Application of disulfide bond isomerase single-domain antibody in preparation of product for treating and / or preventing leukemia

Abstract

The invention discloses an application of a disulfide bond isomerase single-domain antibody in preparation of a product for treating and / or preventing leukemia, the disulfide bond isomerase single-domain antibody comprises a variable region, and the variable region comprises CDR1, CDR2 and CDR3; the amino acid sequences of CDR1-CDR3 of the disulfide bond isomerase single-domain antibody are sequentially as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and the CDR1-CDR3 are defined according to an IMGT definition scheme. The single-domain antibody provided by the invention can be used for remarkably inhibiting the growth of subcutaneous transplanted tumors of Ba / F3BCR-ABLT315I cells in nude mice, and has an obvious inhibiting effect on drug-resistant chronic granulocytic leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of antibody technology, and specifically relates to the use of disulfide isomerase single-domain antibodies in the preparation of products for treating and / or preventing leukemia. Background Art

[0002] The protein disulfide isomerase (PDI) family is a class of oxidoreductases widely distributed in the endoplasmic reticulum (ER) of mammalian cells. Their core function is to regulate the correct folding of proteins by catalyzing the formation, cleavage, and rearrangement of disulfide bonds within and between proteins. This family includes members such as PDI, ERp57, ERp5, and ERp72. PDI, the first member of the family to be identified, possesses typical structural features and catalytic activity. While primarily localized in the ER, small numbers of PDIs are also present in the nucleus, cytoplasm, mitochondria, plasma membrane, and extracellular space.

[0003] PDI is significantly associated with malignant tumor progression and is a potential drug target for malignant tumor treatment. PDI expression is significantly increased in various tumor tissues, such as brain, kidney, prostate, lung, and breast cancers, and is associated with the invasion and metastasis of certain malignancies. The precise mechanism by which PDI promotes tumor progression remains unclear, but PDI is clearly involved in the unfolded protein response (UPR) pathway. The UPR alleviates endoplasmic reticulum (ER) stress by activating ER chaperones, such as PDI, which refolds misfolded proteins and promotes cancer cell survival. Conversely, inhibition of PDI leads to persistent UPR activation, exacerbating ER stress and ultimately triggering tumor cell apoptosis. PDI is secreted by tumor cells into the microenvironment and subsequently into the circulation. Altered PDI levels in these peripheral spaces, such as interstitial fluid and plasma, can serve as biomarkers for early cancer diagnosis. Consequently, PDI inhibitors have become an emerging strategy for targeted tumor therapy. Due to their unique single domain characteristics, single-domain antibodies are easier to design to target specific targets, such as coagulation factors or receptors on the platelet surface, which can reduce off-target effects and improve the safety of treatment. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of a disulfide isomerase single domain antibody in the preparation of a product for treating and / or preventing leukemia, wherein the disulfide isomerase single domain antibody can significantly inhibit Ba / F3 BCR-ABL in nude mice. T315IThe cell subcutaneous transplanted tumor growth has a significant inhibitory effect on drug-resistant chronic myeloid leukemia.

[0005] The present invention also provides the disulfide isomerase single domain antibody for the above application.

[0006] The invention also provides a recombinant protein.

[0007] The present invention also provides a biomaterial related to the above-mentioned disulfide isomerase single-domain antibody or the above-mentioned recombinant protein.

[0008] The present invention also provides a conjugate.

[0009] The present invention also provides a solid phase carrier.

[0010] The present invention also provides an application.

[0011] The present invention also provides a product.

[0012] The present invention also provides a method for preparing the above-mentioned disulfide isomerase single-domain antibody or the above-mentioned recombinant protein.

[0013] According to a first aspect of the present invention, a disulfide isomerase single domain antibody is used in the preparation of a product for treating and / or preventing leukemia, wherein the disulfide isomerase single domain antibody comprises a variable region, wherein the variable region comprises CDR1, CDR2 and CDR3;

[0014] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the CDR1 to CDR3 are defined according to the IMGT definition scheme; or

[0015] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Kabat definition scheme; or

[0016] The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Chothia definition scheme; or

[0017] The amino acid sequences of CDR1-CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively, which are defined according to the Contact definition scheme.

[0018] In some embodiments of the present application, the amino acid sequence of the disulfide isomerase single domain antibody comprises:

[0019] a1) SEQ ID NO: 14; or

[0020] a2) an amino acid sequence obtained by substitution, deletion and / or addition of one or several amino acids of SEQ ID NO: 14 and having the same function as the protein shown in SEQ ID NO: 14; or

[0021] a3) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology with SEQ ID NO: 14 and having the same function as the protein shown in SEQ ID NO: 14.

[0022] In some embodiments of the present application, the disulfide isomerase single domain antibody further comprises a constant region.

[0023] In some embodiments of the present application, the product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip.

[0024] In some embodiments of the present application, the leukemia is chronic leukemia; the chronic leukemia comprises at least one of chronic myelocytic leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia.

[0025] In some embodiments of the present application, the leukemia is drug-resistant chronic myelocytic leukemia.

[0026] According to a second aspect of the present application, a disulfide isomerase single domain antibody used in the application of the first aspect of the present application is provided.

[0027] According to a third aspect of the present application, a recombinant protein is provided, comprising: a disulfide isomerase single domain antibody according to the second aspect of the present application; and

[0028] An optional tag sequence assisting expression and / or purification.

[0029] In some embodiments of the present application, the tag sequence is selected from at least one of the following group: His tag, GGGS sequence, FLAG tag.

[0030] According to a fourth aspect of the present application, a biological material related to the disulfide bond isomerase single domain antibody of the second aspect of the present application, or the recombinant protein of the third aspect of the present application is provided, and the biological material comprises at least one of h1) to h8):

[0031] h1) a nucleic acid molecule encoding the disulfide bond isomerase single domain antibody of the second aspect of the present application, or the recombinant protein of the third aspect of the present application;

[0032] h2) an expression cassette comprising the nucleic acid molecule of h1);

[0033] h3) a vector comprising the nucleic acid molecule of h1);

[0034] h4) a vector comprising the expression cassette of h2);

[0035] h5) a transgenic cell line comprising the nucleic acid molecule of h1);

[0036] h6) a transgenic cell line comprising the expression cassette of h2);

[0037] h7) a transgenic cell line comprising the vector of h3);

[0038] h8) a transgenic cell line comprising the vector of h4).

[0039] In some embodiments of the present application, the transgenic cell line does not comprise reproductive material.

[0040] In some embodiments of the present application, the nucleic acid molecule encoding the disulfide bond isomerase single domain antibody of the second aspect of the present application comprises a nucleic acid molecule encoding the variable region of the disulfide bond isomerase single domain antibody of the second aspect of the present application.

[0041] According to a fifth aspect of the present application, a conjugate is provided, comprising: at least one of the disulfide bond isomerase single domain antibody of the second aspect of the present application, and the recombinant protein of the third aspect of the present application;

[0042] and a coupling moiety, the coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron-dense marker, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein.

[0043] In some embodiments of the present application, the detectable label is a fluorescent or luminescent label.

[0044] In some preferred embodiments of the present invention, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase.

[0045] In some embodiments of the present invention, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 and Re-188.

[0046] In some embodiments of the present invention, the drug is other drugs for preventing or treating leukemia (such as imatinib, nilotinib, dasatinib, ponatinib, etc.).

[0047] In some embodiments of the present invention, the leukemia is chronic leukemia.

[0048] In some embodiments of the present invention, the chronic leukemia includes at least one of chronic myeloid leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia.

[0049] In some embodiments of the present invention, the leukemia is drug-resistant chronic myeloid leukemia.

[0050] According to a sixth aspect of the present invention, a product is provided, comprising at least one of k1) to k3):

[0051] k1) the disulfide isomerase single domain antibody according to the second aspect of the present invention;

[0052] k2) the recombinant protein according to the third aspect of the present invention;

[0053] k3) The conjugate according to the fifth aspect of the present invention.

[0054] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0055] In some preferred embodiments of the present invention, the drug has the function of treating and / or preventing chronic leukemia.

[0056] In some embodiments of the present invention, the chronic leukemia includes at least one of chronic myeloid leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia.

[0057] In some embodiments of the present invention, the leukemia is drug-resistant chronic myeloid leukemia.

[0058] According to the seventh aspect of the present invention, a method for preparing the disulfide isomerase single domain antibody as described in the second aspect of the present invention or the recombinant protein as described in the third aspect of the present invention is proposed, which is obtained by culturing the transgenic cell line described in the fourth aspect of the present invention.

[0059] The present invention has at least the following beneficial effects:

[0060] The present invention screened an anti-PDI single-domain antibody using phage display technology and validated its efficacy against drug-resistant chronic myeloid leukemia. In a drug-resistant chronic myeloid leukemia model, the PDI single-domain antibody provided by the present invention significantly inhibited the growth of nude mouse xenograft tumors, with Western blot results demonstrating downregulation of PDI expression in tumor tissue. This invention provides key experimental evidence for the therapeutic strategy of anti-PDI single-domain antibodies in drug-resistant chronic myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0062] Figure 1 This is a flowchart of the experiment for screening single domain antibodies using phage display technology in Example 1 of the present invention;

[0063] Figure 2 Figure 1 is a diagram showing the identification results of the purified PDI protein in Example 1 of the present invention; wherein A is SDS-PAGE analysis and B is Western blot detection;

[0064] Figure 3 Graph showing the specific phage enrichment results in Example 1 of the present invention; wherein A represents the phage titer determination, and B represents the enrichment results after three rounds of screening;

[0065] Figure 4 This is a diagram showing the screening results of positive monoclonal clones screened by phage ELISA in Example 1 of the present invention;

[0066] Figure 5 The figure is a graph showing the agarose gel electrophoresis results of the positive monoclonal phage in Example 1 of the present invention; wherein, lane M is a marker, and lanes 1 to 9 are bands of PCR products of the positive monoclonal phage colony;

[0067] Figure 6 This is an amino acid sequence alignment analysis diagram in Example 1 of the present invention;

[0068] Figure 7 This is a comparison diagram of the CDRs of the sequences analyzed by the IMGT website in Example 1 of the present invention;

[0069] Figure 8 This is a diagram showing the electrophoresis identification results of the PCR products of the target gene in Example 1 of the present invention;

[0070] Figure 9 This is the plasmid spectrum of SdAb in Example 1 of the present invention;

[0071] Figure 10 This is a diagram showing the electrophoresis identification results of the positive colony PCR products in Example 1 of the present invention;

[0072] Figure 11 Graph showing the expression verification results of the PDI single-domain antibody in Example 1 of the present invention; wherein, M is a marker, lane 1 represents bacteria collected under 16°C induction conditions, lane 3 represents the supernatant after ultrasonic disruption of bacteria at 16°C and centrifugation, lane 4 represents the precipitate after ultrasonic disruption of bacteria at 16°C and centrifugation, lane 5 represents the supernatant after ultrasonic disruption of bacteria at 30°C and centrifugation, and lane 6 represents the precipitate after ultrasonic disruption of bacteria at 30°C and centrifugation;

[0073] Figure 12 Figure 1 is a diagram showing the purification and identification results of the PDI single domain antibody in Example 1 of the present invention; wherein, A is the verification result of the elution of the target protein, lane M is a marker, lane 1 is the supernatant, lane 2 is the flow-through, lanes 3 to 7 are 50 to 100 mM imidazole elution of contaminants, and lanes 8 to 10 are 500 mM imidazole elution of the target protein; B is a diagram showing the results of Western blot identification of the single domain antibody;

[0074] Figure 13 Figures 1 and 2 show the results of antigen-binding ability verification of the PDI single-domain antibody in Example 1 of the present invention; wherein, A is the SDS-PAGE result, lane M is a marker, lane 1 is 10 μg PDI protein loading, and lane 2 is 20 μg PDI protein loading; B is the Western blot result, showing the bands of PDI protein after incubation with PDI single-domain antibody and HRP-protein A and ECL development; C is the ELISA binding ability test, BSA, coated with 5% BSA; PDI, coated with PDI protein;

[0075] Figure 14 This is a graph showing the affinity between the PDI single domain antibody and the PDI protein measured using the Octet system in Example 2 of the present invention;

[0076] Figure 15 This figure shows the effect of 15 days of administration of the PDI single domain antibody in Example 3 of the present invention on the nude mouse xenograft tumor model; wherein A represents the change in nude mouse body weight, B represents the change in nude mouse tumor volume, C represents tumor morphology, and D represents tumor weight;

[0077] Figure 163 is a graph showing the detection results of the PDI protein expression level in tumor tissue after treatment with the PDI single domain antibody in Example 3 of the present invention; wherein A is a Western blot band and B is a quantitative analysis of the corresponding gray value. DETAILED DESCRIPTION

[0078] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0079] Example 1 Screening, expression and purification of disulfide isomerase single domain antibodies

[0080] In this example, a commercial phage display library was used to screen positive colonies with high affinity for PDI protein and sequenced to obtain positive monoclonal phage sequences. Subsequently, the PDI single domain antibody plasmid was constructed, expressed, purified, and identified. The experimental flow chart of phage display technology for screening single domain antibodies is shown in the figure. Figure 1 The specific methods and results are as follows:

[0081] 1. Screening of single domain antibodies

[0082] 1) Expression, purification and identification of PDI protein:

[0083] 1 μL of the preserved PDI recombinant plasmid (the plasmid backbone is pTriEx-4 Neo Vector, and the target gene fragment is the mRNA fragment of the 65-1591 region in the NCBI database NM_000918.4 (P4HB)) was transformed into BL21 (DE3) competent cells, and 200 μL of the bacterial solution was spread on LB solid medium containing antibiotics and incubated in a 37°C constant temperature incubator for 16 hours. The next day, a monoclonal colony with regular morphology was picked and inoculated into 5 mL of LB liquid medium, and after shaking culture at 37°C for 5 hours, it was transferred to 200 mL of fresh culture medium. When the OD 600 When the value reaches 0.6, add 1 mL of IPTG to a final concentration of 0.8 mM, and continue induction at 16°C with shaking for 6 hours. Collect the cells by centrifugation at 8,000 × g for 10 minutes at 4°C, wash with PBS, and repeat the centrifugation. Resuspend the cells in 20 mL of PBS and disrupt them by sonication on ice at 60% power, using a #6 horn, for 3 seconds on, 6 seconds off, for a total of 15 minutes. After disruption, centrifuge at 12,000 × g for 10 minutes, and collect the supernatant to obtain the crude PDI protein solution.

[0084] The His-tagged PDI fusion protein was purified by nickel column affinity chromatography, and the protein concentration of the PDI fusion protein was determined using a BCA protein quantification kit. The purified PDI fusion protein was then identified by SDS-PAGE and Western blot based on the protein concentration. The results are shown in Figure 2. Figure 2 shown.

[0085] Depend on Figure 2 It can be seen that the purified product presents a single target band in the range of 55-70kDa ( Figure 2 A), the target protein has a specific signal at the expected molecular weight position (theoretical value 57kDa) ( Figure 2 B) PDI protein was mainly present in the supernatant, indicating that it was efficiently expressed in a soluble form and its molecular weight was consistent with the theoretical value.

[0086] 2) Screening of antibody library using microplate screening method:

[0087] The helper phage M13 was co-incubated with a frozen antibody library (human domain antibody library (Dab library) purchased from Source Bioscience) to generate a phage display antibody library. The phage display antibody library was then incubated with PDI protein in a microplate for three rounds of "adsorption-elution-amplification" with TG1 as the receptor bacterium. The specific method is as follows:

[0088] ① Dilute PDI protein to 10 μg / mL in carbonate coating buffer. Add 100 μL per well to the ELISA plate and incubate overnight at 4°C (the antigen concentration for the next two rounds is 5 μg / mL). The next day, discard the coating buffer and wash the plate three times with 250 μL of PBS per well. Pat the plate dry each time to remove any remaining liquid. Then, add 200 μL of 5% BSA and block at 37°C for 2 h. Discard the blocking buffer and wash each well five times with 200 μL of PBST and once with 200 μL of PBS. Add 100 μL of the freshly prepared antibody library to each well and incubate at 37°C for 2 h. Discard any phage in the wells and add 200 μL of PBST per well. Wash the plate five times to remove any unbound phage (10 washes for the next two rounds). 100 μL of trypsin elution solution was added to each well and the well was allowed to stand at room temperature for 15 min, during which time the well was pipetted twice to elute the specifically bound phages to obtain the first round of specific phages.

[0089] ② Streak the recipient bacteria TG1 on an M9 plate and incubate at 37°C overnight. Pick a single TG1 colony and place it in 5 mL of 2×TY. After diluting it 100-fold, grow it at 37°C, 250 rpm overnight until the OD 600=0.5. Add 30mL of the above culture medium to the eluted phage and incubate in a 37°C water bath for 1h. Then centrifuge at 4°C, 3200g for 5min. Resuspend the precipitate with 1mL of 2×TY, spread it onto a TYE culture dish supplemented with 4% (wt / vol) glucose and 100μg / mL ampicillin, and incubate it overnight at 37°C. At the same time, use the gradient dilution method on the TYE plate to check the titer of the eluted phage. The next day, scrape the plaque into a test tube containing 5mL of 2×TY culture medium and dilute it with 500mL of 2×TY medium supplemented with 4% (wt / vol) glucose and 100μg / mL ampicillin. Grow in a 2L conical flask at 37°C, 250rpm to OD 600 = 0.5. Phages were purified and precipitated with PEG and the resulting phages were used for two subsequent rounds of repeated screening.

[0090] Among them, during the three rounds of "adsorption-elution-amplification" mentioned above, the titers of input phage and output phage in each round were tested, and the enrichment factor was calculated accordingly (the ratio of output phage / input phage titer in the next round divided by the ratio in the first round). The results are as follows Figure 3 and as shown in Table 1.

[0091] Table 1 Enrichment of specific phages

[0092]

[0093] Combine Figure 3 As shown in Table 1, the input phage titer remained constant during the three rounds of screening (5×10 12 pfu / mL), and the titer of output phage increased significantly with each round: the first round of output phage was 2.5×10 6 pfu / mL (output / input titer ratio = 5.0 × 10 -7 ), the second round of phage output jumped to 1.2×10 8 pfu / mL (output / input titer ratio = 2.4 × 10 -5 , specific phages were enriched 48-fold), and the third round further increased to 1.8×10 8 pfu / mL (output / input titer ratio = 3.6 × 10 -5 , specific phages were enriched 72-fold); compared with the first round, the output / input titer ratio in the third round increased 72-fold, indicating that specific phage clones were effectively enriched during the screening process ( Figure 3 B) After the third round of enhanced washing, the enrichment multiple was only 1.5 times higher than that of the second round, and the increase was not significant, so the fourth round of binding and elution was not performed.

[0094] ③ After the three rounds of screening described above, positive monoclonal phage were screened by ELISA to test the PDI protein binding ability of individual colonies. 80 monoclonal colonies were selected and inoculated into 2×TY medium containing ampicillin and glucose, supplemented with 100 μg / mL ampicillin and 4% (wt / vol) glucose, and incubated at 37°C for 3 hours. After infection with helper phage for 1 hour, the cells were centrifuged and the pellet was resuspended in medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 0.1% (w / v) glucose. The cells were grown at 25°C and 250 rpm for 16-24 hours. The supernatant was centrifuged and the specific phage was collected for later use. The ELISA plate was coated with PDI protein and blocked overnight. The 80 specific phages collected above were mixed with MT buffer (PBS buffer supplemented with 5% BSA and 2% Tween 20) and added to the ELISA plate. The plate was then incubated with HRP-anti M13 antibody, developed, and read at 450-650nm using a microplate reader. The negative group was not coated with PDI protein and directly blocked overnight. The results are shown below. Figure 4 shown.

[0095] right Figure 4 The ELISA results were analyzed, and the OD ratio of the positive group (PDI group) to the negative group (BSA group) was greater than 2.5, which was determined to be a positive monoclonal phage. The top 20 colonies with the highest ratio were screened from the positive monoclonal phage for expansion culture, and 9 colonies were randomly selected for PCR identification. The PCR product bands of the 9 colonies were as follows Figure 5 The above 20 colonies were sent to Shanghai Biotech Co., Ltd. for sequencing (primer 5'-CAGGAAACAGCTATGACCATG-3' (SEQ ID NO: 1)). The sequencing results were analyzed by DNAMAN. The results were as follows: Figure 6 and Figure 7 shown.

[0096] Depend on Figure 6 It was found that among the 20 clones, the G5 sequence was repeated 10 times (50%), the 44 sequence was repeated 6 times (30%), and the 41, 42, A1, and E2 sequences were repeated once each (5% each).

[0097] Depend on Figure 7 It can be seen that after dividing the CDR region using the IMGT database (https: / / www.imgt.org / ), it was found that the CDR3 of G5 has a 12-amino acid sequence, which is longer than other sequences. The six sequence colonies were selected, expanded, and retested by ELISA. The results showed that the OD of the G5 sequence was 450 Based on the cloning frequency (G5 had the highest proportion) and ELISA retest results, the G5 sequence was selected for subsequent experiments.

[0098] 2. Expression and purification of single-domain antibodies

[0099] 1) Construction of single-domain antibody expression vector:

[0100] ① Synthesis of single-domain antibody gene:

[0101] To clone the target gene sequence with a 6xHis tag at the C terminus into the pET-28(+) vector, Ncol (5' end) and Xhol (3' end) restriction sites were first introduced at both ends of the target sequence, and the target gene was amplified by designing 12 primers (Table 2): primers 1-2 cover the Ncol site at the 5' end and the initiation region, primers 3-10 are used to synthesize the main body of the gene, and primers 11-12 introduce the Xhol site, the stop codon, and the 3' end homologous arm; a 20-40 bp homologous overlapping sequence is designed at the end of each pair of primers to achieve seamless splicing between fragments, and then the matching of the overlapping region and the correctness of the reading frame are verified by simulation using Snap Gene software.

[0102] Table 2 Primers for synthesizing the target gene

[0103]

[0104]

[0105] The primers were synthesized by Beijing Genesee Biotech Co., Ltd. After synthesis of the primers, the target gene was synthesized by PCR. Using a 50 μL system, the reagents to be prepared were thawed on ice, and the components in Table 3 were added in proportion on a clean bench. After the reagents were added, the PCR instrument was started, the program was created, and the denaturation, annealing, and extension parameters were set as shown in Table 4, and the instrument was started.

[0106] Table 3 PCR reaction system

[0107]

[0108] Table 4 PCR reaction conditions

[0109]

[0110] ② Amplification of single-domain antibody gene:

[0111] The target gene was amplified by PCR. Using a 50 μL system, the reagents to be prepared were thawed on ice, and the components in Table 5 were added in proportion on a clean bench. After the reagents were added, the PCR instrument was started, the program was created, and the reaction conditions were referred to Table 4.

[0112] Table 5 PCR reaction system

[0113]

[0114] ③ Recovery of single domain antibody gene: After the PCR reaction, the PCR product was separated by electrophoresis using 2% agarose gel. The target band size was about 500 bp (such as Figure 8 After electrophoresis, the gel of the corresponding molecular weight was cut out and recovered using the Beyotime DNA Gel Recovery Kit. The target gene and pET-28a(+) vector were then treated with restriction endonucleases NcoI and XhoI and recovered from the gel to obtain the single-domain antibody target gene and vector with complementary sticky ends.

[0115] ④ Connection between single domain antibody gene and vector:

[0116] The single domain antibody target gene recovered from the gel in step ③ was connected to the pET-28a(+) vector. The connection system is shown in Table 6. Add the reagents in the table in proportion to a sterile centrifuge tube, then place in a 50℃ water bath for 25 minutes for transformation. After standing for 2-3 minutes, the temperature was lowered and the bacterial solution was spread on an LB plate containing kanamycin and incubated at 37℃ overnight. The recombinant plasmid SdAb was constructed (the plasmid map is shown in Figure 9 shown).

[0117] Table 6 Recombination reaction system

[0118]

[0119] ⑤ Identification of single domain antibody recombinant plasmid:

[0120] Pick a single colony from the overnight plate in step ④ and perform colony PCR using primers 1 and 12 in Table 5. Identify positive clones by agarose gel electrophoresis. The results are as follows: Figure 10 As shown; 5 positive colonies were randomly selected and placed in 1.5 mL centrifuge tubes. They were cultured overnight at 37°C in a shaker. Plasmids were extracted from the overnight bacterial culture using the kit protocol. The obtained single-domain antibody plasmid was sequenced to ensure sequence accuracy.

[0121] Depend on Figure 10 It can be seen that all five monoclonal clones showed a single clear band at approximately 500 bp.

[0122] These results demonstrate the successful construction of the PDI single-domain antibody recombinant plasmid SdAb, which can be used for subsequent protein expression. The amino acid sequence of the constructed PDI single-domain antibody is as follows: MAQVQLLESGGGLVQPGGSLRLSCAASGFKISH KSMGWVRQAPGKGLEWVSAIRARGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCATVEVREHQKYLKFWGQGTLVTVSSAAAHHHHHH* (SEQ ID NO: 14). The single-domain antibody has a total of 132 amino acids and a size of 14.5 kDa.

[0123] The CDR1, CDR2, and CDR3 of the three variable regions of the PDI single domain antibody defined by different definition schemes are shown in Table 7.

[0124] Table 7 CDR1, CDR2, and CDR3 defined by different definition schemes

[0125]

[0126]

[0127] 2) Isopropylthiogalactoside (IPTG)-induced expression and identification of recombinant proteins:

[0128] The recombinant plasmid SdAb obtained in step 1) was transformed into Escherichia coli BL21 (DE3), and IPTG was used to induce the expression of the recombinant protein. Two sets of induction conditions were set at 16°C and 30°C, respectively. The specific experimental method is as follows:

[0129] Pick a single colony from the transformation plate and inoculate it into a test tube containing 5 mL of LB medium (containing 50 μg / mL kanamycin) and culture it at 37°C and 220 rpm overnight. The next day, transfer it to 100 mL of LB medium (containing 50 μg / mL kanamycin) at a ratio of 1:100 and culture it at 37°C and 220 rpm until the bacterial OD reaches 0. 600 The pH value is 0.5-0.8. For the negative control sample (without IPTG): Place 1 mL of the culture in a 1.5 mL centrifuge tube and centrifuge at 10,000 × g for 2 minutes at room temperature. Discard the supernatant and resuspend the pellet in 100 μL TBS. Add 25 μL of 5× SDS-PAGE protein loading buffer, incubate at 100°C in a metal bath for 10 minutes, and freeze at -20°C. Add IPTG to the remaining culture to a final concentration of 0.2 mM, then adjust the temperature to 16°C or 30°C and continue shaking for 18 hours to induce expression of the fusion protein. For the positive control sample (with IPTG): Place 1 mL of the induced culture in a 1.5 mL centrifuge tube and centrifuge at 10,000 × g for 2 minutes at room temperature. Discard the supernatant and resuspend the pellet in 100 μL TBS. Add 25 μL of 5× SDS-PAGE protein loading buffer, incubate at 100°C in a metal bath for 10 minutes, and freeze at -20°C. The remaining culture was centrifuged at 4°C, 4000×g for 10 min, and the supernatant was discarded. The cells were resuspended in TBS, and PMSF was added to the resuspension at a final concentration of 1 mM. Ultrasonic disruption was performed, and the precipitate and supernatant were collected after centrifugation. Analyzed by 12% SDS-PAGE and stained with Coomassie Brilliant Blue for band visualization. The results were as follows: Figure 11 shown.

[0130] Depend on Figure 11As can be seen, the target band appeared at 14.5kDa in both induction conditions. In the 30°C induction group, the target protein was mainly present in the precipitate (lane 6), with only a small amount detected in the supernatant (lane 5). In the 16°C induction group, the opposite was true, with a significantly higher proportion of the target protein in the supernatant (lane 3) and less precipitate (lane 4). The soluble protein yield in the 16°C induction group was higher than that in the 30°C group. Because the inclusion body protein (precipitate) requires complex refolding and has a low activity recovery rate, the 16°C low-temperature induction condition was ultimately determined to be the optimal condition for achieving efficient expression of soluble single-domain antibodies.

[0131] 3) Purification of single domain antibodies:

[0132] Purification was performed using a low pressure chromatography system, as follows:

[0133] ① Connect the Ni-NTA affinity chromatography column (pre-filled with 5 mL of medium) to the low-pressure chromatography system and pre-equilibrate it with binding buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) at a flow rate of 1 mL / min. 280 The value reaches the baseline.

[0134] ② Perform preliminary elution with elution buffer containing 50 mM imidazole at a flow rate of 1 mL / min, and collect 1 column volume of the effluent three times in sequence.

[0135] ③ Perform preliminary elution with elution buffer containing 100 mM imidazole at a flow rate of 1 mL / min, and collect 1 column volume of effluent twice.

[0136] ④ Perform preliminary elution with elution buffer containing 500 mM imidazole at a flow rate of 1 mL / min, and collect 1 column volume of effluent three times in sequence.

[0137] ⑤ Transfer 40 μL of the flow-through and each effluent into a 1.5 mL centrifuge tube and add 10 μL of 5× SDS-PAGE protein loading buffer to each tube. Incubate in a metal bath at 100°C for 10 minutes, then freeze at -20°C. Store the remaining effluent at 4°C.

[0138] ⑥ The components collected by the above gradient elution were analyzed by SDS-PAGE. The results were as follows: Figure 12 As shown in A.

[0139] Depend on Figure 12As can be seen from Fig. A, lane 2 (flow-through): no band of the target protein was observed, indicating that the His-tagged protein was effectively bound to the column matrix; lanes 3-5 (50 mM imidazole elution): multiple bands of impurities appeared, but no target band (14.5 kDa) was observed; lanes 6-7 (100 mM imidazole elution): the impurities were reduced, but no target band was observed; lanes 8-10 (500 mM imidazole elution): a trace amount of impurities was observed in lanes 8-9, and lane 10 (500 mM imidazole main peak) showed a single clear target band, with a purity of > 90%.

[0140] 4) Ultrafiltration and concentration of the single-domain antibody:

[0141] The eluate collected in step 3) was placed in a dialysis bag and dialyzed in TBS (20 mM Tris, 0.5 M NaCl, pH 8.0) at a ratio of 1:30, with TBS being replaced every 2 h for 2 times, followed by concentration using a 5 kDa ultrafiltration tube, with centrifugation at a centrifugal force of 3500 x g for 15 min once, until no cut-off volume was observed. The protein concentration after ultrafiltration was detected using a BCA protein quantification kit.

[0142] After the concentrated protein sample was subjected to SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer instrument (20 V, 30 min), and then blocked with 5% BSA at room temperature for 2 h. Then, the diluted primary antibody (His antibody) was added, and the reaction was performed at 4°C overnight. Then, the secondary antibody was added after washing with TBST solution for 3 times on a shaker. After incubation at room temperature for 2 h, the ECL chemiluminescence reagent was exposed and developed, and the results are shown in Fig. B. Figure 12

[0143] As can be seen from Fig. B, a single specific band at 14.5 kDa was observed, indicating that the purified protein was the His-tagged PDI single-domain antibody. Figure 12

[0144] Example 2: Test of the binding ability of the disulfide isomerase single-domain antibody to the antigen

[0145] In this example, the binding ability of the PDI single-domain antibody prepared in Example 1 to the PDI protein was verified in vitro, and the specific experimental methods and results are as follows:

[0146] 1. Western blot:

[0147] The PDI recombinant protein prepared in Example 1 was separated by 12% SDS-PAGE (10 μg and 20 μg per lane, respectively Figure 13 ​​Lanes 1 and 2 in A) were transferred to a PVDF membrane. After blocking for 2 hours, the protein blot was incubated overnight with the single domain antibody prepared in Example 1 (primary antibody, 1:1000 dilution), and then incubated with HRP-protein A (secondary antibody, 1:5000 dilution). ECL color development solution was added to the strips and photographed using an ECL imaging system. The results are shown in Figure 2. Figure 13 As shown in B.

[0148] Depend on Figure 13 B shows that a single specific band appears at 48-63 kDa, confirming that the single domain antibody screened in Example 1 of the present invention can bind to PDI (e.g. Figure 13 B).

[0149] 2. Indirect ELISA:

[0150] The recombinant PDI protein prepared in Example 1 was coated onto a 96-well plate, the PDI single domain antibody screened in Example 1 was used as the primary antibody, and HRP-protein A was used as the secondary antibody. After incubation and color development, the OD value was measured at 450 nm using a microplate reader. The specific steps are as follows:

[0151] (1) Antigen coating: PDI protein solution (5 μg / mL, dissolved in carbonate-bicarbonate coating buffer) was coated into a 96-well plate at a volume of 100 μL per well. A 5% BSA solution was set as a negative control well. After sealing the plate, it was incubated at 4°C overnight (16-18 h). Four replicate wells were set for each sample.

[0152] (2) Blocking: The next day, the coating solution was discarded and 250 μL of blocking buffer (PBST containing 5% BSA, pH 7.4) was added to each well and incubated at 37°C for 2 h.

[0153] (3) Primary antibody binding: Wash 4 times with PBST (300 μL / well each time, soak for 2 min), pat dry the residual liquid, add 100 μL / well of single domain antibody solution (5 μg / mL, diluted in PBST), and incubate on a horizontal shaker at 150 × g at 37°C for 45 min.

[0154] (4) Secondary antibody binding: Recover the primary antibody solution and repeat the PBST washing step. Add 50 μL of HRP-Protein A (1:5000 dilution in PBST) to each well and incubate at 37°C for 45 min in the dark.

[0155] (5) Color development reaction: Recover the secondary antibody solution, wash with PBST, add 50 μL TMB color development substrate to each well, and develop color at 37°C in the dark for 10 min. Add 50 μL / well 2M sulfuric acid stop solution to terminate the reaction, and use a microplate reader to measure the absorbance at 450 nm. The results are as follows: Figure 13 As shown in C.

[0156] Depend on Figure 13 As shown in C, the OD of the recombinant PDI protein-coated wells 450 The mean values ​​were significantly higher than those of the BSA control wells, and the ratios were all greater than 2.5, indicating that the PDI single domain antibody screened by the present invention had affinity for the PDI protein.

[0157] 3. Determination of real-time interaction kinetic parameters:

[0158] The Sartorius Octet molecular interaction instrument (model: R8), and the interaction kinetic parameters between the two were measured in real time by bio-layer interferometry (BLI). This technology is based on the optical interference effect at the tip of the fiber optic sensor probe: when the PDI protein is fixed to the sensor surface and binds to the PDI single-domain antibody, the change in the molecular layer thickness causes the interference light waveform to shift (monitored in real time with nanometer-level precision), thereby directly obtaining the binding rate constant K on , unit: M-1·s -1 (per mole per second) and the dissociation rate constant K off , unit: s -1 (per second), and through the formula KD=K off / K on The equilibrium dissociation constant KD is calculated, and the unit is M (molar concentration). on Characterizes molecular binding efficiency, K off The KD value reflects the dissociation rate of the complex, while the KD value comprehensively characterizes the overall affinity of the interaction between the two. By accurately measuring the KD value, the interaction strength between the single-domain antibody and the PDI protein can be quantitatively assessed.

[0159] Using an anti-streptavidin biosensor, PDI protein was immobilized to a response value of 1 nm (mixed with Thermo21312 biotin at a molar ratio of 1:3 and reacted at room temperature for 30 min). Binding and dissociation assays were performed using a gradient dilution of the single domain antibody (500 nM-18.5 nM). The experimental data were fitted and analyzed using a Langmuir model based on a 1:1 stoichiometric relationship (software: Analysis Studio), calculated KD = 4.5 × 10 -7 M, K on =3.1×10 5 M -1 ·s -1 , K off =1.4×10 -1 s -1 , the results are as follows Figure 14 As shown, Figure 14The results confirmed that the PDI single domain antibody screened by the present invention has a strong affinity with the PDI protein.

[0160] In summary, the present invention successfully screened out single-domain antibodies against PDI for the first time using phage display technology. Through three rounds of "adsorption-elution-amplification" selection, six anti-PDI single-domain antibody sequences were screened from the single-domain antibody library. Among them, the G5 sequence was selected as the research object because of its high sequence repetition rate and ELISA retest results that were better than the other five sequences. A 14.5kDa single-domain antibody was successfully expressed and purified using a prokaryotic expression system (Escherichia coli BL21). Western blot, ELISA, and molecular interaction experiments confirmed its ability to bind to PDI. The PDI single-domain antibody provided by the present invention provides a key guarantee for subsequent experiments and lays the foundation for the development of treatment methods based on anti-PDI single-domain antibodies.

[0161] Example 3: Study of PDI single domain antibody in treating drug-resistant chronic myeloid leukemia

[0162] This example uses a nude mouse xenograft tumor model to explore the in vivo anti-tumor effect of the PDI single domain antibody provided in Example 1 on drug-resistant chronic myeloid leukemia. The specific experimental methods and results are as follows:

[0163] 1. Cell Recovery and Culture

[0164] Ba / F3 BCR-ABL T315I The cell line is a mouse pre-B cell model constructed by expressing the BCR-ABL T315I drug-resistant mutant gene. T315I The cells were taken out from the liquid nitrogen tank and dissolved in a 37°C water bath. After dissolution, they were poured into a centrifuge tube containing 10 mL of preheated culture medium, centrifuged at 500 × g for 5 min, the supernatant was discarded, 2 mL of culture medium was added to resuspend, and the cells were transferred to a 25 cm 2 Place the culture flask in a 5% CO2 incubator and culture at 37°C. When the cell density reaches 80%, centrifuge, discard the supernatant, add 2 mL of 1640 complete medium to resuspend, and subculture at a ratio of 1:2. Collect the cultured Ba / F3 BCR-ABL in a 50 mL centrifuge tube. T315I The cells were centrifuged at 500 × g for 5 min, the supernatant was discarded, and the cells were washed three times with appropriate amount of PBS. The cell concentration was adjusted to 10 × 10 8 cells / mL, set aside.

[0165] 2. Animal modeling, grouping, and sampling

[0166] To evaluate the in vivo antitumor effect of single-domain antibodies against drug-resistant chronic myeloid leukemia, Ba / F3BCR-ABL was injected subcutaneously. T315IA nude mouse xenograft tumor model was established by cell-based approach.

[0167] Add 0.1mL Ba / F3 BCR-ABL T315I The cell suspension was inoculated subcutaneously in the right axilla of nude mice. The tumor growth morphology was observed every day. After 5 days of inoculation, obvious tumors were observed, with a tumor formation rate of 100%. When the tumor volume grew to about 100 mm 3 The experiment was divided into three groups (5 mice each): the control group (Veh) was intraperitoneally injected with TBS, the positive group (HHT) was intraperitoneally injected with 1 mg / kg of homoharringtonine every day, and the single-domain antibody group (SdAb) was intraperitoneally injected with 10 mg / kg of PDI single-domain antibody every three days for 15 consecutive days. The body weight and tumor volume of nude mice were measured every three days (using a vernier caliper to measure the longest diameter L and the shortest perpendicular diameter W of the tumor, according to the formula V = L × W). 2 After the administration, the nude mice were anesthetized and killed, and the tumor tissues were removed, weighed, and photographed. Figure 15 shown.

[0168] Depend on Figure 15 A and Figure 15 As shown in B, the body weight and tumor volume of nude mice in the positive group and single-domain antibody group were lower than those in the control group.

[0169] Depend on Figure 15 As shown in Figure C, the tumor volume in the single-domain antibody group was significantly smaller than that in the control group, but there was significant variability in tumor volume between individuals within the group. This phenomenon may be related to the different pharmacokinetic responses of nude mice to single-domain antibodies.

[0170] Depend on Figure 15 D shows that the tumor volumes of the single-domain antibody group and the positive group were significantly reduced compared with the control group (P≤0.05), and the inhibitory effect of the single-domain antibody group was slightly better than that of the positive group.

[0171] 3. Western Blot Analysis

[0172] Three tumor tissue samples were collected from each of the control group and the single-domain antibody group (a total of 6 cases) for protein immunoblotting analysis.

[0173] Western blot was used to detect the expression level of PDI protein in the tumor tissues of nude mice in the control group and the single domain antibody group (10 mg / kg). Figure 16 A). The grayscale value analysis of the bands was performed using Image J software. The results showed that the relative expression of PDI protein in the single-domain antibody group was significantly lower than that in the control group (P≤0.05) ( Figure 16 B).

[0174] In summary, the present application focuses on the regulatory function of PDI in thrombus formation, and through phage display technology, an anti-PDI single domain antibody is screened, and using a drug-resistant chronic myeloid leukemia nude mouse xenograft tumor model, it is confirmed that the single domain antibody can significantly inhibit tumor progression. The present application provides key experimental evidence for the treatment strategy of anti-PDI single domain antibody in drug-resistant chronic myeloid leukemia.

[0175] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a disulfide isomerase single domain antibody in the preparation of a product for treating and / or preventing leukemia, characterized in that: The disulfide isomerase single domain antibody comprises a variable region, wherein the variable region comprises CDR1, CDR2 and CDR3; The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the CDR1 to CDR3 are defined according to the IMGT definition scheme; or The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Kabat definition scheme; or The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 20, respectively, and the CDR1 to CDR3 are defined according to the Chothia definition scheme; or The amino acid sequences of CDR1 to CDR3 of the disulfide isomerase single domain antibody are shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and the CDR1 to CDR3 are defined according to the Contact definition scheme.

2. The use according to claim 1, characterized in that The amino acid sequence of the disulfide isomerase single domain antibody comprises: a1) SEQ ID NO: 14; or a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 14 and having the same function as the protein shown in SEQ ID NO: 14; or a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% identical to SEQ ID NO: 14 and has the same function as the protein shown in SEQ ID NO:

14.

3. The use according to claim 1 or 2, characterized in that The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the leukemia is chronic leukemia; the chronic leukemia includes at least one of chronic myeloid leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia; More preferably, the leukemia is drug-resistant chronic myeloid leukemia.

4. The disulfide isomerase single domain antibody for use according to claim 1 or 2.

5. A recombinant protein comprising: the disulfide isomerase single domain antibody according to claim 4; and Optional tag sequence to facilitate expression and / or purification.

6. A biomaterial related to the disulfide isomerase single-domain antibody according to claim 4 or the recombinant protein according to claim 5, wherein the biomaterial comprises at least one of h1) to h8): h1) a nucleic acid molecule encoding the disulfide isomerase single domain antibody according to claim 4 or the recombinant protein according to claim 5; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) A transgenic cell line comprising the vector described in h4).

7. A conjugate comprising: at least one of the disulfide isomerase single-domain antibody according to claim 4 and the recombinant protein according to claim 5; and a coupling moiety, the coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein; Preferably, the detectable marker is a fluorescent or luminescent marker; Preferably, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase; Preferably, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 and Re-188.

8. A product comprising at least one of k1) to k3): k1) The disulfide isomerase single domain antibody according to any one of claims 4; k2) the recombinant protein according to claim 5; k3) The conjugate according to claim 7.

9. The product according to claim 8, characterized in that: The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has the function of treating and / or preventing chronic leukemia; the chronic leukemia includes at least one of chronic myeloid leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia; More preferably, the leukemia is drug-resistant chronic myeloid leukemia.

10. A method for preparing the disulfide isomerase according to any one of claims 4 or the recombinant protein according to claim 5, which is obtained by culturing the transgenic cell line according to claim 6.

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