CN114957470B

CN114957470B - RAGE-targeting nanobody and application thereof

Info

Publication number: CN114957470B
Application number: CN202210391071.3A
Authority: CN
Inventors: 李志杰; 肖炜; 王复婧; 王继刚; 孙继超
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2023-01-17
Anticipated expiration: 2041-11-12
Also published as: CN114716551A; CN114031687A; CN114957470A; CN114716551B; CN114031687B

Abstract

The invention provides a RAGE-targeting nanobody and application thereof, wherein the complementarity determining regions of the amino acid sequence of the RAGE-targeting nanobody are CDR1 shown as SEQ ID NO. 9, CDR2 shown as SEQ ID NO. 11 and CDR3 shown as SEQ ID NO. 13. The nano antibody of the technical scheme of the invention has stronger binding with RAGE, can block the interaction of the RAGE and various receptors, and has therapeutic effect on AD and cancer.

Description

RAGE-targeting nanobody and application thereof

Technical Field

The invention belongs to the technical field of immunology or molecular biology, and particularly relates to a RAGE-targeted nano antibody and application thereof.

Background

RAGE (the receptor for advanced glycation end products), the cell surface receptor referred to herein as advanced glycation end products (AGEs), is the advanced glycation end products (AGEs). AGEs accumulation in cells and tissues is an important molecular feature of aging, and AGE protein modification occurs when intracellular proteins survive for too long, resulting in loss of function of the modified proteins by cross-linking with each other. AGE modification is an irreversible process, and as time goes by, AGEs accumulate, the RAGE pathway is activated, and RAGE expression is increased. In addition to AGEs, important proteins in the development of diseases such as the Alzheimer's Disease (AD) -associated Abeta protein and the cancer-associated HMGB1 protein have been identified as ligands for RAGE, and these ligand-receptor signaling pathways are closely involved in disease prediction and disease progression.

RAGE plays a role in the progression of AD disease primarily in the blood circulation system and central nervous system. In the blood circulation, RAGE may act as an Abeta receptor promoting its transport into the brain, causing Abeta deposition; activation of RAGE in the central nervous system may promote neuroinflammatory responses, increased Abeta production, and increased tau phosphorylation, key regulators of pathological changes in AD. RAGE expression levels are significantly elevated in both AD animal models and AD patients, and evidence suggests that elevated RAGE expression occurs prior to Abetaplaque. These results suggest that RAGE activation may be a key factor in the pathological alteration of AD by age-related physiological changes.

The nano-antibody can play a role in treatment by targeting important targets of various diseases and blocking or activating corresponding target proteins and signal pathways, and the Capacizumab is the first nano-antibody drug approved by the FDA in the United states and used for treating Thrombotic Thrombocytopenic Purpura (aTPP). In addition, the nanobody can be used as a targeting tool to help the drug or diagnostic molecule to locate a disease-related target protein in vivo, wherein the nanobody targeting a tumor biomarker is continuously identified, such as the nanobodies of proteins such as EGFR1 or EGFR2 (or HER1 and HER 2), VEGFR2, c-Met and CXCR7, and the like, have been reported. However, to date, no nanobodies targeting RAGE have been discovered.

In addition, in malignant tumors (including kidney cancer, prostate cancer, stomach cancer, breast cancer, colon cancer and the like), the expression level of RAGE is remarkably increased, and the phenomenon is proved to be related to the metastasis and poor prognosis of cancer, so the RAGE can also be used as a cancer diagnosis marker, and the RAGE nano antibody is combined with a nano material with an imaging effect, so that the RAGE has an application value of cancer imaging diagnosis. HMGB1 highly expressed in the process of tumor development is proved to activate signal pathways such as p38 MAPK, p42/p44, JNK and the like by combining with RAGE receptors, and further activate fibrinolytic enzymes, so that cancer cells are more prone to migration and metastasis. AGE-RAGE signaling pathways in cancer cells also affect apoptosis, necrosis, and autophagy, and by inhibiting RAGE receptor activation, apoptosis may be increased and the development of drug resistance in cancer cells may be reduced. Therefore, the targeting drug based on the RAGE nano antibody also has the application value of clinical cancer treatment.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a nano antibody targeting RAGE and its application, which has strong binding force with RAGE and can be used as a drug for various imaging and treatments of RAGE associated with Alzheimer's disease and cancer.

In contrast, the technical scheme adopted by the invention is as follows:

the nano antibody targeting RAGE has a complementarity determining region (CDR 1) shown as SEQ ID NO. 9, a CDR2 shown as SEQ ID NO. 11, and a CDR3 shown as SEQ ID NO. 13; specifically, the CDR1 sequence shown in SEQ ID NO. 9 is: EVSGGAFSRYAVG, the CDR2 sequence shown in SEQ ID NO:11 is: FAAAISWSGNNTY, the CDR3 sequence shown in SEQ ID NO:13 is: MLVSTYDY.

As a further improvement of the invention, the framework region of the amino acid sequence of the nano antibody targeting RAGE is FR1 shown in SEQ ID NO. 8, FR2 shown in SEQ ID NO. 10, FR3 shown in SEQ ID NO. 12 and FR4 shown in SEQ ID NO. 14.

As a further improvement of the invention, the amino acid sequence of the nano antibody targeting RAGE is shown as SEQ ID NO. 23.

The large amount of selected nano antibodies targeting RAGE have the potential of blocking the combination of RAGE and ligands, can effectively relieve the pathological changes of Abeta and tau, and have the application value of clinical treatment. In addition, the nanometer antibody is assembled with the nanometer material, the blood brain barrier permeability of the nanometer antibody is increased, meanwhile, the target imaging of the RAGE nanometer antibody in the brain can be realized by means of the fluorescence and CT imaging characteristics of the nanometer material and the targeting property of the nanometer antibody, and the RAGE nanometer antibody has the application value of AD clinical diagnosis.

The invention also discloses a nucleic acid, which is: a nucleic acid sequence encoding a nanobody targeting RAGE as described in any preceding paragraph.

The invention also discloses an expression vector which comprises the nucleic acid.

The invention also discloses a host cell which comprises the expression vector.

The invention also discloses application of the nano antibody targeting the RAGE in preparing a detection reagent for targeting the RAGE of the Alzheimer's disease and the cancer, a medicament, an in vivo imaging probe, a chimeric immune cell or a bispecific antibody for targeting the RAGE of the Alzheimer's disease and the cancer.

Compared with the prior art, the invention has the beneficial effects that:

the nano antibody of the technical scheme of the invention has stronger binding with RAGE, can block the interaction of RAGE and various receptors, can effectively relieve the pathological changes of Abeta and tau, and has therapeutic effect on AD and cancer; besides, the nano antibody targeting RAGE is modified on nano materials with various imaging characteristics, so that AD and cancer can be specifically diagnosed through imaging; and the nano antibody has smaller molecular weight and better tissue penetrability, can be expressed and purified by using an escherichia coli system, and has low preparation cost.

Drawings

FIG. 1 is a graph of RAGE expression levels in wild-type and AD mice in accordance with an embodiment of the present invention.

FIG. 2 is a graph comparing the expression levels of RAGE in normal tissues and renal clear cell carcinoma, in accordance with an embodiment of the present invention.

FIG. 3 is a graph comparing the expression levels of RAGE in different renal cancer cell lines in an example of the present invention.

FIG. 4 is a graph of RAGE expression levels versus renal cancer patient prognosis in an example of the present invention.

FIG. 5 is a schematic illustration of the extracellular domain of RAGE in an embodiment of the present invention.

FIG. 6 is a graph of protein expression analysis of a RAGE-targeting nanobody of an embodiment of the present invention versus a comparative antibody.

FIG. 7 is a graph comparing in vitro ELISA affinities of RAGE-targeting nanobodies of the present invention with comparative antibodies, wherein (a) is NbH nanobodies, (b) is NbF8 nanobodies, (c) is NbF9 nanobodies, (d) is NbD nanobodies, (e) is NbS1 nanobodies, (f) is NbS2 nanobodies, and (g) is NbS3 nanobodies.

Detailed Description

Preferred embodiments of the present invention are described in further detail below.

(1) Analysis of RAGE expression in tumor cells and AD brain tissue

As RAGE has been reported to be expressed in elevated amounts in AD disease states, this example first compares RAGE expression in the brain of normal and AD mice. In addition, RAGE may be expressed in a plurality of tumor cell lines, and the examples select renal clear carcinoma cells and cell lines for which RAGE expression is being analyzed, comprising the steps of:

(1) tissue expression analysis of RAGE:

a) Collecting tissue, and dehydrating in 30% sucrose solution at 4 deg.C overnight;

b) OCT embedding is carried out on the next day, and the mixture is stored at minus 80 ℃;

c) Placing the frozen tissue block in a frozen microtome for re-warming for 10 minutes and then slicing, wherein the thickness of the slice is set to be 10 mu M;

d) After the slices were slightly air-dried, they were placed in a precooled acetone/methanol (4: 1) Mixing the solution, fixing at-20 deg.C for 5 min;

e) TBS rinsing and rewarming the slices for 10 minutes, and sealing 4% donkey serum for 1 hour;

f) Incubating anti-HA primary antibody for 1 hour, and rinsing with TBS buffer solution for 3 times;

g) Alexa 488-conjugated secondary antibody was incubated for 1 hour and TBS rinsed 3 times;

h) And sealing the chip by using a sealant containing DAPI, and performing machine detection.

(2) Cellular expression analysis of RAGE: culturing renal clear cancer cell lines A498, A704, caki-1, KMRC1, KMRC20, OS-RC2, UT16, UT33a, UT44, VMRC-RCW, VMRC-RCZ,769P,786O; collecting cells, and extracting proteins in the cells by using RIPA tissue lysate under the condition of 4 ℃; WB measures RAGE expression in different cell lines.

The expression level of RAGE is detected in a brain model of a normal mouse with the age of 6 months, a 5xFAD mouse model with the age of 4 months and the age of 6 months respectively by adopting the method, and the result is shown in figure 1, and the expression of RAGE in AD mice is obviously increased.

This example also compares the expression levels of RAGE in normal tissues and renal clear cell carcinoma (KIRC), and as shown in FIG. 2, RAGE expression levels in tumor tissues are significantly elevated.

This example also examined the expression of RAGE in various renal cancer cell lines, as shown in FIG. 3, where RAGE was highly expressed in KMRC1, KMRC20, OS-RC2, UT16, UT33a, UT44, VMRC-RCW, VMRC-RCZ,769P,786O cell lines. As shown in FIG. 4, by comparing the amount of RAGE expression to the prognosis for patients with renal cancer, the survival of patients with high RAGE expression was significantly reduced compared to patients with low RAGE expression.

(2) Screening of RAGE Nanobodies

The extracellular domain of purified RAGE was expressed in E.coli, and a schematic is shown in FIG. 5.

Screening natural alpaca phage display nano antibody library by using immune tube method, and selecting phage display library with capacity of 2 x 10 ⁹ . The screening steps are as follows:

a) Coating the target protein on an immune tube according to the concentration of 10 mu g/mL, and carrying out 3 rounds of enrichment screening;

b) Using a third round of phage eluate plating, randomly picking 96 monoclonals for ELISA verification, and taking the positive standard that the ELISA reading is 3 times larger than the corresponding BSA reading and the reading is 0.5 or more;

c) Sending the positive monoclone identified by phage ELISA for 2 times to a company for sequencing to determine sequence information, and randomly selecting 3 negative monoclonals as a control for sequencing;

d) And designing and synthesizing the screened nano antibody according to the sequencing information, and performing expression and purification by using escherichia coli.

In the experiment, although we find 17 potential positive clones through phage ELISA analysis, phage ELISA is only verified through 192 randomly picked clones, and there is a potential disadvantage of missing some high-binding-force clones, so that in the experiment, PCR amplification is further performed on the screening result, then deep sequencing is performed, through the generation and analysis of 30M data, all gene sequences encoding the nano-antibodies are extracted, six million specific sequences are obtained in total, finally, through a large number of experiments, three nano-antibodies are screened (an HA tag is introduced at the C end of the nano-antibody, which is convenient for the detection of the subsequent experiments), and are expressed and purified through escherichia coli, and the three nano-antibodies are NbH nano-antibody, nbF8 nano-antibody and NbF9 nano-antibody.

5363 the complementarity determining region of the amino acid sequence of the nanometer NbH antibody is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4, CDR3 shown in SEQ ID NO. 6, the framework region is FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5, and FR4 shown in SEQ ID NO. 7. The NbH nanometer antibody has the amino acid sequence shown in SEQ ID NO:22, namely MAVQLVESGGGSVEAGGSLRLSCAAAGIAFSSYILAWFRQAPGKERELVSASWRGDSTYYLPSVKGRFTISRDNAKNMVYLQMNDLKPEDTAVYICAAKEFPRWLTLSPLDYDHWGQGTQVTVSS.

The complementary determining region of the amino acid sequence of the NbF8 nano antibody is CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 13; the framework region is FR1 shown in SEQ ID NO. 8, FR2 shown in SEQ ID NO. 10, FR3 shown in SEQ ID NO. 12 and FR4 shown in SEQ ID NO. 14. The amino acid sequence of the NbF8 nano antibody is shown in SEQ ID NO. 23, namely MAVQLVESGGGLVQAGGSLRLSCEVSGGAFSRYAVGWFRQAPGKEREFAAAISWSGNNTYYAASVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAMLVSTYDYWGQGTQVTVSS.

The complementary determining region of the amino acid sequence of the NbF9 nano antibody is CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 18 and CDR3 shown in SEQ ID NO. 20, the framework region is FR1 shown in SEQ ID NO. 15, FR2 shown in SEQ ID NO. 17, FR3 shown in SEQ ID NO. 19 and FR4 shown in SEQ ID NO. 21. The amino acid sequence of the NbF9 nano antibody is shown in SEQ ID NO. 24, namely MAVQLVESGGGLVQPGGSLRLSCVVSGFTFRSYAMSWVRQAPGKGLEWVSAITSGGESTSYAGSVKGRFTISRDNAKNTVYLQMNSLEPEDTAIYYCNARRVGSPGSWGQGTQVTVSS.

And comparing the three nano antibody sequences with a phage ELISA positive clone sequence, wherein the three nano antibody sequences are not matched with the phage ELISA positive clone.

(3) Nanobody affinity assay

The affinity of the nanobodies was identified below using an ELISA affinity assay for the three RAGE nanobodies described above.

In this example, the sequences of NbH nm antibody, nbF8 nm antibody and NbF9 nm antibody were subjected to gene cloning and protein expression analysis, and the sequences of the other four antibodies (NbD, nbS1, nbS2 and NbS3 nm antibody) were subjected to gene cloning and protein expression analysis, and the results are shown in fig. 6. Wherein, the sequence of the NbD nano antibody is shown as SEQ ID NO. 25, the sequence of the NbS1 nano antibody is shown as SEQ ID NO. 26, the sequence of the NbS2 nano antibody is shown as SEQ ID NO. 27, and the sequence of the NbS3 nano antibody is shown as SEQ ID NO. 28.

The results of in vitro ELISA affinity verification of the seven antibody sequences are shown in fig. 7, the ELISA negative control is the binding strength of the nanobody and GFP protein, as shown in fig. 7 (a) to 7 (c), the nanobodies NbH, nbF8, nbF9 and RAGE have strong binding; as shown in the results of FIGS. 7 (d) to 7 (g), the nanobodies NbD, nbS1, nbS2, nbS3 bind weakly to RAGE or also to the negative control, GFP.

The embodiment of the invention also discloses a nucleic acid, which is: nucleic acid encoding the NbH, nbF8, nbF9 nanobody or its complementary sequence.

The embodiment of the invention also discloses an expression vector which comprises the nucleic acid.

The embodiment of the invention also discloses a host cell which comprises the expression vector.

The embodiments of the present invention also disclose the use of the RAGE-targeting nanobodies described above as detection reagents for RAGE targeting alzheimer's disease and cancer, drugs targeting RAGE for alzheimer's disease and cancer, in vivo imaging probes, chimeric immune cells, or bispecific antibodies.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

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115

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115

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Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

Claims

1. A nanobody targeting RAGE characterized by: the complementarity determining regions of the amino acid sequence of the nano antibody targeting RAGE are CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 13.

2. The nanobody targeting RAGE as claimed in claim 1, characterized in that: the framework region of the amino acid sequence of the nano antibody targeting RAGE is FR1 shown as SEQ ID NO. 8, FR2 shown as SEQ ID NO. 10, FR3 shown as SEQ ID NO. 12 and FR4 shown as SEQ ID NO. 14.

3. The nanobody targeting RAGE of claim 2, wherein: the amino acid sequence of the nano antibody targeting RAGE is shown as SEQ ID NO 23.

4. A nucleic acid, wherein said nucleic acid is: a nucleic acid sequence encoding the nanobody targeting RAGE of any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the expression vector of claim 5.