Anti-cd4 nanobodies with longer plasma half-life and uses thereof
By constructing NbCD4-NbHSA-NbCD4 nanobodies, the problem of short plasma half-life of nanobodies has been solved, enabling their effective application in the treatment of HIV infection and CD4-positive T-cell tumors, with a longer plasma half-life and high affinity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-11
- Publication Date
- 2026-03-24
AI Technical Summary
The small molecular weight of nanobodies results in a short plasma half-life, limiting their application in the therapeutic field.
A "staff hat" type nanobody of NbCD4-NbHSA-NbCD4 was constructed. By introducing domains targeting CD4 and HSA into the nanobody, the long circulating half-life of HSA was utilized to increase the in vivo retention time of the nanobody.
It significantly prolonged the plasma half-life of nanobodies, improved their stability and therapeutic efficacy in vivo, and showed high neutralizing activity and affinity, especially in the treatment of HIV infection and CD4-positive T-cell tumors.
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Figure CN118290589B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine. More particularly, it relates to an anti-CD4 nanobody with a longer plasma half-life and its applications. Background Technology
[0002] CD4 is an important receptor molecule expressed on the surface of T cells, but it is also a target for viruses such as HIV to attack T cells. It has been reported that HIV mainly affects the process of viral entry into cells by contacting specific receptors (such as CD4) on the surface of host cells through the glycoprotein gp120 on its envelope. Inhibiting the binding of gp120 and CD4 can effectively inhibit HIV-1 infection of host cells. Furthermore, since T-cell tumors also express CD4, anti-CD4 antibodies could be considered as a target for treating HIV infection or CD4-positive T-cell tumors.
[0003] The antibodies in camel serum are single-domain antibodies containing only one heavy chain variable region, namely VHH antibodies. VHH antibodies have an elliptical crystal structure of 4nm × 2.5nm × 3nm, and a molecular weight only 1 / 10 that of ordinary antibodies, approximately 12-14kDa. They are the smallest intact antigen-binding fragments and are therefore also known as nanobody (Nb). Nanobody antibodies are characterized by high stability (they do not degrade even at 90℃), high affinity, over 80% homology with human antibodies, and low toxicity and immunogenicity.
[0004] However, due to their extremely small molecular weight, significantly below the glomerular filtration limit, nanobodies have a very short plasma half-life of only a few minutes, which restricts their application in the therapeutic field. For protein drugs, the glomerular filtration limit is generally around 60 kDa. Therefore, improving the half-life of nanobodies is a major bottleneck for their in vivo application.
[0005] X-ray crystal structure of HSA reveals it to be a heart-shaped molecule containing 585 amino acids. It is primarily composed of α-helices, lacking β-sheets, and consists of three homologous domains: DI, DI I, and DI II. Each of these is further divided into A and B subdomains (DIA, DIB, DI IA, DI IB, DI I IA, and DI I IB), connected by long, flexible rings. HSA is the most abundant and structurally stable protein in human plasma, with a concentration of approximately 45 mg / ml (0.6 mM) and a circulating half-life of up to 20 days. When nanobodies bind to HSA, their hydrodynamic radius and molecular weight increase, glomerular filtration decreases, and in vivo retention time is significantly prolonged. A series of bispecific nanobodies based on anti-tumor antigens, anti-CD16, and anti-HSA nanobodies have been reported. Among them, the humanized, trivalent bispecific nanobodies Ozoral izumab developed by Ablynx were approved in Japan in September 2022 (English trade name: This is also the world's first approved bispecific nanobody. Ozoral izumab contains three nanobody domains, two of which target TNF-α, and one binds to HSA to prolong the drug's half-life. The average half-life (t1 / 2) after a single dose is 18.2 days. Summary of the Invention
[0006] This invention obtains camel-derived nanomonoantibodies targeting CD4 and HSA and their VHH by immunizing camels with CD4 and HSA antigens respectively, and constructs a "hat-shaped" nanobody of NbCD4-NbHSA-NbCD4 for the treatment of HIV-infected patients.
[0007] Based on these studies, the present invention provides an anti-CD4 nanobody with a longer plasma half-life, comprising a CD4 targeting domain and an HSA targeting domain;
[0008] The CD4 targeting domain includes three complementary determinant regions (CDRs) 1-3, the sequences of which are shown in SEQ ID NO: 1-3; the HSA targeting domain includes three complementary determinant regions (CDRs) 1-3, the sequences of which are shown in SEQ ID NO: 4-6.
[0009] In one specific implementation, the HSA targeting domain is located in the middle, with a CD4 targeting domain on each side.
[0010] In one specific implementation, the CD4 targeting domain further includes four backbone regions FR1-4, whose sequences are shown in SEQ ID NO:7-10.
[0011] In one specific implementation, the HSA targeting domain further includes four backbone regions FR1-4, whose sequences are shown in SEQ ID NO:11-14.
[0012] The present invention also provides the application of the above-mentioned nanobody in the preparation of a therapeutic drug for HIV CD4-positive T-cell tumors.
[0013] The present invention also provides the application of the above-mentioned nanobody in the preparation of CAR-T therapeutic agents for HIV or CD4-positive T-cell tumors.
[0014] This invention also provides the application of the above-mentioned nanobody in the preparation of CD4 detection agents.
[0015] The present invention also provides a nucleic acid encoding the above-mentioned nanobody.
[0016] The present invention also provides the application of the above-mentioned nucleic acid in the preparation of therapeutic drugs for HIV infection or CD4-positive T-cell tumors.
[0017] This invention develops nanobody drugs targeting the HIV attack target CD4. By introducing the anti-HSA VHH, a "hat-shaped" NbCD4-NbHSA-NbCD4 nanobody was constructed. In vivo experiments showed that the plasma half-life of this antibody was significantly prolonged compared to NbCD4 alone. Simultaneously, pseudovirus neutralization experiments were used to evaluate the efficacy of single nanobodies and the "hat-shaped" nanobody in treating HIV infection. This invention provides a potentially effective novel nanobody drug for the clinical treatment of HIV. Attached Figure Description
[0018] Figure 1 This is a schematic diagram of the "official hat" type nanobody of NbCD4-NbHSA-NbCD4.
[0019] Figure 2 To detect the binding of "official hat" type nanobodies and CD4 nanomonoantibodies of different purified concentrations to CD4 protein using ELISA, Nb15 was used as an isotype control antibody.
[0020] Figure 3 The curves show the inhibition of global HIV pseudovirus infection of ghost cells by "official hat" type nanobody and CD4 nanomonobody. Nb15 is the isotype control antibody.
[0021] Figure 4 SPR detection statistics for eukaryotically expressed "official hat" type nanobodies and CD4 nanomonoantibodies; Detailed Implementation
[0022] 1. Eukaryotic expression of "official hat" type nanobody
[0023] Using molecular cloning technology, the VHH genes of two CD4 nanoclone antibodies were fused with the VHH gene of an HSA nanoclone antibody and inserted into the pCDNA3.4 eukaryotic expression vector to construct the NbCD4-NbHSA-NbCD4-pCDNA3.4 expression plasmid. The constructed NbCD4-NbHSA-NbCD4-pCDNA3.4 was transfected into 293f cells to express the "official hat" type nanobody for producing NbCD4-NbHSA-NbCD4. Figure 1 Cell supernatant was collected and antibody purified. ELISA analysis showed that the antibody had excellent binding capacity. Figure 2 The corresponding sequence numbers of the constructed "official hat" type nanobodies are shown in Table 1.
[0024] Table 1. CDR and FR sequences corresponding to "official hat" type nanobodies.
[0025]
[0026] 2. "Official hat" type nanobody neutralizes HIV-infected ghost cells
[0027] Twenty cell supernatants were selected for in vitro neutralization experiments. 20 μl of cell supernatant was incubated with HIV pseudovirus at 5% CO2 and 37°C for 1 hour, followed by the addition of 1.0 x 10⁻⁶ ppm of [a specific substance / method / etc.]. 4 After incubating Ghost cells at 5% CO2 and 37°C for 48 hours, the cell supernatant was removed, and 100 μl / well of Glos's buffer (pre-warmed to room temperature) was added. The culture plate was gently shaken to allow for complete cell lysis. Then, 50 μl / well of the cell lysate was transferred to a 96-well white fluorescent microplate (Costar), followed by 50 μl / well of Britite-Gloluxinitiative substrate (pre-warmed to room temperature). The mixture was vortexed and immediately placed in a GloMax chemiluminescence immunoassay reader (Promega) for detection. Neutralization titer (ID) was measured. 50 or ND 50 This is expressed as a dilution factor at which 50% inhibition rate is achieved.
[0028] The results are as follows Figure 3 As shown, the "official hat" type nanobody NbCD4-NbHSA-NbCD4 has good neutralizing activity, and its virus inhibition rate is generally higher than that of CD4 nanomonoclonal antibodies.
[0029] 3. Affinity Identification of "Official Hat" Type Nanobody
[0030] Affinity assays were performed on the NbCD4-NbHSA-NbCD4 antibody and CD4 protein. The Fortebio biomolecular interaction platform was used to detect affinity. The antibody was immobilized onto the Anti-human IgG Fc Capture Biosensors (AHC) probe for 400 s, then bound to the CD4 protein antigen for 180 s, followed by dissociation for 180 s. The antibody-antigen binding and dissociation were observed, and the data were derived by fitting a curve to the instrument. The antibody-protein binding and dissociation curves are shown below. Figure 4 As shown in the figure, the NbCD4-NbHSA-NbCD4 antibody exhibits good affinity.
[0031] The "Hood-like" type nanobody NbCD4-NbHSA-NbCD4 of this invention can not only bind to HAS, thereby greatly prolonging the plasma half-life, but also specifically recognize and bind to CD4, blocking the binding of CD4 ligands to CD4, thereby inhibiting diseases related to the CD4 pathway, such as HIV infection and CD4-positive T-cell tumors. Because the "Hood-like" type nanobody NbCD4-NbHSA-NbCD4 can recognize CD4 molecules on the cell surface, its sequence can also be applied to CAR (Chimeric Antigen Receptor, composed of a VHH sequence fused with a third- or fourth-generation CD28-4-1BB-CD3zeta molecule sequence) cell therapy for tumors or HIV infection.
[0032] Furthermore, because the "hat-shaped" nanobody NbCD4-NbHSA-NbCD4 can recognize CD4 molecules on the cell surface, it can also be used for ADC (antibody-drug conjugate) therapy by conjugation with drugs or for antibody-dependent molecular imaging diagnostics by conjugation with isotopes. The nucleic acid encoding the "hat-shaped" nanobody NbCD4-NbHSA-NbCD4 can also be carried in an AAV system for therapeutic purposes.
[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. An anti-CD4 nanobody with a longer plasma half-life, characterized in that, It includes two CD4 targeting domains and one HSA targeting domain; The CD4 targeting domain includes three complementarity-determining regions CDR1-3, the sequences of which are shown in SEQ ID NO:1-3; the HSA targeting domain includes three complementarity-determining regions CDR1-3, the sequences of which are shown in SEQ ID NO:4-6. The HSA targeting domain is located in the middle, with a CD4 targeting domain connected to each of the two sides.
2. The nanobody according to claim 1, characterized in that, The CD4 targeting domain also includes four backbone regions FR1-4, whose sequences are shown in SEQ ID NO:7-10.
3. The nanobody according to claim 1, characterized in that, The HSA targeting domain also includes four backbone regions FR1-4, whose sequences are shown in SEQ ID NO:11-14.
4. The use of the nanobody according to any one of claims 1-3 in the preparation of a therapeutic drug for HIV infection.
5. The use of the nanobody according to any one of claims 1-3 in the preparation of CD4 detection reagent.
6. A nucleic acid, characterized in that, The nanobody is encoded according to any one of claims 1-3.
7. The use of the nucleic acid according to claim 6 in the preparation of a therapeutic drug for HIV infection.