other radioactive isotopes of iodine, such as 123 I. 125 I. or 131 I, the salt used is 123 I salt, 125 I salt, or 131 I. Salt.

[0030] The present invention screens E6scFv-hFc through phage library display technology and performs eukaryotic expression through mammalian cells (HEK293), and further radiolabels it to obtain a novel radionuclide probe targeting PD-L2. The probe is stable in vitro and in vivo, easy to prepare, and has good affinity for PD-L2. In vivo micro-PET / CT imaging shows that the probe can effectively detect tumors with high PD-L2 expression, which indicates that it has the potential to develop into a powerful tool for non-invasive quantification of tumor PD-L2 expression, providing a new strategy for screening potential beneficiaries of ICI treatment and evaluating the efficacy.

[0031] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0033] Figure 1a -b:E6scFv( Figure 1a ) and E6scFv-hFc( Figure 1b )’s MALDI-TOF-MS results.

[0034] Figure 2a -b: SEC-HPLC of E6scFv-hFc ( Figure 2a ) and SPR affinity experiment results ( Figure 2b ).

[0035] Figure 3a -b: After purification by PD-10 column 89 Zr-DFO-E6scFv-hFc( Figure 3a )and 124 I-E6scFv-hFc( Figure 3b )'s radiochemical purity analysis results.

[0036] Figure 4a -b: 89 Zr-DFO-E6scFv-hFc( Figure 4a )and 124 I-E6scFv-hFc( Figure 4b ) of the cellular uptake experiment.

[0037] Figure 5a -b: 89 Zr-DFO-E6scFv-hFc( Figure 5a )and 124 I-E6scFv-hFc( Figure 5b ) Biodistribution results in normal Kunming mice.

[0038] Figure 6a -b: 89 Zr-DFO-E6scFv-hFc( Figure 6a )and 124 I-E6scFv-hFc( Figure 6b ) micro-PET / CT imaging results in tumor-bearing mice.

[0039] Figure 7 : Process diagram of radionuclide labeling of the engineered antibody fragment E6scFv-hFc targeting PD-L2 of the present invention and imaging results of its effect in mice. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0041] The amino acid and DNA sequence information involved in the present invention are shown in Table 1 and Table 2 respectively:

[0042] Table 1 Amino acid sequence

[0043]

[0044]

[0045] Table 2 DNA sequences

[0046]

[0047]

[0048]

[0049] Example 1: Screening and preparation of E6scFv-hFc

[0050] 1. Construction of scFv phage library

[0051] The phage library was commissioned by Wuhan Dai'an Biological Company to build. The steps of library construction include: collecting human peripheral blood or lymphocytes to extract lymphocytes, extracting mRNA, reverse transcription to obtain cDNA, designing suitable primers, using cDNA as a template to amplify VH and VL genes and cut gel recovery. The vector and VL fragments are double-digested and recovered, and the competent cells are electrotransformed, and then DNA sequence analysis (diversity analysis) is performed. The vector and VH fragments are double-digested and recovered, and the heavy chain T4 is connected and the product is purified. After electrotransformation of competent cells, DNA sequence analysis is performed, and the phage antibody library preparation is finally completed.

[0052] 2. Antibody enrichment and selection

[0053] (1) The immunotube was coated with antigen (PD-L2 standard protein) at 20-30 μg / tube, supplemented with 0.1 M NaHCO3 to 2 mL, and incubated at 37°C for 2 h;

[0054] (2) After discarding the antigen coating solution, wash three times with PBST, add 5 mL of 4% skim milk for blocking, and incubate at 37°C for 1.5 h;

[0055] (3) After discarding the 4% skim milk, wash three times with PBST. Add 5×10 12 ~10×10 12 The phages were washed, shaken at room temperature for 30 min, and allowed to stand at 37°C for 1.5 h;

[0056] (4) After discarding the phage, wash 10 times with PBST;

[0057] (5) Add 1 mL of Gly-HCl (pH 2.0), shake at room temperature for 8 min, and add 200 μL of Tris-HCl (pH 9.5) to neutralize, which is used as the eluent;

[0058] (6) Measure the elution titer and transform 700 μL of phage from the elution solution into 1 mL of TG1 and spread on three large plates for amplification;

[0059] (7) Scrape off the bacteria from the three large plates and save them. Take 1 mL for amplification. The titer should be greater than 10 12 pfu / mL;

[0060] (8) Repeat the above steps 3-5 times.

[0061] 3. Screening of positive clones by ELISA

[0062] (1) Phage preparation

[0063] (1-1) Randomly pick a single colony from the SOBAG plate and inoculate it into a 96-well bacterial culture plate. Add 200 μL 2YT-AG to each well and culture at 37°C overnight.

[0064] (1-2) Pipette 25 μL of bacterial solution into a new bacterial culture plate, add 175 μL of 2YT-AG medium, and culture at 37°C for 3 h;

[0065] (1-3) Centrifuge at 3500 rpm for 10 min, remove the supernatant, resuspend the bacterial pellet in 200 μL 2YT-AI (100 μg / mL Amp, 1 mM IPTG) medium, and induce at 30°C overnight;

[0066] (1-4) Centrifuge at 3500 rpm for 10 min and store the supernatant at 4°C for later use.

[0067] (2) Indirect ELISA screening of positive clones

[0068] (2-1) Add the antigen protein expressed by the purified 293 cells to the ELISA plate, coat it at 4°C overnight, and set the ELISA-labeled well as the positive well; add an equal amount of BSA to the ELISA plate, coat it at 4°C overnight, and set the ELISA-labeled well as the negative control well;

[0069] (2-2) After discarding the coating solution, wash with PBS three times and block with 4% PBSM (PBS containing 4% skim milk) for 1 h. After washing with PBS once, add 50 μL of the prepared single-chain antibody supernatant and 50 μL of 4% PBSM to each well and react at 37°C for 1 h.

[0070] (2-3) After washing three times with PBS and PBST, 100 μL of anti-M13 phage / HRP conjugation (diluted 1:5000 in 4% PBSM) was added to each well and incubated at 37°C for 1 h;

[0071] (2-4) Wash three times with PBST and PBS, add 100 μL TMB substrate solution, react for 15 min in the dark, add 25 μL 2 mol / L H2SO4 to terminate the reaction, and measure the OD450nm value with a microplate reader. Calculate the OD450nm positive well reading value / OD450nm negative well reading value, and select the phage with a high ratio as the positive clone; amplify the screened positive ScFv phage to obtain a sufficient number for subsequent experiments.

[0072] 4. Expression and purification of scFv-hFc protein

[0073] (1) Extracting the screened positive ScFv phage DNA, performing PCR amplification, and obtaining the display sequence; then performing Sanger sequencing on the PCR product and analyzing the sequencing results, and finally identifying and determining the scFv sequence (VH+linker+VL);

[0074] (2) The ScFv gene was fused with the hFc fragment gene (CH2+CH3) of human IgG1 to obtain the target gene (scFv-hFc), which was then amplified by PCR and inserted into the corresponding expression vector. The sequence of the constructed vector was sequenced to verify that the correct plasmid was amplified;

[0075] (3) Mix the plasmid and transfection reagent in an optimal ratio and add to the bioreactor containing HEK293. The cells are cultured in serum-free medium and maintained in an orbital shaker or conical flask on a bioreactor at 37°C and an appropriate stirring speed for 6 days;

[0076] (4) After centrifuging the cell culture medium, the supernatant is loaded onto an affinity purification column at an appropriate flow rate to purify the target antibody, and the purified antibody is subjected to quality control analysis by SDS-PAGE and SEC-HPLC.

[0077] MALDI-TOF-MS of E6scFv and E6scFv-hFc were as follows Figure 1a and Figure 1b As shown, SEC-HPLC of E6scFv-hFc is as follows Figure 2a As shown. The results of SEC-HPLC of E6scFv-hFc showed that after HEK293 cell expression and Protein A purification, the purity of the antibody fragment exceeded 90%. The results of MALDI-TOF-MS and SDS-PAGE showed that the average molecular weight of E6scFv-hFc was about 103.03 kDa, which was consistent with the theoretical molecular weight value. It can be seen that based on the above method, the engineered antibody fragment E6scFv-hFc with high specificity targeting PD-L2 was successfully prepared. The affinity of E6scFv-hFc for PD-L2 protein was detected by SPR, and the results are shown in Figure 2b As shown in Figure 2, the antibody fragment has a high affinity for PD-L2 protein (KD = 1.03 × 10 -9 M,Ka=6.23×10 5 M -1 ·S -1 , Kd=6.44×10 -4 1 / s).

[0078] Embodiment 2: 89 Zr-DFO-E6scFv-hFc / 124 Preparation of I-E6scFv-hFc

[0079] 89 Preparation of Zr-DFO-E6scFv-hFc: Add DFO equivalent to about 8-10 times the molar equivalent of E6scFv-hFc to a solution of the antibody fragment E6scFv-hFc at a concentration of 3 mg / mL, adjust the pH value of the reaction system to 8.5-9.0 with a buffer (Na2CO3 / NaHCO3); react at 37°C for 0.5-1h to obtain the labeled precursor DFO-E6scFv-hFc;

[0080] Take 1.0-1.5mCi 89 The Zr solution was mixed with mixed A (0.25M HEPES and 2M Na2CO3, volume ratio of 10:1), and pH = 7 was adjusted by 1M oxalic acid. Then 100-300μg of DFO-E6scFv-hFc precursor was added to the above solution and reacted at 37°C for 1h, mixed up and down 5-10 times every 10min, and reacted for half an hour for intermediate quality control. When the labeling rate is greater than 90%, it was separated and purified with a PD-10 column, and the PD-10 column needs to be activated with 25mL 0.01M PBS for standby use. After the sample solution is loaded on the column, 0.01M PBS is used to elute the target compound 89 Zr-DFO-E6scFv-hFc.

[0081] 124 Preparation of I-E6scFv-hFc: 0.5-1.0 mL of 60-90 KBq / μL Na 124 I solution was added with an appropriate amount of 0.1M pH=7.2 PB buffer and 0.1-0.3mg antibody fragment E6scFv-hFc, and then 20-100μg N-bromosuccinimide was added to react at room temperature for 1min. After the reaction was completed, 0.1-0.3mL 10% human serum albumin was added to the reaction system to terminate the reaction; the obtained reaction solution was purified by PD-10 column to obtain 124 I-labeled antibody fragment 124 I-E6scFv-hFc.

[0082] The labeling rate and radiochemical purity were determined by Radio-TLC. The labeling rate was determined by radio-thin layer chromatography (Radio-TLC) analysis. The analysis method was: 20 μL saturated EDTA and 5 μL 89Mix the Zr-DFO-E6scFv-hFc reaction solution, take 2 μL and spot it on the silica gel-stained glass fiber strip, 1 cm from the bottom, and develop it to the upper 1 / 3 with 0.5 M citric acid / sodium citrate developer (pH = 5.0) for radio-TLC quality control. 124 I-E6scFv-hFc was developed with organic solvent acetone, and the spotting operation was the same as above. The results showed: 89 Zr-DFO-E6scFv-hFc and 124 The labeling rates of I-E6scFv-hFc were all greater than 85%; Figure 3a and Figure 3b As shown, the radiochemical purity after purification was greater than 99%. In addition, the labeled product could maintain high in vitro stability in 0.01M PBS and 5% HSA.

[0083] Embodiment 3: 89 Zr-DFO-E6scFv-hFc and 124 In vitro cellular uptake experiment of I-E6scFv-hFc

[0084] 1×10 6 cells (A549 / A549-PDL2), cultured overnight and ensured that the cells adhered to the wall; 60 μCi 89 Zr-DFO-E6scFv-hFc / 124 I-E6scFv-hFc was diluted to 15 mL (single plate) with serum-free 1640 medium, 50 μg E6scFv-hFc was added to each well of the block group, and 2 μCi / 500 μL of 89 Zr-DFO-E6scFv-hFc / 124 After I-E6scFv-hFc, the cells were incubated at 37°C for 30 min, 60 min, 120 min (120 min block), and 240 min (240 min block) to terminate the reaction. The culture medium was aspirated, and 1 mL × 2 of PBS was used to wash the cells. Then, 200 μL of NaOH solution was added to digest the cells. After the last time point, the NaOH in the well plate was aspirated into the γ-Counter tube, and a small amount of PBS was aspirated to wash the well plate, and the well plate was added to the counting tube, and the well plate was measured (5 μL of the dilution solution was taken as the marker). The results are shown in Figure 2. Figure 4a and Figure 4b As shown. It can be seen that 89 Zr-DFO-E6scFv-hFc and 124 I-E6scFv-hFc was significantly highly taken up in positive cells A549-PDL2.

[0085] Embodiment 4: 89 Zr-DFO-E6scFv-hFc and 124 Biodistribution of I-E6scFv-hFc in normal Kunming mice

[0086] Fifteen normal female Kunming mice (n=3) aged 5 weeks were injected with 0.2 mL of 89 Zr-DFO-E6scFv-hFc (0.37MBq) was injected and the animals were killed by cervical dislocation 2, 8, 24, 48 and 96 hours after injection. The blood, heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, muscle, bone, brain and other related tissues and organs were collected, cleaned and weighed, and the radioactivity count was measured with a γ-Counter to calculate the percentage of injected dose per gram of tissue, recorded as %ID / g. The results are shown in Figure 5a As shown, 89 Zr-DFO-E6scFv-hFc is relatively slowly cleared from the blood and is mainly metabolized by the liver. Its uptake in other major organs such as the heart, spleen, and brain is low. Compared with monoclonal antibodies (whole antibodies), it is metabolized faster and is more suitable as an in vivo tracer. 124 The experimental steps and statistical methods of I-E6scFv-hFc were the same as above, and the results were as follows Figure 5b shown. 89 Zr-DFO-E6scFv-hFc and 124 The biodistribution results of I-E6scFv-hFc in normal Kunming mice showed that the radiotracer was mainly cleared through the liver.

[0087] Embodiment 5: 89 Zr-DFO-E6scFv-hFc and 124 Micro-PET / CT imaging of I-E6scFv-hFc in tumor-bearing mice picture

[0088] Female 5-week-old Balbc / nude mice were injected subaxillary with 1 × 10 7 Tumor cells (A549 / A549-PDL2) were used to construct a tumor-bearing mouse model. 0.2 mL of 89 Zr-DFO-E6scFv-hFc and 124 I-E6scFv-hFc (about 3.7MBq / mouse) was injected and Micro-PET / CT imaging was performed at 2 hours, 8 hours, 24 hours, 48 ​​hours and 96 hours after injection. Before imaging, the mice were anesthetized with oxygen mixed with 3% (volume fraction) isoflurane. During imaging, oxygen anesthesia containing 1.5% (volume fraction) isoflurane was maintained. Micro-PET / CT used a 10-minute static scan. The imaging results are shown in Figure 6a and Figure 6b The results showed that compared with the negative model A549 group, the positive model A549-PDL2 group had a high specific uptake in the tumor region, indicating that the above tracer has a good detection ability for tumors with high PD-L2 expression.

[0089] like Figure 7As shown, the present invention provides a PD-L2-targeted single-chain antibody E6scFv and an engineered antibody fragment E6scFv-hFc prepared by screening through phage library display technology, and a 89 Zr and 124 I-labeled radiotracer targeting PD-L2 89 Zr-DFO-E6scFv-hFc and 124 I-E6scFv-hFc. This series of tracers is easy to prepare, has good stability, has high affinity and specificity for PD-L2, and has good detection ability for tumors with high PD-L2 expression. Preclinical in vivo and in vitro evaluations have confirmed that it can be used as a PET / CT clinical tracer for non-invasive quantification of PD-L2 by molecular imaging.

[0090] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An immune checkpoint PD-L2 targeting single-chain antibody, characterized in that: The single-chain antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:

2.

2. The immune checkpoint PD-L2 targeting single-chain antibody according to claim 1, wherein: A linker is also provided between the heavy chain variable region (VH) and the light chain variable region (VL).

3. The immune checkpoint PD-L2 targeting single-chain antibody according to claim 2, wherein: The amino acid sequence of the linker is shown in SEQ ID NO: 3, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO:

4.

4. An immune checkpoint PD-L2 targeted engineered antibody fragment, characterized in that: The engineered antibody fragment is a fusion protein of the single-chain antibody according to any one of claims 1 to 3 and an hFc fragment.

5. The immune checkpoint PD-L2 targeting engineered antibody fragment according to claim 4, wherein: The amino acid sequence of the hFc fragment is shown in SEQ ID NO:

5.

6. The immune checkpoint PD-L2 targeting engineered antibody fragment according to claim 4, wherein: The engineered antibody fragment is a bivalent antibody, and the sequence of each chain is shown in SEQ ID NO:

6.

7. A DNA fragment encoding the single-chain antibody according to any one of claims 1 to 3 or the engineered antibody fragment according to any one of claims 4 to 6.

8. A radionuclide-labeled engineered antibody fragment, characterized in that: The engineered antibody fragment is the engineered antibody fragment according to any one of claims 4-6.

9. The radionuclide-labeled engineered antibody fragment according to claim 8, wherein: The radionuclide-labeled engineered antibody fragment is obtained by radioactively labeling the engineered antibody fragment mediated by N-bromosuccinimide or coupled with a bifunctional chelating agent.

10. The radionuclide-labeled engineered antibody fragment according to claim 9, wherein: The bifunctional chelating agent is at least one of NOTA, DOTA, DFO, RESCA, DTPA, THP, HYNIC and SHNH.

11. The radionuclide-labeled engineered antibody fragment according to claim 8, wherein: The radioactive nuclide is a radioactive isotope of iodine, 68 Ga, 64 Cu, 89 Zr, 18 F. 177 Lu, 225 Ac, 86 Y. 90 Y. 111 In, 11 C. 211 At 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 213 Bi, 212 Bihe 99m At least one of Tc.

12. The radionuclide-labeled engineered antibody fragment according to claim 11, wherein: The radioactive isotope of iodine is 124 I. 123 I. 125 I or 131 I.

13. Use of the radionuclide-labeled engineered antibody fragment according to any one of claims 8 to 12 in the preparation of a PET / CT molecular diagnostic imaging agent targeting PD-L2.

14. A diagnostic or therapeutic radiopharmaceutical targeting PD-L2, characterized in that: An engineered antibody fragment labeled with a radionuclide according to any one of claims 8 to 12.

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