Humanized anti-human CXCL16 neutralizing antibody as well as preparation method and application thereof

Abstract

The invention discloses a humanized anti-human CXCL16 neutralizing antibody as well as a preparation method and application thereof. The sequence of a heavy chain HC of the antibody is shown as SEQ ID NO.25, the sequence of a light chain LC of the antibody is shown as SEQ ID NO.26, the sequence of a heavy chain variable region VH of the antibody is shown as SEQ ID NO.31, and the sequence of a light chain variable region VL of the antibody is shown as SEQ ID NO.32. The invention further discloses a preparation method of the humanized anti-human CXCL16 neutralizing antibody. According to the invention, the humanized anti-human CXCL16 neutralizing antibody is established, the neutralizing effect is strong, the production is easy, the cost is low, the treatment of diseases related to the CXCL16 is facilitated, the defects of poor neutralizing capability, high price and the like of the CXCL16 antibody at present are overcome, and an effective tool is provided for subsequent in-vivo and in-vitro experiments of the CXCL16.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a humanized anti-human CXCL16 neutralizing antibody, its preparation method, and its application. Background Technology

[0002] Chemokines play crucial roles in various pathogenesis mechanisms, participating in the migration of immune cells to inflammatory and angiogenic sites, as well as tumorigenesis and metastasis. Chemokines are involved in multiple pathways, such as angiogenesis, the migration and invasion of endothelial and immune cells at tumor sites, and the proliferation and survival of tumor cells. CXC motif chemokine ligand 16 (CXCL16) belongs to the α-chemokine subfamily. mCXCL16 also contains both a CXC chemokine domain and a transmembrane domain, while sCXCL16 contains only a chemokine domain. The two forms of CXCL16 have different functions. sCXCL16 is a chemokine responsible for the chemotaxis of cells carrying the CXCR6 receptor, while mCXCL16 is a transmembrane protein regulated by pro-inflammatory cytokines and plays an important role in the accumulation of immune cells at inflammatory sites. mCXCL16 also mediates the adhesion of Gram-negative and Gram-positive bacteria, guiding cells expressing mCXCL16 (such as macrophages and dendritic cells) to phagocytose bacteria. mCXCL16 can also act as a receptor to transduce extracellular signals into the cell.

[0003] The latest epidemiological statistics on malignant tumors in my country show that there are 3.929 million new cancer cases annually, with lung cancer, breast cancer, and esophageal cancer accounting for about one-third of these new cases, and the number of cases continues to increase. Radiotherapy is one of the main treatment methods for these common malignant tumors. However, radiation-induced pulmonary fibrosis (RIPF) is one of the major limiting factors to consider when planning chest radiotherapy for these diseases, affecting treatment outcomes. The incidence of RIPF in patients receiving chest radiotherapy is approximately 9-30%, and once it occurs, it significantly reduces the patient's quality of life, and in severe cases, can lead to death. The mechanism of RIPF development remains unclear, and there is a lack of effective therapeutic targets and drugs; therefore, research on RIPF has always been a hot topic in radiobiology research. Due to the recent application of immunotherapy in lung and esophageal cancer, especially the combination of PD-1 inhibitors and radiotherapy, the incidence of grade 2 or higher radiation pneumonitis can increase. Radiation pneumonitis is closely related to the occurrence of RIPF, and late-stage RIPF is irreversible. Therefore, the prevention and treatment of RIPF is currently particularly urgent! The incidence of radiation-induced pulmonary fibrosis (RIPF) is 9-30%, and once it occurs, it is difficult to reverse. The symptoms it causes, such as cough and difficulty breathing, severely impact the patient's quality of life. Furthermore, there is currently no effective drug treatment.

[0004] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic interstitial lung disease with no clear cause. It is identified as one of the most common and serious forms of idiopathic interstitial pneumonia, characterized by dyspnea, cough, and deteriorating lung function. IPF has a reported prevalence of approximately 58.7 per 100,000 patients and a high mortality rate. Respiratory failure is the leading cause of death in IPF patients, followed by coronary artery disease, pulmonary embolism, and lung cancer. Currently, only pirfenidone and nintedanib are approved by the U.S. Food and Drug Administration for the treatment of IPF. Both interfere with fibroblast proliferation and migration, and reduce the contraction of collagenous gel embedded in fibroblasts and excessive extracellular matrix (ECM) secretion. However, gastrointestinal and skin-related adverse events (AEs) have been reported with pirfenidone treatment, and diarrhea and elevated liver enzymes are common adverse events with nintedanib treatment, hindering the widespread use of these drugs. Therefore, there is an urgent need to explore other appropriate treatments for IPF.

[0005] CXCL16 plays a crucial role in both types of fibrotic diseases. Previous studies have found that CXCL16 neutralizing antibodies can treat pulmonary fibrosis in mouse RIPF and BIPF models, and the efficacy of these antibodies has been experimentally confirmed. However, while non-human antibodies against CXCL16 (such as mouse and rabbit antibodies) exist, their application in humans has significant drawbacks: firstly, they easily trigger human anti-mouse antibody responses (HAMA responses), leading to a shortened antibody half-life, reduced efficacy, and even inducing immune side effects such as allergies; secondly, their affinity and specificity still have room for improvement, making it difficult to meet the precision requirements of clinical treatment.

[0006] Humanized antibodies can effectively reduce immunogenicity while maintaining or optimizing affinity by retaining the key antigen-binding regions of non-human antibodies and replacing the non-human backbone sequence. Currently, in research on humanized antibodies against CXCL16, the published antibodies exhibit low affinity, insufficient binding activity, poor CDR region sequence stability, and susceptibility to degradation. Therefore, there is an urgent need to develop novel humanized antibodies with higher affinity and lower immunogenicity. Summary of the Invention

[0007] The purpose of this invention is to provide a humanized anti-human CXCL16 neutralizing antibody, its preparation method, and its application, which has a strong neutralizing effect and is helpful in the treatment of CXCL16-related diseases.

[0008] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a humanized anti-human CXCL16 neutralizing antibody, wherein the sequence of the heavy chain HC of the antibody is shown in SEQ ID NO. 25 and the sequence of the light chain LC is shown in SEQ ID NO. 26.

[0009] In the above technical solution, the sequence of the heavy chain variable region VH of the antibody is shown in SEQ ID NO. 31, and the sequence of the light chain variable region VL is shown in SEQ ID NO. 32.

[0010] In a second aspect, the present invention provides a pharmaceutical composition comprising the antibody described above.

[0011] In the above technical solutions, the pharmaceutical composition also includes excipients.

[0012] In the above technical solutions, the excipients include any one or a combination of at least two of the following pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0013] Thirdly, the present invention provides a nucleic acid molecule that encodes the aforementioned antibody.

[0014] Fourthly, the present invention provides a recombinant vector containing the aforementioned nucleic acid molecules.

[0015] Fifthly, the present invention provides a recombinant cell containing the above-mentioned recombinant vector.

[0016] In a sixth aspect, the present invention provides the use of the above-mentioned antibody or nucleic acid molecule or recombinant vector or recombinant cell in the preparation of a medicament for treating radiation-induced and idiopathic pulmonary fibrosis.

[0017] In a seventh aspect, the present invention provides a method for preparing the above-mentioned antibody, comprising the following steps: Step 1: Preparation of CXCL16 protein antigen: Using the human CXCL16 gene sequence as a template, the 30-205 aa full gene was synthesized into an expression vector for protein expression. After identification, the expression of the vector was confirmed, and the CXCL16 protein antigen was thus prepared. Step 2, Animal Immunization and Polyclonal Antiserum Detection: The experimental animals were immunized with the CXCL16 protein antigen, and then the antiserum of the experimental animals was detected by ELISA. Step 3, Flow Cytometry Sorting: Flow cytometry was used to sort cells using biotin-human CXCL16(30-205aa)-His tag. Antigen-specific single B cells were collected for the first round of detection. Routine ELISA was performed using human CXCL16(30-205aa)-His tag as the detection base. Multiple positive clones were selected based on the ELISA OD values ​​from the first round of detection and their neutralization function was tested. Step 4, Linear Expression Module (LEM): Based on the experimental data from the B cell screening stage of Phase IIb, multiple positive clones were selected to construct the linear expression module LEM. At the same time, CH2 and CH3 of the rabbit heavy chain Fc were replaced with CH2 and CH3 of the human IgG1 Fc. For the selected multiple positive clones, ELISA was performed using human CXCL16 (30-205aa)-His tag as the detection source. Multiple clones were selected for Gator affinity sorting. Then, the LEM supernatant was extracted, and multiple antibodies with better performance were selected for humanization modification based on the SPR results. Step 5, Humanized Antibody Library Design and Construction: First, sequence analysis is performed on non-human antibodies, and their 3D structure is simulated using technical means. Based on the structural and sequence characteristics, a humanized antibody library is designed, and a humanized phage display library is further constructed. Step 6: Screening of high-affinity humanized clones: The constructed library is screened using phage display technology to preliminarily identify antibody clones with high affinity at the phage level; Step 7, Validation of high-affinity clones and selection of optimal clones: The antibody clones screened in Step 6 are experimentally confirmed to have the affinity of antibody-antigen interactions at the protein level. After comprehensive evaluation, the antibody clone with the best affinity is finally selected as the antibody.

[0018] The beneficial effects of this invention are as follows: This invention establishes a humanized anti-human CXCL16 neutralizing antibody with strong neutralizing effect, easy production and low cost, which is helpful for the treatment of CXCL16-related diseases. It overcomes the shortcomings of current CXCL16 antibodies such as poor neutralizing ability and high price, and provides an effective tool for subsequent in vivo and in vitro experiments on CXCL16. Attached Figure Description

[0019] Figure 1 This is a diagram showing the experimental results of the CXCL16 humanized antibody inhibiting fibroblast activation, as presented in this invention. Figure 1 A is the IF result chart. Figure 1 B and 1C are WB result images. Figure 1 D is the antibody performance parameter. Detailed Implementation

[0020] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0022] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein are to be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0023] Example 1 Antibody Production and Expression Includes the following steps: Step 1: Preparation of CXCL16 protein antigen: The human CXCL16 gene sequence was retrieved from NCBI and used as a template. 30-205 amino acids were directly synthesized into an expression vector for protein expression. Once the vector expression was confirmed, antigen preparation was considered complete.

[0024] Step 2, Animal Immunization and Polyclonal Antiserum Detection: Two white rabbits were immunized with immunogens, and the rabbit antiserum was tested by ELISA after four immunizations.

[0025] Step 3, Flow Cytometry Sorting: Flow cytometry was used to sort cells using biotin-human CXCL16 (30-205 aa)-His tag, collecting antigen-specific B cells from 8*96 plates. The collected 8*96 plates underwent initial testing using a routine ELISA assay with the human CXCL16 (30-205 aa)-His tag as the detection source. Based on the ELISA OD values ​​from the initial test, 134 positive clones were expected to be selected for neutralization function testing.

[0026] Step 4: Linear Expression Module (LEM): Based on the experimental data from the B cell screening phase of Stage IIb, 10 positive clones were selected to construct the linear expression module LEM. Simultaneously, the CH2 and CH3 regions of the rabbit heavy chain Fc were replaced with the CH2 and CH3 regions of the human IgG1 Fc, resulting in a higher success rate for subsequent humanization. For the selected 10 positive clones, ELISA was performed using the human CXCL16 (30-205aa)-His tag as the detection source. The 10 clones were then ranked for Gator affinity. The LEM supernatant was extracted, and the three antibodies with the best performance were selected for humanization based on the SPR results.

[0027] Step 5, Humanized Antibody Library Design and Construction: First, sequence analysis is performed on non-human antibodies, and their 3D structure is simulated using technical means. Based on the structural and sequence characteristics, a humanized antibody library is designed, and then a humanized phage display library is constructed.

[0028] Step 6, Screening of high-affinity humanized clones (phage level): The constructed library is screened using phage display technology to preliminarily identify antibody clones with high affinity at the phage level; Step 7, Validation of high-affinity clones and selection of the optimal clone (protein level): The high-affinity clones screened in Step 6 are expressed as monoclonal antibodies of IgG1. The affinity of the antibody-antigen (human cxcl16(30-205aa)-His tag) interaction at the protein level is confirmed by ELISA and BLI (Gator) experiments. After comprehensive evaluation, the antibody clone with the best affinity is finally selected, which is the antibody of this invention.

[0029] This invention has developed a humanized anti-human CXCL16 neutralizing antibody and confirmed its neutralizing effect. The CXCL16 neutralizing antibody developed in this invention exhibits strong neutralizing efficacy, is easy to produce, and is inexpensive, which is beneficial for the treatment of CXCL16-related diseases. It overcomes the shortcomings of current CXCL16 antibodies, such as poor neutralizing ability and high price, and provides an effective tool for subsequent in vitro and in vivo experiments on CXCL16.

[0030] Example 2: CXCL16 humanized antibody inhibits fibroblast activation 1. Experimental Procedure IF staining Human primary lung fibroblasts (HLF) and the human lung fibroblast line MRC5 were extracted, plated onto slides, and cultured with antibodies for 24 hours. After fixing the cells onto the slides, they were stained and scanned using α-SMA and COL1A1 antibodies.

[0031] Western blot Human primary lung fibroblasts (HLF) and the human lung fibroblast line MRC5 were extracted, plated onto slides, and cultured with antibody for 24 hours. Protein extraction was then performed on the cells. Lung tissue was added to RIPA (G2002, Xavier Biotech) and PMSF (G2007, Xavier Biotech), homogenized, sonicated, and centrifuged, then boiled at 100°C with 5× loading buffer (G2013, Xavier Biotech). Electrophoresis was performed using a one-step PAGE gel preparation kit (12%) (E304-01, Novizan, Nanjing, China). Protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA) and blocked with 5% BSA or nonfat milk for 1 hour. The PVDF membrane was incubated overnight at 4°C with the following primary antibodies: anti-COL1A1 (1:1000, A5786, Ibrex), anti-GAPDH (1:10000, A00227-1, Boster, Hubei, China), anti-α-SMA (1:1000, A17910, Ibrex), and anti-Fn (1:1000, A12977, Ibrex). After incubation with appropriate secondary antibodies at room temperature for 1 hour, the membrane was developed using the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA), and protein bands were observed. The blot signal was detected using the G:BOX Chemi X system (Syngene, Cambridge, UK).

[0032] Table 1: 3F11 Humanized Clones - Full-Length Sequence

[0033] Table 2: 3F11 Humanized Clones - Variable Region Sequences

[0034] Table 3: 4C9 humanized clones - full-length sequences

[0035] Table 4: 4C9 humanized clones - variable region sequences

[0036] Table 5: 6A9 Humanized Clones - Full-Length Sequence

[0037] Table 6: 6A9 Humanized Clones - Variable Region Sequences

[0038] 2. Experimental Results: IF results are as follows Figure 1 As shown in Figure A, 24 hours after antibody addition, the activation of primary human lung fibroblasts (HLF) decreased. Cell morphology shrank, and the fluorescence intensity of COL1A1 (green fluorescence) and α-SMA (pink fluorescence) decreased, along with decreased protein expression, indicating inhibited fibroblast activation. Antibody 4899 exhibited the lowest fluorescence intensity and was considered the most effective neutralizing antibody in this batch. A similar trend was observed in the human lung fibroblast cell line (MRC5). 24 hours after antibody addition, MRC5 activation decreased. Cell morphology shrank, and the fluorescence intensity of COL1A1 (green fluorescence) and α-SMA (pink fluorescence) decreased, along with decreased protein expression, indicating inhibited fibroblast activation. Antibody 4899 exhibited the lowest fluorescence intensity and was considered the most effective neutralizing antibody in this batch.

[0039] WB results are as follows Figure 1 B and Figure 1 As shown in Figure C, 24 hours after antibody addition, HLF and MRC5 activation decreased. Cell morphology shrank, and Fn and α-SMA protein expression decreased, indicating that fibroblast activation was inhibited. Furthermore, after treatment with antibody 4899, both Fn and α-SMA protein expression decreased, suggesting that it was the antibody with the best neutralizing effect in this batch.

[0040] Analysis of antibody performance parameters ( Figure 1 (D) The binding affinity of an antibody to its target is measured by the dissociation constant (KD). The smaller the KD, the stronger the binding affinity, the tighter the antibody binds to the target, and the easier it is to exert an inhibitory effect. Among them, 4893 has a relatively strong binding affinity; while 4894 has a relatively weak binding affinity; 4899 is in between.

[0041] The biological activity of antibodies is measured by EC50; the smaller the EC50, the higher the biological activity. 4899 and 4897 have high biological activity; C4900 has relatively low biological activity.

[0042] From the perspective of humanization rate, the humanization rates of these nine antibodies are all at a high level, mostly between 88% and 94%. The advantage of humanized antibodies lies in their reduced heterology nature, decreasing the likelihood of triggering an immune response in the human body, thus allowing them to exert their biological functions (such as inhibiting fibrosis) more safely and effectively. The high humanization rate of these antibodies provides immunogenicity assurance for their better targeting of fibrosis-related targets and inhibition of fibrosis in subsequent research or potential clinical applications. This means that the risk of them triggering adverse reactions in the human body is relatively low, which is more conducive to their functional performance.

[0043] In summary, all nine antibodies exhibited varying degrees of potential to inhibit fibrosis and can be applied to the treatment of radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis. Among them, antibody 4899 showed a high humanization rate, good binding affinity and biological activity, and significant neutralizing effect, making it the best antibody.

[0044] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A humanized anti-human CXCL16 neutralizing antibody, characterized in that: The sequence of the heavy chain HC of the antibody is shown in SEQ ID NO. 25, and the sequence of the light chain LC is shown in SEQ ID NO.

26.

2. The antibody according to claim 1, characterized in that: The sequence of the heavy chain variable region VH of the antibody is shown in SEQ ID NO. 31, and the sequence of the light chain variable region VL is shown in SEQ ID NO.

32.

3. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody according to any one of claims 1-2.

4. The pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition also includes excipients.

5. The pharmaceutical composition according to claim 4, characterized in that: The excipients include any one or a combination of at least two of the following pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-2.

7. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule described in claim 6.

8. A recombinant cell, characterized in that: The recombinant cells contain the recombinant vector of claim 7.

9. The use of the antibody of any one of claims 1-2, the nucleic acid molecule of claim 6, the recombinant vector of claim 7, or the recombinant cell of claim 8 in the preparation of a medicament for treating radiation-induced and idiopathic pulmonary fibrosis.

10. A method for preparing the antibody according to any one of claims 1-2, characterized in that: Includes the following steps: Step 1: Preparation of CXCL16 protein antigen: Using the human CXCL16 gene sequence as a template, the 30-205 aa full gene was synthesized into an expression vector for protein expression. After identification, the expression of the vector was confirmed, and the CXCL16 protein antigen was thus prepared. Step 2, Animal Immunization and Polyclonal Antiserum Detection: The experimental animals were immunized with the CXCL16 protein antigen, and then the antiserum of the experimental animals was detected by ELISA. Step 3, Flow Cytometry Sorting: Flow cytometry was used to sort cells using biotin-human CXCL16(30-205aa)-His tag. Antigen-specific single B cells were collected for the first round of detection. Routine ELISA was performed using human CXCL16(30-205aa)-His tag as the detection source. Multiple positive clones were selected based on the ELISA OD values ​​from the first round of detection and their neutralization function was tested. Step 4, Linear Expression Module (LEM): Based on the experimental data from the B cell screening stage of Phase IIb, multiple positive clones were selected to construct the linear expression module LEM. At the same time, CH2 and CH3 of the rabbit heavy chain Fc were replaced with CH2 and CH3 of the human IgG1 Fc. For the selected multiple positive clones, ELISA was performed using human CXCL16 (30-205aa)-His tag as the detection source. Multiple clones were selected for Gator affinity sorting. Then, the LEM supernatant was extracted, and multiple antibodies with better performance were selected for humanization modification based on the SPR results. Step 5, Humanized Antibody Library Design and Construction: First, sequence analysis is performed on non-human antibodies, and their 3D structure is simulated using technical means. Based on the structural and sequence characteristics, a humanized antibody library is designed, and a humanized phage display library is further constructed. Step 6: Screening of high-affinity humanized clones: The constructed library is screened using phage display technology to preliminarily identify antibody clones with high affinity at the phage level; Step 7, Validation of high-affinity clones and selection of optimal clones: The antibody clones screened in Step 6 are experimentally confirmed to have the affinity of antibody-antigen interactions at the protein level. After comprehensive evaluation, the antibody clone with the best affinity is finally selected as the antibody.

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