CN119119277B

CN119119277B - CD 38-targeted nano antibody, pharmaceutical composition and application thereof

Info

Publication number: CN119119277B
Application number: CN202411496580.8A
Authority: CN
Inventors: 贾琳; 戴凌云; 李志杰; 黄丕英; 丁有斌
Original assignee: Shenzhen Technology University
Current assignee: Shenzhen Technology University
Priority date: 2024-10-24
Filing date: 2024-10-24
Publication date: 2025-09-09
Anticipated expiration: 2044-10-24
Also published as: CN119119277A

Abstract

The invention provides a CD 38-targeted nano antibody, a pharmaceutical composition and application thereof, and relates to the technical field of biological medicine. The targeting CD38 nanobody is any one of a first antibody and a second antibody and comprises three complementarity determining regions, the CDR 1-3 sequences of the complementarity determining regions of the first antibody are shown as SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the CDR 1-3 sequences of the complementarity determining regions of the second antibody are shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 6. The nanometer antibody has higher affinity with purified and cell surface CD38 protein, provides an effective new method for targeting CD38 for anti-tumor intervention, can be used for preparing detection reagents such as molecular probes and the like based on the nanometer antibody, and provides a new way for tumor specific imaging and diagnosis through specific combination with CD38 on the tumor surface.

Description

CD 38-targeted nano antibody, pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a CD 38-targeted nano antibody, a pharmaceutical composition and application thereof.

Background

Tumor targeted therapy is a method of treating tumors by specifically targeting molecules or signaling pathways that play an important role in tumorigenesis and progression. Administration of corresponding targeted therapies based on tumor specific molecular phenotypes is increasingly becoming the current choice of tumor therapy. Tumor targeted drugs can be broadly divided into two broad classes, monoclonal antibodies and small molecule compounds. With the deep research, new targets are continuously emerging, the development of anti-tumor drugs is subjected to a plurality of breakthrough progress, and the speed of tumor targeting drugs is continuously increased by 5-6 per year. However, the effectiveness of targeted therapies is limited by the heterogeneity of tumor cells, which may vary significantly from patient to patient in response to the same drug, and in addition, tumor cells may develop resistance through multiple mechanisms, resulting in reduced therapeutic efficacy. Thus, the development of targeted drugs is still inadequate at the present time, the wide application of existing therapeutic approaches is limited, and most patients do not benefit from it. Therefore, there is an urgent need to discover new molecular targets and develop more effective molecular targeted therapeutic strategies to improve the accuracy and effectiveness of the treatment, reducing the treatment burden on patients.

CD38 (Cluster of Differentiation) is a type II transmembrane glycoprotein with a molecular weight of about 46kD. CD38 was first discovered by scientists in e.l.reinherez and s.f.schlossman et al in 1980 as a specific marker of T cell activation. The CD38 protein sequence includes a short cytoplasmic tail at the N-terminus, a single transmembrane domain and a longer extracellular region at the C-terminus. The present study proves that the CD38 protein is a difunctional extracellular enzyme, has the activities of cyclase and hydrolase and participates in nucleotide metabolism. CD38 uses NAD ⁺ as a substrate to form nucleotide metabolites such as cyclic ADP ribose (cADPR), which is a potent second messenger that can regulate the mobilization of Ca ²⁺ in the cytoplasm, activating signaling pathways that control various biological processes. It has also been found that CD38 degrades ATP, NAD ⁺, cADPR, AMP, thereby producing adenosine (adenosine), inducing an inhibitory immune microenvironment. In summary, the activity of CD38 is important for maintaining the homeostasis of NAD ⁺, nicotinamide, etc. substances in the body.

Based on TCGA data analysis, CD38 is highly expressed in a number of non-solid and solid tumors, the former including chronic B-lymphocytic leukemia, multiple myeloma, mantle cell lymphoma, etc., and the latter including lung cancer, stomach cancer, melanoma, glioma, esophageal cancer, cervical cancer, etc. While CD38 is expressed in a variety of human tissues, it is expressed at very low or low levels in normal other organs. Furthermore, after secondary metastasis of primary cancer with high expression of CD38, tumor tissues in metastasis still maintain high-level expression of CD 38. Several studies have shown that CD38 has great potential as a new therapeutic target for malignant tumors and metastases thereof, and is a very potential target protein molecule.

Currently, antibodies against CD38 have been subject to some research progress. Currently, monoclonal antibodies directed against CD38 that have been used in clinical therapy include daratumumab, isatuximab and MOR202, among others. Researchers have analyzed the eutectic structures of these antibodies and CD38, constructed fluorescent antibodies based on these antibodies, developed a method for rapid quantification of CD38 on the cell surface using flow cytometry, and constructed immunotoxins against CD38, which demonstrated high selective killing effects on Multiple Myeloma (MM) cells, EC ₅₀ reached pM levels with good efficacy, and high specificity and safety.

Nanobodies (nanobodies), also known as single domain antibodies, are the smallest antigen binding fragments obtained from heavy chain antibodies naturally occurring in camelid blood. Compared with the traditional antibody, the nano antibody has the advantages of smaller volume (about 15 kD), good stability and solubility, easy transformation, low production cost, capability of recognizing hidden or unusual antigenic sites and the like. While there has been some progress in recent years in nanobodies of CD38, there remains a need for new targeting antibodies or combinations thereof that are more selective, more potent, less toxic and more patient-friendly to continue to improve the clinical outcome of patients with related tumors.

Disclosure of Invention

The invention provides a CD 38-targeting nanobody, which is any one of the following two antibodies:

A first antibody and a second antibody;

The first antibody and the second antibody each comprise three complementarity determining regions;

wherein the sequence of CDR1 of the complementarity determining region of the first antibody is shown as SEQ ID NO.1, the sequence of CDR2 is shown as SEQ ID NO.3, and the sequence of CDR3 is shown as SEQ ID NO. 5;

The sequence of CDR1 of the complementarity determining region of the second antibody is shown as SEQ ID NO.2, the sequence of CDR2 is shown as SEQ ID NO.4, and the sequence of CDR3 is shown as SEQ ID NO. 6.

Preferably, the amino acid sequence of the first antibody is shown in SEQ ID No. 7.

Preferably, the amino acid sequence of the second antibody is shown in SEQ ID NO. 8.

In addition, the invention also provides a pharmaceutical composition comprising the CD 38-targeting nanobody or antigen-binding fragment thereof as described above.

In addition, the invention also provides an application of the nano antibody for targeting CD38 in preparing medicines for diagnosing and/or treating tumors, wherein the tumors comprise one or more of epidermal tissue tumors, connective tissue tumors, lymphatic and hematopoietic system tumors, organ epithelial cell tumors, reproductive system and urinary system tumors, digestive system tumors, nervous system tumors and endocrine system tumors.

Preferably, the tumor is a respiratory tumor in the organ epithelial cell tumor.

Preferably, the tumor is non-small cell lung cancer in the respiratory tumor.

In addition, the invention also provides application of the CD 38-targeting nano antibody in preparation of products for detecting tumors.

Preferably, the tumor detection product comprises at least one of a detection reagent, a detection kit and a detection device.

The invention provides a CD 38-targeted nano antibody, a pharmaceutical composition and application thereof, wherein the CD 38-targeted nano antibody is any one of a first antibody and a second antibody, the first antibody and the second antibody comprise three complementarity determining regions, the sequence of CDR1 of the complementarity determining region of the first antibody is shown as SEQ ID NO.1, the sequence of CDR2 is shown as SEQ ID NO.3, the sequence of CDR3 is shown as SEQ ID NO.5, the sequence of CDR1 of the complementarity determining region of the second antibody is shown as SEQ ID NO.2, the sequence of CDR2 is shown as SEQ ID NO.4, and the sequence of CDR3 is shown as SEQ ID NO. 6. The invention screens and obtains two nano antibodies with high affinity with CD38 protein based on phage nano antibody library, and verifies the binding condition of the nano antibodies and CD38 protein. The two nano antibodies targeting CD38 provided by the invention have higher affinity with purified and cell surface CD38 proteins. The specific treatment and/or delivery strategy developed based on the two nanobodies (including the derivative antibodies modified by adding, reducing or replacing the original sequences of the two nanobodies) provides an effective new method for targeting CD38 for anti-tumor intervention. Meanwhile, detection reagents such as molecular probes can be prepared based on the nano antibody, and a new way is provided for tumor specific imaging and diagnosis through specific binding with CD38 on the tumor surface.

Drawings

FIG. 1 shows the result of immunofluorescence staining of CD38 protein on PC9 cells in example 1 of the present invention;

FIG. 2 shows the result of immunofluorescence staining of CD38 protein on A549 cells in example 1 of the present invention;

FIG. 3 is a Western Blot detection of CD38 expression on PC9 and A549 cells in example 1 of the invention;

FIG. 4 shows the coomassie blue staining (approximately 16kD in size) of a nanobody (fusion His tag and HA tag) expressing and purifying targeting CD38 in example 2 of the invention;

FIG. 5 shows the Western Blot detection results of CD 38-targeting nanobodies (using HA and His tag antibodies) in example 2 of the invention;

FIG. 6 shows the results of detecting the binding activity of CD 38-targeting nanobody and CD38 protein by ELISA in example 2 of the invention (both results show binding activity);

FIG. 7 shows the result of the detection of the affinity of the CD38 protein with the first antibody NbD (38.26 nM) in the Biacore SPR platform-based CD 38-targeting nanobody of example 2 of the present invention;

FIG. 8 shows the result of detecting the affinity between the second antibody NbD and the CD38 protein (21.07 nM) in the Biacore SPR platform-based CD 38-targeting nanobody of example 2 of the present invention;

FIG. 9 is a graph showing the results of CD 38-targeting nanobody localization on PC9 and A549 cells (CD 38 that nanobody can be localized to the cell surface) based on a confocal laser microscope in example 2 of the invention;

FIG. 10 shows the binding results of different concentrations of CD 38-targeting nanobodies of example 2 of the invention to CD38 antigen on PC9 cells;

FIG. 11 shows the binding results of different concentrations of CD 38-targeting nanobodies with CD38 antigen on A549 cells in example 2 of the invention.

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

In an embodiment of the present application, a CD 38-targeting nanobody is provided, where the CD 38-targeting nanobody is any one of the following two antibodies:

A first antibody and a second antibody;

The complementarity determining regions (complementary determining region, CDRs) of the amino acid sequences described above are the most frequent and diverse portions of the antibody molecule. CDRs are located at the convergence region of the antibody structure and consist essentially of three hetero-chains (Hypervariable Region, HVR). Residues with antigen specificity that interact with these regions are referred to as variable regions (V).

The main function of the CDR is that the highest diversity functional region in the antibody molecule directly performs corresponding physical and chemical reaction with specific antigen molecules, so that the antigen is identified and combined in the immune process. Thus, CDRs are important building blocks to ensure a high degree of specificity of antibody molecules.

Further, the amino acid sequence of the first antibody is shown as SEQ ID NO. 7.

Further, the amino acid sequence of the second antibody is shown as SEQ ID NO. 8.

Therefore, the CD 38-targeting nanobody provided by the invention comprises a first antibody (named NbD) and a second antibody (named NbD 6), wherein the two antibodies are respectively provided with 3 complementarity determining regions, namely CDR1, CDR2 and CDR3, the corresponding sequence numbers of the two complementarity determining regions are shown in the table 1, and the specific sequence is shown in the table 2.

TABLE 1 sequence number correspondence table of complementarity determining region sequences and amino acid sequences in CD 38-targeting nanobodies

CD 38-targeting nanobodies	CDR1	CDR2	CDR3	Amino acid sequence
					First antibody	SEQ.ID NO.1	SEQ.ID NO.3	SEQ.ID NO.5	SEQ.ID NO.7
Second antibody	SEQ.ID NO.2	SEQ.ID NO.4	SEQ.ID NO.6	SEQ.ID NO.8

TABLE 2 complementarity determining region sequences and amino acid sequences of the first and second antibodies in CD 38-targeting nanobodies

In addition, in an embodiment of the present application, there is also provided a pharmaceutical composition comprising the CD 38-targeting nanobody or antigen-binding fragment thereof as described above.

Further, the tumor is a respiratory tumor in the organ epithelial cell tumor.

Further, the tumor is non-small cell lung cancer in respiratory system tumor.

In addition, the invention also provides application of the CD 38-targeted nano antibody in preparation of detection products for detecting tumors.

Further, the tumor detection product comprises at least one of a detection reagent, a detection kit and a detection device.

The detection reagent may be a reagent for detecting a specific biomarker or chemical component, and may be designed to bind to a nanobody to improve the specificity and sensitivity of detection.

The above-described detection kit may contain all components necessary for performing the detection, and may include, for example, but not limited to, reagents, buffers, standards, and sometimes detection consumables. These kits aim to simplify the detection procedure making it more user-friendly.

The detection device may be a device for reading and analyzing the results of the detection reagent or the kit, or may be a device that performs detection of tumor using the nanobody described above, or that functions as a detection reagent or a detection kit. These devices may be manual or automated and may include software for data processing and interpretation of results.

Example 1 verification of expression of CD38 protein

1. Immunofluorescent staining

A549 and PC9 cells were resuscitated and after 3 passages the cells were seeded in 24 well plates with slide for subsequent experiments. The method comprises the following specific steps:

(1) The cells were placed on ice, washed 3 times with PBS, and fixed with 4% paraformaldehyde for 10min;

(2) Washing 3 times with PBS, blocking with 3% bsa for 1 hour;

(3) Diluting CD38 antibody with blocking solution at 1:300, incubating with cells overnight at 4 ℃;

(4) Pre-cooling PBS for 3 times, diluting Alexa 488-conjugated secondary antibody with a blocking solution according to a ratio of 1:1000, and incubating with cells for 1 hour in a dark place;

(5) The cells were washed 3 times with pre-chilled PBS, blocked with DAPI-containing blocking agents, and confocal microscopy was used to detect CD38 protein expression.

2、Western Blot

A549 and PC9 cells were resuscitated and after 3 passages the cells were inoculated in 100mm dishes for subsequent experiments. The method comprises the following specific steps:

(1) Digesting for 3-4min with pancreatin, diluting pancreatin with 4-5mL PBS, stopping digestion, centrifuging, and collecting cells;

(2) A549 and PC9 cells were lysed by addition of 200. Mu.L of Lysis Buffer (RIPA+1× Protease inhibitor);

(3) Centrifuging at 14000rpm/20min/4 ℃ for 30s (25% of energy, 3s of work and 7s of interval), sucking 90% of supernatant (180 mu L) into a new EP tube, and measuring the protein concentration by a BCA method (simultaneously preparing the BCA for measuring the protein concentration, taking a new common EP tube with the same amount as the number of samples, and adding 54 mu L of PBS+6 mu L of samples);

(4) 12.5% SDS-PAGE gel, transfer membrane (0.45 μm PVDF membrane, 200mA,120 min), 5% BSA blocking 1h followed by incubation overnight;

(5) TBST was washed 5 times for 5min each, secondary antibody incubated for 1h, TBST was washed 5 times for 5min each, and developed.

Experimental results:

In this example, the expression profile and prognosis of CD38 in multiple cancer species were first analyzed using TCGA database, and this example focused on the validation of CD38 expression in non-small cell lung cancer a549 cells and PC9 cells. In this example, the surface of PC9 was detected as CD 38-expressing by immunofluorescent staining (see FIG. 1), and the surface of A549 was detected as CD 38-expressing (see FIG. 2). Further verification using Western Blot detection, the results of fig. 3 show that CD38 is significantly expressed in both a549 and PC9 cells.

Example 2 screening and validation of extracellular Domain nanobodies of CD38 protein

1. Nanobody screening

Screening the natural alpaca-derived phage display nanobody library by adopting an immune tube method, wherein the selected phage display library capacity is 2 multiplied by 10 ⁹. The screening steps are as follows:

(1) Coating target protein on an immune tube according to the concentration of 50 mug/mL, and carrying out enrichment screening for 3 rounds;

(2) Using third phage eluent to plate, randomly picking 96 monoclonal antibodies for ELISA verification, wherein ELISA reading is 3 times greater than corresponding BSA reading and reading is greater than 0.5 as positive standard;

(3) Sequencing and determining sequence information by 2 times of positive monoclonal sent company identified by phage ELISA;

(4) Designing and synthesizing the screened nano antibody according to the sequencing information, and carrying out expression purification by escherichia coli;

(5) The affinity of the nanobody is initially identified by ELISA affinity experiments, the nanobody with better affinity is selected, and the affinity constant is determined by surface plasmon resonance (surface plasmon resonance, SPR) after expression and purification.

2. Purification expression of nanobodies

The nanobody gene sequence was cloned into pCold-II vector with a fused 6 xhis tag at its C-terminus, while fusion expressed hemagglutinin tag (hemagglutinin HA tag) was used for subsequent detection. The expression purification steps are as follows:

(1) To prevent inclusion body formation and protein degradation, induction was performed at 16 ℃ using 0.2mM IPTG;

(2) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition 1300W of the high-pressure bacteria breaker;

(3) Centrifuging at 4 ℃ for 45min by 12000g, taking supernatant and incubating with Ni-NTA filler at 4 ℃ for 1 hour;

(4) After purification of the Ni-NTA column, the eluate was eluted with different imidazole gradients, each gradient approximately 5mL. Purity >95% as determined by SDS-PAGE running. The concentration of nanobody NbD was 5.4mg/mL and the concentration of NbD6 was 3.97mg/mL by BCA assay in a volume of about 3mL after concentration.

3. ELISA assay for nanobodies

ELISA plates were coated with CD38 protein overnight, blocked, then various concentrations of nanobody with HA tag were added for 1h incubation at room temperature, PBST was rinsed 3 times, and after 1h incubation of anti-HA antibody at room temperature, horseradish peroxidase (HRP) -labeled anti-HA antibody amplified signal, TMB developed, and control of irrelevant nanobody and blank control of irrelevant protein antigen were made.

4. Surface plasmon resonance experiments

This experiment was used to verify the direct interaction between the in vitro expression of purified nanobodies and the in vitro purified antigen proteins and calculate the equilibrium constants of both. Purified antigen protein (CD 38) was immobilized on a chip, different concentrations of nanobodies (NbD and NbD) were sequentially added to analyze affinity with the antigen protein, record the reaction signal within 300s, make a kinetic curve, and calculate each relevant parameter.

5. Targeting detection of nanobodies on cells

PC9 and a549 cells were resuscitated and the cells were seeded in 96-well plates after 3 passages for subsequent experiments. The method comprises the following specific steps:

(2) Washing 3 times with PBS, blocking with 5% BSA for 1h;

(3) Sequentially diluting the nano antibody into different concentration gradients (0-1000 nM) by using 1% BSA, and incubating with the cells for 1h;

(4) Pre-chilling PBST for 3 times, diluting anti-HA antibody with 1% BSA according to a ratio of 1:1000, and incubating with cells for 1h;

(5) Pre-cooling PBST, washing 3 times, diluting Alexa 488-conjugated secondary antibody with 1% BSA according to a ratio of 1:1000, and incubating with cells in dark for 1h;

(6) Pre-cooling PBST, washing for 3 times, and detecting the combination condition of the cell surface nano antibody by using a full-automatic electrophoresis fluorescence immunoassay instrument.

Experimental results:

(1) First, screening of phage nanobody library was performed on CD38, and 2 positive clones were initially identified, designated NbD (primary antibody) and NbD6 (secondary antibody), respectively (specific sequences and complementarity determining region CDRs of NbD and NbD6 are shown in tables 1 and 2).

(2) Purified nanobodies (first antibody NbD and second antibody NbD 6) and control nanobody C9 were expressed, and the correctness of the antibody protein was detected and confirmed (fig. 4 and 5). As can be seen from FIG. 4, coomassie brilliant blue staining results show that high-purity nanobodies (NbD and NbD) with the size of about 16kD are obtained by expressing and purifying the nano-antibody protein fused with the His tag and the HA tag, and as can be seen from FIG. 5, western Blot detection is performed by using the HA and the His tag antibodies, so that the nano-antibody protein is proved to be correctly expressed.

(3) As demonstrated by ELISA, nanobody NbD (primary antibody) and NbD6 (secondary antibody) were confirmed to have binding activity to recombinant CD38 protein as shown in fig. 6.

(4) Using the Biacore SPR platform, the affinity constants of NbD and NbD clones to recombinant CD38 protein were detected and determined, and the results showed that the affinities of NbD (primary antibody), nbD (secondary antibody) to CD38 protein were 38.26nM (see fig. 7) and 21.07nM (see fig. 8), respectively.

(5) Immunofluorescence and CELL ELISA were used to detect whether the nanobody could bind to CD38 protein on the surface of cell membrane. Nanobody NbD (first antibody, at a concentration of 1 μm) and NbD6 (second antibody, at a concentration of 1 μm) were localized to CD38 on the surface of PC9 cells and a549 cells under laser confocal microscope observation with reference to fig. 9, wherein the left column is first antibody NbD5 and the right column is second antibody NbD6, detection of binding of different concentrations of first antibody NbD5, second antibody NbD and control nanobody C9 to CD38 antigen on PC9 cells with reference to fig. 10 is CELL ELISA, and detection of binding of different concentrations of first antibody NbD5, second antibody NbD6 and control nanobody C9 to CD38 antigen on a549 cells with reference to fig. 11 is CELL ELISA. The results both show the specific recognition capability of the nano antibodies NbD and NbD to the CD38 protein on the surface of PC9 and A549 cell membranes.

In summary, both CD 38-targeting nanobodies provided in the present invention have higher affinity to purified and cell surface CD38 proteins. The specific treatment and/or delivery strategy developed based on the two nanobodies (including the derivative antibodies modified by adding, reducing or replacing the original sequences of the two nanobodies) provides an effective new method for targeting CD38 for anti-tumor intervention. Meanwhile, detection reagents such as molecular probes can be prepared based on the nano antibody, and a new way is provided for tumor specific imaging and diagnosis through specific binding with CD38 on the tumor surface.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.

Claims

1.A CD 38-targeting nanobody, wherein the CD 38-targeting nanobody is any one of the following two antibodies:

A first antibody and a second antibody;

2. The CD 38-targeting nanobody of claim 1, wherein the amino acid sequence of the first antibody is set forth in seq id No. 7.

3. The CD 38-targeting nanobody of claim 1, wherein the amino acid sequence of the second antibody is set forth in seq id No. 8.

4. A pharmaceutical composition comprising a CD 38-targeting nanobody or antigen-binding fragment thereof according to any one of claims 1-3.

5. Use of a CD 38-targeting nanobody according to any of claims 1-3 for the preparation of a product for detecting CD 38.

6. The use of claim 5, wherein the CD38 detecting product comprises at least one of a detection reagent, a detection kit, and a detection device.