Anti-cd38 protein antibodies, methods of making and using the same

Abstract

The application relates to the field of biological medical technology, in particular to an anti-CD38 protein antibody and a preparation method and application thereof. The application provides a nanobody, namely an anti-CD38 protein antibody; the anti-CD38 protein antibody is obtained by screening a phage display natural nanobody library, the preparation method process is simple, and the cost is low; the anti-CD38 protein antibody can be combined with CD38 with high affinity, and can be applied to the fields of CD38 related disease immune detection, antigen enrichment and purification and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to an anti-CD38 protein antibody and a preparation method and application thereof. BACKGROUND

[0002] CD38 is a 46kDa single-chain type II transmembrane glycoprotein widely distributed in various cells and tissues of mammals, including immune cells (such as T cells, B cells, natural killer cells and macrophages), nervous system cells, endocrine system and metabolism-related tissues. CD38 catalyzes the degradation of NAD + to generate cyclic ADP ribose (cADPR) and nicotinamide mononucleotide adenosine diphosphate (NAADP), and regulates intracellular calcium signaling and metabolic activity. However, the activity of CD38 will significantly increase with age. In addition, CD38 is considered to be closely related to many pathological processes, such as aging, metabolic disorders, neurodegenerative diseases, autoimmune diseases and some tumors, etc. Abnormal up-regulation or down-regulation of its activity will trigger the occurrence and development of a series of diseases, especially in the aging process, the high expression of CD38 significantly reduces the NAD + level in vivo, further aggravating mitochondrial dysfunction and inflammatory response, thereby accelerating the progression of age-related diseases. These characteristics make CD38 an important target in basic medicine and drug development research in recent years, and its inhibitors and related antibody drugs show broad application prospects in metabolic regulation, immunotherapy and anti-aging fields.

[0003] At present, the monoclonal small molecule antibody Daratumumab targeting CD38 has been approved for the treatment of multiple myeloma and shows good clinical efficacy. Preclinical studies have shown that Daratumumab induces multiple myeloma cell death through multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis. However, the monoclonal antibody has a large molecular weight, poor tissue penetration, and limited direct impact on NAD + metabolism. SUMMARY

[0004] Based on this, the present application provides an anti-CD38 protein antibody and a preparation method and application thereof, which at least solve one problem in the prior art.

[0005] In a first aspect, the present application provides an anti-CD38 protein antibody, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO. 2, the amino acid sequence of CDR2 is shown in SEQ ID NO. 4, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 6. The three sequences are the complementarity determining region amino acid sequences of the anti-CD38 protein antibody, which are mainly responsible for the recognition of CD38.

[0006] The anti-CD38 protein antibody is a nanobody, which can bind to CD38 with high affinity. The anti-CD38 protein antibody can be a monovalent antibody, a bivalent antibody or a multivalent antibody. The amino acid sequence of the anti-CD38 protein antibody can be used as a precursor to be modified by random or site-directed mutagenesis technology, and a mutant with better properties (yield, water solubility, affinity, specificity, stability, etc.) can be obtained.

[0007] In some embodiments, the anti-CD38 protein antibody further comprises the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7. The four sequences are the framework region amino acid sequences of the anti-CD38 protein antibody, which are mainly responsible for maintaining the structure of the nanobody.

[0008] In some embodiments, the anti-CD38 protein antibody comprises the amino acid sequence shown in SEQ ID NO. 8. The amino acid sequence of SEQ ID NO. 8 is EVQVVESGGGLVQAGGSLRLSCAASGRDFRAIAMAWFRQAPGKEREFVAAINRVTGSTYYAGSAKGRFTISRDSAANTAYLQMNSLKPEDTAAYYCAATNRAIVLTTPAYNYWGQGTQVTVSS.

[0009] In a second aspect, the present application provides a preparation method of the anti-CD38 protein antibody, which comprises the following steps:

[0010] The anti-CD38 protein antibody is obtained by panning a phage display natural nanobody library with CD38 protein as an antigen.

[0011] In some embodiments, the phage display natural nanobody library is AlpSDab-P produced by Sichuan Apark Biotechnology Co., Ltd.

[0012] In some embodiments, the panning includes four rounds of panning in a 96-well plate, wherein the antigen amount of the first round of panning is 2 µg / well, the blocking solution is a 3% BSA solution, the input amount of the phage display natural nanobody library is 10 11 pfu / well, the binding time of the antigen and the phage display natural nanobody library is 1.5 h; the antigen amount of the second round of panning is 1.5 µg / well, the blocking solution is a 3% BSA solution, the input amount of the phage display natural nanobody library is 10 11 pfu / well, the binding time of the antigen and the phage display natural nanobody library is 1 h; the antigen amount of the third round of panning is 1 µg / well, the blocking solution is a 3% BSA solution, the input amount of the phage display natural nanobody library is 10 11 pfu / well, the binding time of the antigen and the phage display natural nanobody library is 1 h; the antigen amount of the fourth round of panning is 0.5 µg / well, the blocking solution is a 3% BSA solution, the input amount of the phage display natural nanobody library is 10 11 pfu / well, and the binding time of the antigen and the phage display natural nanobody library is 1 h.

[0013] In a third aspect, the present application provides use of the anti-CD38 protein antibody in preparing an immunodetection product.

[0014] In some embodiments, the immunodetection product includes an enzyme-linked immunosorbent assay kit, an immunochromatographic assay kit, a SEA immunodetection kit, or an immunodetection chip.

[0015] In a fourth aspect, the present application provides an immunodetection product including the anti-CD38 protein antibody.

[0016] In some embodiments, the immunodetection product is used for detecting CD38 protein.

[0017] By adopting the above technical scheme, the embodiment of the present application has at least the following beneficial effects: a kind of nanobody, i.e., anti-CD38 protein antibody is provided;The anti-CD38 protein antibody is obtained by screening phage display natural nanobody library, and the preparation method is simple and low in cost;The anti-CD38 protein antibody can be combined with CD38 with high affinity, and can be applied to the fields of CD38 related disease immune detection, antigen enrichment and purification. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure for phage-ELISA identification result of anti-CD38 positive clone phage in the embodiment of the present application.

[0019] Figure 2 Figure for amino acid sequence and domain schematic diagram of CD38-1-36 in the embodiment of the present application.

[0020] Figure 3 Figure for competitive ELISA experimental result of CD38-1-36 in the embodiment of the present application.

[0021] Figure 4 Figure for western blot result of CD38-1-36 in the embodiment of the present application.

[0022] Figure 5 Figure for SDS-PAGE result of CD38-1-36 in the embodiment of the present application. DETAILED DESCRIPTION

[0023] The concept and technical effects of the present application will be described clearly and completely below to fully explain the purpose, scheme and effects of the present application.

[0024] According to the embodiment of the present application, CD38 is used as an antigen, and anti-CD38 protein antibody is obtained by screening phage display natural nanobody library AlpSDab-P. Specifically, the screening method comprises the following steps:

[0025] S1, dilute CD38 antigen to 10 μg / mL by phosphate buffer solution with pH=8.6, take 100 μL CD38 antigen and add it into 96-well plate, and incubate at 4℃ for 12 h;After incubation, wash the 96-well plate with PBST solution for 3 times;

[0026] S2, after washing, add 300 μL protein blocking solution to each well, the protein blocking solution is 3% BSA solution, and incubate at 37℃ for 2 h;After incubation, wash the 96-well plate with PBST solution for 5 times;

[0027] S3, after washing, 100 μL of phage-displayed natural nanobody library AlpSDab-P was added to each well, and incubated at 37°C for 1 h; after incubation, the 96-well plate was washed 10 times with PBST solution;

[0028] S4, after washing, 100 μL of Gly-HCl eluent with pH=2.2 was added to each well, and incubated on a horizontal shaker for 8 min;

[0029] S5, after incubation, 100 μL of eluent in each well was neutralized with 10 μL of neutralization buffer Tris-HCl with pH=9.0, to obtain 110 μL of neutralized solution;

[0030] S6, 10 μL of the neutralized solution was taken to plate to measure the phage titer; the remaining 100 μL of the neutralized solution was used to infect E. coli ER2738, amplify the phage, and proceed to the next round of selection.

[0031] The selection condition was gradually stringent in each round to promote the selection of high-affinity nanobodies, and a total of four rounds of selection were performed. The selection condition and experimental parameters of each round are shown in Tables 1 and 2.

[0032] Table 1. Selection condition of anti-CD38 protein antibody

[0033]

[0034] Table 2. Selection experimental parameters of anti-CD38 protein antibody

[0035]

[0036] After four rounds of selection and phage ELISA identification, the nanobody against CD38 protein was successfully obtained, and was named CD38-1-36. The phage vector displaying CD38-1-36 was pcomb3xss, and was named pcomb3xss-CD38-1-36. Further, CD38-1-36 was expressed by auto-induction, which specifically included the following steps:

[0037] D1, the phage vector pcomb3xss-CD38-1-36 was transformed into chemically competent cells by heat shock at 42°C for 90 s E. coli In Rosetta, obtain bacterial solution one;

[0038] D2, 50 μL of bacterial solution and 5 μL of ampicillin (Amp) were added to 5 mL of LB liquid medium, the final concentration of Amp was 100 mg / mL, and the culture was incubated at 37°C and 220 rpm on a shaker for 12 h. After incubation, bacterial solution two was obtained;

[0039] D3, after the completion of the culture, the bacteria liquid was inoculated into 100 mL of 2YT medium at a 1% (v / v) inoculation amount, and 100 μL of Amp was added, and the culture was incubated at 37°C, 250 rpm for 3.5 h to the logarithmic phase (OD 600 to 0.5-0.7), to obtain culture one;

[0040] D4, culture one was added to 0.1 mM IPTG solution, and was placed in a 25°C, 180 rpm shaker for 12 h to induce protein expression, to obtain culture two;

[0041] D5, culture two was centrifuged at 12000 rpm / min, 4°C for 20 min, and the precipitate obtained by centrifugation was resuspended in 20 mL of PBS buffer to obtain a bacterial resuspension;

[0042] D6, 20 mg of lysozyme was added to the resuspension, and was enzymatically hydrolyzed at 25°C for 30 min to obtain an enzymatic hydrolysate;

[0043] D7, 15 μL of PMSF was added to the enzymatic hydrolysate, and the final concentration of PMSF was 1 mmol / L, and the solution was broken by a 300 W cell sonicator at 4°C for 30 min. The solution was observed to become clear and transparent during the breaking, and the breaking was stopped, to obtain a broken product;

[0044] D8, the broken product was centrifuged at 10000 rpm / min, 4°C for 20 min, and the supernatant was collected; the supernatant was filtered using a 0.22 μm water filter to remove impurities, and finally purified.

[0045] The purification in step D8 includes the following sub-steps:

[0046] D81, 2 mL of Ni-NTA column material was added to the purification column, and after the Ni-NTA was naturally settled, 5-10 times the column volume of distilled water was used to wash and remove impurities, and 15 mL of equilibration buffer was used to equilibrate the purification column;

[0047] D82, the supernatant was added to the purification column in batches, and was allowed to slowly flow out, and the flow-through of each time was collected;

[0048] D83, 15 mL of equilibration buffer (containing 50 mM imidazole) was used to equilibrate the purification column, and to wash the impure proteins in the purification column;

[0049] D84, after the washing was completed, 5 mL of elution buffer (containing 300 mM imidazole) was used to elute the target protein bound in the purification column, and the target protein liquid was collected in a 1.5 mL centrifuge tube for storage;

[0050] D85, transfer the collected target protein solution to a 3 kDa dialysis bag, and dialyze at 4°C using PBS dialysate with a concentration of 10 mmol / L at pH=7.4, replacing the dialysate every 8 h, and the dialysis time is 1 d;

[0051] D86, after dialysis, take the protein solution and measure the protein concentration using Nano Drop 2000; after the measurement is completed, use a 1.5 mL centrifuge tube to dispense and store in a refrigerator at -20°C.

[0052] During the panning process, positive clone phages were identified by phage-ELISA. Specifically, when the third and fourth rounds of panning were performed, 48 clones were randomly selected on the plate for measuring the phage titer for phage amplification, and were divided into an experimental group and a blank group:

[0053] (1) Experimental group: dilute the CD38 antigen to a concentration of 0.5 μg / mL using PBS buffer, add 100 μL per well to a 96-well plate, and coat at 4°C for 12 h;

[0054] (2) Blank group: dilute the BSA to a concentration of 10 μg / mL using PBS buffer, add 100 μL per well, and coat at 4°C for 12 h;

[0055] After coating, wash 3 times with PBST solution, wherein the PBST solution is PBS buffer with the addition of 0.5% Tween-20, and the final concentration of PBS in the PBST solution is 10 mmol / L; after washing, add 300 μL of 3% BSA per well, and block at 37°C for 2 h; after blocking, wash 3 times with PBST; in the 96-well plates of the experimental and control groups, add 100 μL of phage amplification solution per well, and incubate at 37°C for 1 h; after incubation, add 100 μL of diluted HRP-M13 secondary antibody per well, and incubate at 37°C for 1 h; after incubation, wash 4 times with PBST; after washing, add 100 μL of TMB color developing solution per well, and develop color at 37°C for 10 min in the dark; after color development, add 50 μL of 2 mol / L H2SO4 stop solution per well; measure the absorbance value at 450 nm using an enzyme marker.

[0056] As shown in Figure 1 , 48 positive clones were sequenced, and 7 different amino acid sequences were obtained; the nanobody CD38-1-36 gene with better performance was selected for sequencing and subsequent identification.

[0057] Figure 2The amino acid sequence of CD38-1-36 is IMGT numbered and domain schematic, and the amino acid sequence of CD38-1-36 includes framework regions (FR1-FR4) and complementarity determining regions (CDR1-CDR3). Among them, the amino acid sequences of the framework regions FR1-FR4 of the nanobody CD38-1-36 are shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7, respectively; and the amino acid sequences of the complementarity determining regions CDR1-CDR3 are shown in SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6, respectively. The complementarity determining region is mainly responsible for the recognition of CD38; the framework region structure is relatively stable, and is mainly responsible for maintaining the structure of the nanobody.

[0058] The six positive phage-displayed CD38 antibodies obtained in the panning process were randomly numbered as 1-36, 3-25, 4-1, 5-14, 6-28 and 7-12, and were divided into six experimental groups for nanobody competitive ELISA experiments. CD38 protein, NAD and CD38 antibody were added in the experimental groups, and only CD38 protein and CD38 antibody were added in the control group. The purpose of this experiment is to detect whether the CD38 antibody has a competitive effect when combined with the CD38 protein + If there is a competitive effect, the OD 450 signal will be weakened, which can prove that this antibody can compete with NAD + for the binding site. As shown in Figure 3 , the binding signal of the CD38 antibody numbered 1-36 (i.e. CD38-1-36) gradually weakens, indicating that this CD38 antibody can compete with NAD + for the binding site and effectively block the binding of NAD + .

[0059] The expression of the nanobody CD38-1-36 was identified by polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Western blot). The results of SDS-PAGE are shown in Figure 4 , wherein lane 1 is the blank control supernatant; lane 2 is the self-induced supernatant; lane 3 is the 0.1 mM IPTG supernatant; lane 4 is the 0.3 mM IPTG supernatant; lane 5 is the 0.6 mM IPTG supernatant; lane 6 is the 0.9 mM IPTG supernatant; lane 7 is the blank control precipitate; lane 8 is the self-induced precipitate; lane 9 is the 0.1 mM IPTG precipitate; lane 10 is the 3 mM IPTG precipitate; lane 11 is the 0.6 mM IPTG precipitate; and lane 12 is the 0.9 mM IPTG precipitate. It can be seen that a large amount of protein can be expressed under the induction of 0.1 mM IPTG.

[0060] Figure 3 , Figure 4 The results indicate that the CD38-1-36 nanobody was successfully expressed and purified, and the nanobody has high purity. The molecular weight of the nanobody is approximately 16 kDa.

[0061] The protein was induced to express in *E. coli* under 0.1 mM IPTG conditions. Lysozyme was added and the protein was sonicated to release it. The mixture was centrifuged at 13000 rpm for 30 minutes to obtain the supernatant. This supernatant may contain the target protein; the precipitate was retained after centrifugation. Further purification of the protein was performed. The histidine (His) residues in the expressed protein can react with nickel ions (Ni... 2+ This protein binds to the nickel column by forming coordinate bonds, while other contaminating proteins cannot bind or only bind weakly. Therefore, proteins in the supernatant can bind to the nickel column. Competitive elution is achieved by increasing the concentration of imidazole in the protein washing buffer (imidazole binds to Ni). 2+ (Binding) elutes the target protein, thus obtaining a high-purity His-tagged protein. For example... Figure 5 As shown, lanes 1-5 contain 500 mM imidazole elution buffer; lane 6 contains protein standards; lanes 7-10 contain 250 mM imidazole elution buffer; lane 11 contains 50 mM imidazole elution buffer; lane 12 contains the flow-through liquid after the supernatant has passed through the nickel column; lane 13 contains the precipitate after high-speed centrifugation of the cell lysis buffer; and lane 14 contains the supernatant after high-speed centrifugation of the cell lysis buffer. It is evident that the protein collection solutions in lanes 7 to 10 contain fewer contaminants and a large amount of the target protein, indicating they are the target protein solutions.

[0062] In the embodiments of this invention, all culture media are commercially available, with LB liquid culture medium from Beijing Solarbio Technology Co., Ltd., and self-inducing culture medium from Sichuan Apak Biotechnology Co., Ltd.

[0063] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. An anti-CD38 protein nanobody, characterized in that, The amino acid sequence of the anti-CD38 protein nanobody CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.4, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

6.

2. The anti-CD38 protein nanobody according to claim 1, characterized in that, The anti-CD38 protein nanobody also includes the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.

7.

3. The anti-CD38 protein nanobody according to claim 1, characterized in that, The anti-CD38 protein nanobody comprises the amino acid sequence shown in SEQ ID NO.

8.

4. The application of the anti-CD38 protein nanobody according to any one of claims 1-3 in the preparation of immunoassay products.

5. The application according to claim 4, characterized in that, The immunoassay products include enzyme-linked immunosorbent assay (ELISA) kits, immunochromatographic assay kits, SEA immunoassay kits, or immunoassay chips.

6. An immunoassay product, characterized in that, Includes the anti-CD38 protein nanobody as described in any one of claims 1-3.

7. The immunoassay product according to claim 6, characterized in that, The immunoassay product is used to detect CD38 protein.

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