Novel fully human antibody based on fully human antibody library and application thereof

Abstract

The invention provides a novel fully-humanized antibody and application thereof, and particularly provides a fully-humanized antibody QC99 for resisting yellow fever virus. A heavy chain variable region and a light chain variable region of the humanized antibody respectively have amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.2. Affinity analysis shows that the antibody disclosed by the invention has good affinity, and cell experiments prove that the antibody has excellent antiviral capacity, has an excellent prevention or treatment effect on yellow fever viruses, and has a wide clinical application prospect.

Description

Novel fully human antibodies based on fully human antibody libraries and their applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fully human antibody QC99 for treating yellow fever virus, its preparation method, and its use in antiviral infection preparations. Background Technology

[0002] Yellow fever virus (YFV) is a typical mosquito-borne, highly virulent pathogen, prevalent in Africa and South America. It can cause acute hepatitis, hemorrhagic fever, and multiple organ failure, with a mortality rate as high as 20–50% during outbreaks. Although the classic 17D attenuated vaccine has been widely used for over 80 years and provides durable and highly protective immunity in most healthy individuals, significant gaps remain in the current public health system: limited vaccine production capacity and insufficient global reserves make it difficult to respond to sudden outbreaks; vulnerable populations such as immunocompromised patients, pregnant women, and the elderly are unsuitable for live vaccines; coverage rates in endemic areas have long been inadequate; and with global travel, climate change, and vector spread, the risk of YFV spillover and urban outbreaks continues to increase. Therefore, the development of neutralizing antibodies that can be rapidly deployed during outbreaks, applied to specific populations, and used for post-exposure prophylaxis or early treatment has irreplaceable strategic value.

[0003] However, antibody development for yellow fever virus (YFV) is particularly challenging compared to other flaviviruses such as dengue, Zika, and encephalitis. Firstly, the envelope protein E of YFV exhibits high conformational plasticity, dynamically switching between mature virus, respiratory state, sub-mature virus, and post-fusion trimer. The structural differences between these conformations significantly affect the exposure of key epitopes, meaning that many potential neutralizing sites can only be captured by antibodies during transient states of the viral life cycle, thus greatly increasing the difficulty of obtaining highly effective antibodies.

[0004] Secondly, YFV infection often produces a large number of immature / partially mature particles. The prM protein in these particles is not completely cleaved, and it tightly covers key regions on the surface of the E protein, forming what is known as prM shielding. A significant proportion of antibodies induced by natural infection or 17D vaccination target immunodominant but non-functional regions of the prM–E complex, such as fusion loops (FLs). While these antibodies have strong binding affinity, their neutralizing ability is limited due to a lack of stereochimeric characteristics, and they may mediate ADE risk in the presence of immature viruses, further narrowing the pool of viable antibody candidates.

[0005] Third, the dominant immune response of YFV itself constitutes a natural barrier. Innate immunity strongly favors structurally feature-poor planar epitopes such as FL and prM contact interfaces, while responding weakly to truly protective, cross-conformally conserved neutralizing "supersites." This means that it is extremely difficult to obtain high-affinity, highly neutralizing candidate antibodies from conventional immunization animal or human memory banks, and deep screening, single-cell cloning, or strongly structure-driven design strategies are necessary.

[0006] Fourth, although there has been considerable structural research in the field of flaviviruses, high-resolution cryo-EM data for YFV have only gradually become complete in recent years. In particular, the structures involving pre-fusion E dimers, post-fusion E trimers, and prM–E complexes have long been lacking, making it difficult for researchers to determine which epitopes are stably exposed, which highly overlap in different conformations, and which can block key steps in the viral life cycle. This lag in structural information directly limits the realization of rational design and has resulted in most previously screened antibodies having limited potency and unclear mechanisms.

[0007] In addition, truly strong neutralizing antibodies usually need to possess a "double-lock mechanism":

[0008] (1) In the early stages of entry, it blocks viral adsorption, conformational change, or dimer recombination;

[0009] (2) Locking the E trimer during the membrane fusion stage to prevent the release of viral nucleic acid.

[0010] However, antibodies capable of recognizing the same supersite across both pre-fusion and post-fusion conformations are extremely rare, requiring exceptionally high levels of recognition angle, affinity, interfacial depth, and spatial adaptability. Consequently, existing natural antibody libraries have struggled to provide sufficient template molecules.

[0011] Finally, from a drug development perspective, YFV outbreaks mostly occur in areas with limited medical resources. Antibodies used clinically must possess characteristics such as broad conservation, low escape risk, controllable cost, rapid deployment, and stable stockpiling. This requires that antibody epitopes be conserved across viral strains and preferably avoid hypervariable regions; the antibody molecule format must support a long half-life and simple production; and structural engineering must be used to reduce the risk of ADE and improve stability in advance.

[0012] In summary, the development of yellow fever virus antibodies faces multiple obstacles, including complex structure, misdirected immunity, interference from immature particles, epitope scarcity, and difficulty in achieving a dual mechanism. Novel antibodies are urgently needed. Summary of the Invention

[0013] The purpose of this invention is to study the fully human antibody QC99, its preparation method, and its use against yellow fever virus, based on the above research background, that is, to provide a novel fully human antibody, its preparation method, and its use.

[0014] In a first aspect, the present invention provides a fully human antiviral antibody QC99, wherein the heavy chain variable region of the fully human antibody is composed of a FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 region, and the light chain variable region is composed of a FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 region. The heavy chain variable region and the light chain variable region of the antibody each have the amino acid sequences shown in SEQ ID NO. 1-2.

[0015] The amino acid sequence (SEQ ID NO.1) of the variable region of the fully human antibody heavy chain is as follows:

[0016] EVQLVESGGGLQKPYPHTKPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAIFWTISLIMHCMRYYMFTANGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVNHICTPLYNECYQDNNIDTWGQGTLVTVSS

[0017] The amino acid sequence (SEQ ID NO.2) of the variable region of the light chain of the fully human antibody is as follows:

[0018] IQMTQSPSSSLSASVGDRVTITCRANTMAFSSIRHKERPAYSMQWYQQKPGKAPKLLIYSLQLLTHLKSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCNGMNRNYPCAEGFDLNSREQIFGQGTKVEIK

[0019] To obtain fully human antibodies, a phage display library of fully human antibodies was first constructed. Then, the fully human antibodies were screened, and specific positive clones were screened using phage enzyme-linked immunosorbent assay (ELISA). After sequence analysis, the fully human antibodies were obtained. After expression and purification of the fully human antibodies in the 293 system, high-purity fully human antibodies were obtained.

[0020] A second aspect of the present invention provides a method for preparing the fully human antibody, comprising the following steps:

[0021] (A) Synthesize the full-length, fully human antibody based on the entire genome of the antibody variable region gene;

[0022] (B) The full-length fully human antibody obtained in step (A) was cloned into an expression vector using PCR technology, and the correct clone was confirmed by sequencing.

[0023] (C) The above expression vector is introduced into the host cell to express the fusion protein.

[0024] In this invention, any suitable vector is applicable, preferably pGEM-T, Pet32a, pcDNA3.1, pEE6.4, pEE12.4, pDHFR or pDR1, wherein the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0025] In this invention, mammalian or insect host cells or prokaryotic cell culture systems can be used for the expression of the fusion protein of this invention. The usable host cells are prokaryotic cells containing the aforementioned vector, and can be one of DH5a, Top10, BL21(DE3), or TG1.

[0026] The fusion protein of the present invention can be readily produced in the following cells: mammalian cells, such as CHO, NSO, HEK293, BHK or COS cells; bacterial cells, such as Escherichia coli, Bacillus subtilis or Pseudomonas fluorescens; insect cells, or fungal or yeast cells, said cells being cultured using techniques known in the art.

[0027] The method for preparing the fusion protein disclosed in this invention involves culturing the aforementioned host cells under expression conditions to express, isolate, and purify the fusion protein. Using this method, antibodies can be purified into a substantially homogeneous substance, for example, appearing as a single band on SDS-PAGE electrophoresis.

[0028] The fusion protein disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the fusion protein peptide bound to the affinity column.

[0029] Various protein purification methods can be used, and such methods are known in the art and described, for example (Wilchek and Bayer, 1990, Methods in enzymology) (Scopes, 2013, Proteinpurification: principles and practice).

[0030] Biacore analysis showed that the fully human antibody of this invention has good affinity. Small animal experiments demonstrated that mice in the protection group pre-injected with the fully human antibody of this invention showed no signs of neurotoxicity after injection of the viral antigen, and no toxin-induced deaths were observed after one month of continuous monitoring. This indicates that the fully human antibody of this invention has excellent antiviral antigen activity.

[0031] Therefore, a third aspect of the present invention provides a pharmaceutical composition comprising the fully human antibody. In addition to the fully human antibody, the pharmaceutical composition also includes a pharmaceutically acceptable drug carrier.

[0032] The fully human antibody of the present invention, together with pharmaceutically acceptable excipients, forms a pharmaceutical formulation composition, thereby exerting a more stable therapeutic effect. These formulations can ensure the conformational integrity of the amino acid core sequence of the fully human antibody disclosed in the present invention, while also protecting the multifunctional groups of the protein and preventing their degradation (including but not limited to aggregation, deamination or oxidation).

[0033] Under normal circumstances, liquid formulations can be stored stably at 2℃-8℃ for at least one year, while lyophilized formulations remain stable at 30℃ for at least six months. The formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, or lyophilized formulations, with injections or lyophilized formulations being preferred.

[0034] For the aqueous injection or lyophilized formulation of the fully human antibody disclosed in this invention, pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic adjusters, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., and their addition should minimize the granulation tendency of the bifunctional, bispecific antibody protein. Solution stabilizers can be sugars, including reducing and non-reducing sugars; amino acids, including monosodium glutamate or histidine; alcohols, including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol, and the amount of solution stabilizer added should be such that the final formulation remains stable within a time considered stable by those skilled in the art. Isotonic adjusters can be one of sodium chloride or mannitol. Buffers can be one of TRIS, histidine buffer, or phosphate buffer.

[0035] The above-mentioned formulation is a composition containing the fully human antibody, and it exhibits significant antiviral antigen effects after administration to animals, including humans. Specifically, it is effective in the prevention and / or treatment of tetrodotoxin poisoning and can be used as an antiviral antigen drug.

[0036] When the fully human antibodies and their compositions described in this invention are administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage should be determined with reference to animal experimental results and various other factors, but the total dosage should not exceed a certain range. Specifically, the intravenous injection dose is 1~1800 mg / day.

[0037] In a fourth aspect, the present invention provides a use of the fully human antibody, specifically in the preparation of an antiviral drug for the prevention or treatment of yellow fever virus.

[0038] The role and effect of invention

[0039] This invention develops a fully human antibody targeting the characteristics of yellow fever virus. The preparation process is simple, and it has excellent affinity for the antigen, with an IC50 value that is significantly better than existing antibodies, resulting in stronger antiviral capabilities. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0041] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1. Preparation and expression of fully human antibodies

[0043] The fully human antibodies were derived from a phage display antibody library. The methods for constructing and expressing the antibodies themselves are standard experimental techniques in the field, briefly described below:

[0044] (1) The heavy and light chains of the whole gene synthesized antibody, wherein the amino acid sequences of the variable regions of the heavy chain and the variable regions of the light chain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0045] (2) Antibody expression and purification

[0046] The antibody was expressed and purified according to the method described in the literature [Hu S, Fu W, Li T, et al. Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency[J]. Science translational medicine, 2017, 9(380)]. The antibody purity was identified as over 95% by SDS-PAGE.

[0047] Example 2. Biacore Analysis

[0048] Antiviral antigens were coated onto a CM5M5 chip (GE), and the antibodies to be tested were captured. The affinity of each fusion protein was then detected using a Biacore T100 (GE Healthcare). The specific affinity values ​​are shown in Table 1.

[0049] Table 1. Biacore Analysis Results

[0050] The parameter values ​​and units represent the affinity / kinetics of the combination: 14.56 KD (nM). surface

[0051] Example 3. Virus IC50 Calculation

[0052] Recombinant pseudoviruses carrying the target viral envelope protein were used, with luciferase or fluorescent protein reporter genes inserted into their genomes. Monoclonal antibodies or serum samples were serially diluted and mixed with a fixed dose of pseudovirus under suitable conditions to ensure complete binding. The mixture was then added to pre-coated target cells stably expressing the corresponding receptor / co-receptor. After further culturing for a certain period, cells or supernatant were collected, and the infection efficiency of the pseudovirus was reflected by detecting luciferase activity / fluorescence intensity. Wells containing only pseudovirus and no antibody were used as 0% inhibition controls, while background wells without pseudovirus or positive controls that completely blocked infection were used as 100% inhibition references. Based on the infection signal intensity corresponding to each antibody dilution (or mass concentration), the percentage of infection or inhibition rate relative to the control wells was calculated, and a dose-response curve of "inhibition rate – antibody concentration" was plotted. Curve fitting was performed to obtain the antibody concentration (or serum dilution factor) that reduced the infection signal by 50%, which was the IC50 value of that sample against the pseudovirus. Specific results are shown in Table 2.

[0053] Table 2 Results of Antibody Antitoxic Activity Detection

[0054] IC50 (μg / mL): Fully human antibody 1.22, Control antibody 9.51 surface

[0055] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A fully human antibody QC99 against yellow fever virus, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.

2.

2. The nucleic acid encoding the fully human antibody QC99 against yellow fever virus as described in claim 1.

3. The method for preparing the fully human antibody QC99 against yellow fever virus as described in claim 1, characterized in that, Includes the following steps: (A) Synthesize the full-length humanized antibody based on the entire variable region gene of the antibody; (B) Use PCR technology to clone the full-length humanized antibody obtained in step (A) into an expression vector, and confirm the correct clone after sequencing verification; (C) Introduce the above expression vector into host cells to express the fusion protein.

4. The method for preparing the fully human antibody QC99 against yellow fever virus according to claim 3, characterized in that: in, The expression vector is pGEM-T, Pet32a, pcDNA3.1, pEE6.4, pEE12.4, pDHFR, or pDR1, and the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences. The host cell is a prokaryotic cell, mammalian cell, insect cell, or fungal cell.

5. A pharmaceutical composition containing the fully human antibody QC99 against yellow fever virus as described in any one of claims 1 to 2, characterized in that, It also includes pharmaceutically acceptable drug carriers.

6. The pharmaceutical composition according to claim 5, characterized in that: in, The pharmaceutical composition is an aqueous injection or a lyophilized formulation, and the pharmaceutically acceptable drug carrier includes one or a combination of surfactants, solution stabilizers, isotonic modifiers, and buffer solutions.

7. The use of the fully human antibody QC99 against yellow fever virus as described in any one of claims 1 to 2 in the preparation of drugs against yellow fever virus.