CN115925934A - Humanized monoclonal antibody with improved stability and application thereof - Google Patents

Humanized monoclonal antibody with improved stability and application thereof Download PDF

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CN115925934A
CN115925934A CN202210883244.3A CN202210883244A CN115925934A CN 115925934 A CN115925934 A CN 115925934A CN 202210883244 A CN202210883244 A CN 202210883244A CN 115925934 A CN115925934 A CN 115925934A
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Abstract

The invention provides a humanized monoclonal antibody or an antigen binding fragment thereof with improved stability and specifically binding to human ACE2, a related product thereof, and a preparation method and application thereof. The humanized monoclonal antibody or the antigen binding fragment thereof resisting human ACE2 can be specifically bound with a human ACE2 receptor with high affinity, has high stability and good drug formation property, and is expected to become a therapeutic antibody medicine for coronavirus infection with ACE2 as a receptor.

Description

Humanized monoclonal antibody with improved stability and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a humanized monoclonal antibody or an antigen binding fragment thereof with improved stability and specifically binding to human ACE2, a related product thereof, a preparation method and application thereof.
Background
Coronaviruses belong phylogenetically to the order of the nested viruses (Nidovirales) the family of Coronaviridae (Coronaviridae) the genus coronaviruses (Coronavirus). Viruses of the genus coronavirus are enveloped, single-stranded, linear, positive-stranded RNA viruses in their genome, and are a large group of viruses widely occurring in nature. Coronaviruses only infect vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cows, birds.
At present, 7 kinds of coronavirus which are known to infect humans are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome), MERS-CoV (causing middle east respiratory syndrome), and novel coronavirus (SARS-CoV-2), respectively. Among these coronaviruses, SARS-CoV-2 and HCoV-NL63 are those which have angiotensin converting enzyme 2 (ACE 2) as a receptor and which infect host cells by binding the RBD region of the major glycosylated spike protein (S protein) on their surface to the host cell surface receptor ACE2, thereby infecting human host cells and humans.
The antibody, especially the blocking antibody, can block the combination of the virus and the cell receptor by combining with the receptor protein of the host cell, thereby blocking the virus infection, achieving the process of blocking the virus from invading the host cell and realizing the prevention and treatment effects. Therefore, antibodies that target the human ACE2 receptor and block its binding to the S protein RBD of coronaviruses such as SARS-CoV, SARS-CoV-2 and HCoV-NL63 are highly likely to be effective antibodies for inhibiting infection by these viruses.
The natural stability of the antibody protein medicine is obviously lower than that of a small molecular compound, and amino acid mutation introduced by humanized modification can also influence the physicochemical properties of antibody molecules, so that the antibody molecules have undesirable phenomena of chemical modification, breakage, aggregation and the like in the production, storage and administration processes, the yield and activity of the antibody medicine are influenced, and the high molecular aggregate can also cause the medicine safety problems in the aspects of enhancing the immunogenicity and the like.
Therefore, the development of therapeutic antibody drugs against coronavirus with higher stability and better druggability has potential clinical application value and prospect.
Disclosure of Invention
Object of the Invention
The invention aims to provide a humanized monoclonal antibody or an antigen binding fragment thereof, a related product thereof, a preparation method and application thereof, wherein the humanized monoclonal antibody or the antigen binding fragment thereof not only has higher antigen binding activity, but also has higher stability and better drug property, and specifically binds to human ACE 2.
Solution scheme
In order to achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides a humanized monoclonal antibody or antigen-binding fragment thereof that specifically binds human ACE2, comprising a set of heavy chain variable regions and light chain variable regions selected from the group consisting of:
(1) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively; or
(2) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.
In a preferred embodiment (I):
the humanized monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 8, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity with the amino acid sequence shown as SEQ ID NO. 8; and also,
the light chain variable region has an amino acid sequence shown as SEQ ID NO. 9, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity with the amino acid sequence shown as SEQ ID NO. 9.
In a specific embodiment of this preferred embodiment, the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 8, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 9.
For this preferred embodiment (I), it is further preferred that the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as follows:
the heavy chain has an amino acid sequence as shown in SEQ ID NO. 11, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 11; and the number of the first and second electrodes,
the light chain has an amino acid sequence as set forth in SEQ ID NO. 12, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12.
In a further preferred specific embodiment of preferred embodiment (I), the humanized monoclonal antibody or antigen binding fragment thereof comprises a heavy chain and a light chain as follows:
the heavy chain has an amino acid sequence shown as SEQ ID NO. 11, and the light chain has an amino acid sequence shown as SEQ ID NO. 12.
In a preferred embodiment (II):
the humanized monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 10, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity with the amino acid sequence shown as SEQ ID NO. 10; and the number of the first and second electrodes,
the light chain variable region has an amino acid sequence shown as SEQ ID NO. 9, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity with the amino acid sequence shown as SEQ ID NO. 9.
In a specific embodiment of this preferred embodiment, the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 10, and the light chain variable region has an amino acid sequence shown as SEQ ID NO. 9.
For this preferred embodiment (II), it is further preferred that the humanized monoclonal antibody or antigen-binding fragment thereof comprises heavy and light chains as follows:
the heavy chain has an amino acid sequence as shown in SEQ ID NO. 13, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 13; and the number of the first and second electrodes,
the light chain has an amino acid sequence as set forth in SEQ ID NO. 12, or an amino acid sequence with at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12.
In a further preferred specific embodiment of preferred embodiment (II), said humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as follows:
the heavy chain has an amino acid sequence shown as SEQ ID NO. 13, and the light chain has an amino acid sequence shown as SEQ ID NO. 12.
In addition, the humanized monoclonal antibody or an antigen-binding fragment thereof is preferably selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, and diabody.
In a second aspect, the present invention provides a bispecific antibody comprising a humanized monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above.
In a third aspect, the present invention provides a polynucleotide encoding a humanized monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, or a bispecific antibody as described in the second aspect above.
In particular embodiments, the polynucleotide is DNA or mRNA.
In a fourth aspect, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.
In a fifth aspect, the present invention provides an expression vector comprising the nucleic acid construct according to the fourth aspect described above; preferably, the expression vector is a eukaryotic expression vector.
In a sixth aspect, the present invention provides a transformed host cell comprising a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above or an expression vector as described in the fifth aspect above.
Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
In a seventh aspect, the present invention provides a method of preparing a humanized monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, the method comprising:
1) Culturing the transformed host cell of the sixth aspect above under conditions suitable for expression of the humanized monoclonal antibody or antigen-binding fragment thereof, such that the humanized monoclonal antibody or antigen-binding fragment thereof is expressed;
2) Recovering the expressed humanized monoclonal antibody or antigen-binding fragment thereof from the host cell or culture thereof.
In an eighth aspect, the present invention provides a drug conjugate comprising a humanized monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above or a bispecific antibody as described in the second aspect above, and an effector molecule conjugated directly or indirectly via a spacer to the humanized monoclonal antibody or antigen-binding fragment thereof;
preferably, the effector molecule is a detectable label, toxin, and/or chemotherapeutic agent;
further preferably, the detectable label is an enzyme label, a fluorescein label, an isotope label, a biotin label, a chemiluminescent group, or a metal particle.
In a ninth aspect, the present invention provides a pharmaceutical composition comprising a humanized monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a bispecific antibody as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a transformed host cell as described in the sixth aspect above and/or a drug conjugate as described in the eighth aspect above, and a pharmaceutically acceptable carrier and/or excipient.
In particular embodiments, the pharmaceutical composition may be in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;
preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.
The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the administration target, the target organ, the symptom, the administration method, and the like, and can be determined according to the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.
In a tenth aspect, the present invention provides the use of a humanized monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, a bispecific antibody as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a transformed host cell as described in the sixth aspect above, a drug conjugate as described in the eighth aspect above and/or a pharmaceutical composition as described in the ninth aspect above for the manufacture of a medicament for the prevention and/or treatment of an infection with a coronavirus which has ACE2 as a receptor;
preferably, the ACE 2-receptor coronavirus is selected from the group consisting of: SARS-CoV and SARS-CoV-2;
wherein, the SARS-CoV-2 can be SARS-CoV-2 original strain and/or SARS-CoV-2 variant strain;
alternatively, the SARS-CoV-2 variant strain is Alpha (b.1.1.7), beta (b.1.351), gamma (p.1), kappa (b.1.617.1), delta (b.1.617.2) strain, omicron (b.1.1.529/ba.1) subtype strain, omicron ba.1.1 subtype strain, omicron ba.2 subtype strain, omicron ba.2.12.1 subtype strain, omicron ba.2.75 subtype strain, omicron ba.3 subtype strain, omicron ba.4 subtype strain or omicron.5 subtype strain, further preferably Delta (b.1.617.2) strain, omicron (b.1.1.529/ba.1 subtype) strain, omicron ba.2 strain, omicron ba.12.12 subtype strain, omicron (b.1.1.529/ba.1 subtype strain), omicron ba.2 strain, omron ba.1.1.12.75 subtype strain or Omicron ba.5 subtype strain.
In an eleventh aspect, the present invention provides a method for preventing or treating an ACE2 receptor coronavirus infection, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of a nanobody or antigen-binding fragment thereof as described in the above first aspect, a bispecific antibody as described in the above second aspect, a polynucleotide as described in the above third aspect, a nucleic acid construct as described in the above fourth aspect, an expression vector as described in the above fifth aspect, a transformed host cell as described in the above sixth aspect, a drug conjugate as described in the above eighth aspect and/or a pharmaceutical composition as described in the above ninth aspect.
The "prophylactically or therapeutically effective amount" is determined by the judgment of the doctor, taking into consideration the type of the dosage form, the method of administration, the age and weight of the patient, the symptoms of the patient, and the like, and varies depending on the subject, the organ to be treated, the symptoms, the method of administration, and the like.
Advantageous effects
The humanized monoclonal antibody or the antigen binding fragment thereof aiming at the human ACE2 can be specifically bound with the human ACE2, so that the binding of the human ACE2 and an RBD (radial basis function) region of a coronavirus S protein is blocked, the coronavirus taking the ACE2 as a receptor loses the capability of invading a host, and the effects of preventing and treating the virus infection are achieved; furthermore, the humanized monoclonal antibody or the antigen binding fragment thereof resisting human ACE2 can be specifically bound with human ACE2 receptors with high affinity, has high stability and good druggability, and provides a new effective selection for preventing and/or treating coronavirus infection with ACE2 as a receptor.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.
The present invention will be described in detail below.
Definition of
"antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of an antibody, which typically include at least a portion of the antigen-binding or variable region, e.g., one or more CDRs, of a parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Antigen binding fragments include those selected from Fab, fab '-SH, fv, scFv, F (ab') 2, diabodies, CDR-containing peptides, and the like.
The "Fab fragment" consists of the CH1 and variable regions of one light and one heavy chain.
The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.
An "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form the F (ab ') 2 molecule.
An "F (ab') 2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.
"Single chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which linker enables the scFv to form the desired structure for antigen binding.
A "bispecific antibody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using linkers that are so short that they cannot pair between two domains of the same chain, the domains pair with complementary domains of another chain and form two antigen binding sites.
"humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies are largely human immunoglobulins in which residues from a hypervariable region of the recipient antibody are replaced by residues from a hypervariable region of a non-human species, such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some cases, fv framework residues of the human immunoglobulin are substituted for corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not present in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance.
"specific" binding, when referring to a ligand/receptor, antibody/antigen or other binding pair, refers to determining the presence or absence of a binding reaction of a protein, such as human ACE2, in a heterogeneous population of proteins and/or other biological agents. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.
The invention also provides a pharmaceutical composition containing the anti-human ACE2 antibody or the antigen-binding fragment thereof. To prepare a pharmaceutical composition, the antibody or antigen-binding fragment thereof can be prepared into various desired dosage forms by mixing with a pharmaceutically acceptable carrier or excipient. Examples of the dosage form of the pharmaceutical composition of the present invention include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments, which are oral preparations, and examples of non-oral preparations include injections, suppositories, transdermal preparations, ointments, plasters, and external liquid preparations, and those skilled in the art can select an appropriate dosage form according to the administration route and the administration target.
The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject, the target organ, the symptom, the administration method, and the like, and can be determined by the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.
The pharmaceutical compositions of the present invention may also contain other agents, including but not limited to cytotoxic, cytostatic, antiangiogenic or antimetabolic agents, tumor-targeting agents, immunostimulants or immunomodulators or antibodies that bind to cytotoxic, cytostatic or other toxic agents.
The invention is further illustrated by the following examples; in the following examples, unless otherwise specified, all the biological and chemical materials used are commercially available products.
Example 1: acquisition and humanization of murine monoclonal antibody m11B11, and construction of light and heavy chain expression plasmids of murine and humanized monoclonal antibodies
In previous studies by the inventors, BALB/C mice were immunized with a recombinant protein of the human ACE2 extracellular domain (hACE 2-ecto), spleen cells of the immunized mice were fused with myeloma cells to prepare hybridoma cells, ELISA was performed using the purified hACE2 recombinant protein, and an anti-hACE 2 monoclonal antibody hybridoma cell line m11B11 was selected by testing the blocking ability against pseudovirus infection of a novel coronavirus, and further, an m11B11 antibody coding sequence was obtained from the anti-hACE 2 monoclonal antibody hybridoma cell line m11B11 by the method of 5' RACE.
The amino acid sequences of the variable regions of the m11B11 antibodies prepared and selected according to the above methods are shown in Table 1 below.
TABLE 1 amino acid sequence of variable region of m11B11 antibody
Figure BDA0003765028910000081
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Humanizing a murine m11B11 antibody by using an IMGT database and a computer-aided drug design technology to form a humanized monoclonal antibody H11B11 of the anti-hACE 2; the amino acid sequence of the variable region of the humanized monoclonal antibody H11B11 is shown in Table 2 below.
TABLE 2 amino acid sequence of variable region of H11B11 antibody
Figure BDA0003765028910000082
The coding sequence of the constant region (SEQ ID NO: 17) of the human IgG1 mutant antibody was added to the 3 'end of the heavy chain variable region gene of each of the monoclonal antibodies shown in tables 1 and 2, and the coding sequence of the human kappa light chain constant region (SEQ ID NO: 18) was added to the 3' end of the light chain variable region gene thereof, to form the heavy chain and the light chain of each monoclonal antibody, wherein the amino acid sequence of the heavy chain (m 11B 11-HC) of m11B11 is shown in SEQ ID NO:19, the amino acid sequence of the light chain (m 11B 11-LC) of m11B11 is shown in SEQ ID NO:20, the amino acid sequence of the heavy chain (H11B 11-HC) of H11B11 is shown in SEQ ID NO:21, and the amino acid sequence of the light chain (H11B 11-LC) of H11B11 is shown in SEQ ID NO: 12; the artificial synthesis method is adopted for the whole gene synthesis.
The synthesized light and heavy chain total genes of the antibody are cut by EcoRI (NEB, product number R0101S) and NotI (NEB, product number R0189S), and are respectively connected to an expression vector pTT5 (the main elements of which sequentially comprise a BSPQI cutting site, a CMV Promoter (target gene expression Promoter), an ampR (ampicillin resistance gene), a pMB1ori (replication initiation), an ori p, a signal peptide (target gene expression signal peptide) and a target gene) to obtain complete light and heavy chain expression plasmids of monoclonal antibodies m11B11 and H11B 11.
Example 2: expression of murine monoclonal antibody m11B11 and humanized monoclonal antibody H11B11
Extracting complete light and heavy chain expression plasmids of the monoclonal antibodies m11B11 and H11B11 prepared in example 1, co-transfecting CHO-18 cells (the CHO-18 cells are self-prepared by Suzhou Jun) according to the light and heavy chain equal proportion, and performing suspension serum-free domestication and screening on CHO-K1 cells (ATCC, CCL-61) to make the CHO-18 cells suitable for transient expression; after the cotransfection, the cells were cultured for 7 days, and then the cell culture fluid was centrifuged at a high speed, vacuum-filtered through a microporous filter membrane, loaded onto a monoclonal antibody purification pre-packed column (HiTrap MabSelectSuRe column), the target protein was eluted in one step with a buffer solution containing 100mM acetic acid-sodium acetate at ph3.6 as an eluent, and the target sample was recovered and dialyzed into PBS. Using NanoDrop TM One/OneC micro UV-Vis Spectrophotometer (Thermo Scientific) TM The goods number is: ND-ONE-W) the antibody protein concentration and the protein amount in the eluate were measured, and the expression level was converted based on the transfection volume. The results of measurement of the expression levels of the monoclonal antibodies m11B11 and H11B11 are shown in Table 3 below.
TABLE 3 light and heavy chain compatibility and expression level of m11B11 and H11B11 antibodies
Figure BDA0003765028910000091
The results in table 3 show that the expression level of the humanized antibody H11B11 is significantly higher than that of the murine antibody m11B11; that is, the expression level of the murine antibody m11B11 was significantly increased after undergoing humanization.
Example 3: high-temperature stability detection of murine monoclonal antibody m11B11 and humanized monoclonal antibody H11B11
The antibody sample prepared in example 2 was replaced with a buffer system (20 mM histidine-histidine hydrochloride buffer containing 230mM sucrose/trehalose) at pH5.5, and the antibody sample concentration was controlled to about 10mg/ml, and the sample was dispensed into vials at 500. Mu.l/vial. Vials loaded with antibody samples were placed in a 40 ℃ incubator and examined for antibody stability at 0, 2 and 4 weeks.
Antibody stability was assessed by the following parameters: (a) Detecting the purity of the antibody by an R-CE-SDS (reduction electrophoresis method) method and an NR-CE-SDS (non-reduction electrophoresis method); and (b) detecting the binding activity of the antibody and ACE2 by an ELISA method.
The specific detection method comprises the following steps:
detection of antibody purity by NR-CE-SDS method
The final volume of 100. Mu.l was measured by sequentially adding 1% SDS, 40mM phosphate buffer (pH 6.5), 5. Mu.l 0.25M NEM and the sample to a 1.5mL EP tube, and mixing them to give a final concentration of 1.0mg/mL; centrifuging at 3000rpm for 30s at room temperature, and then incubating at 70 ℃ for 5min; after the incubation is finished, cooling to room temperature, and centrifuging at 12000rpm for 5min; and respectively taking 75 mu l of sample solution out of the sample tubes to sample injection bottles to avoid bubbles, and detecting by using a capillary electrophoresis apparatus (PA 800PLUS, SCIEX). The antibody sample purity calculation method comprises the following steps: antibody protein purity (%) = antibody protein peak area/total peak area.
Detection of antibody purity by R-CE-SDS method
To a final volume of 100. Mu.l, 1% SDS, 40mM phosphate buffer (pH 6.5), a sample, and 5. Mu.l of beta-mercaptoethanol were sequentially added to a 1.5mL EP tube, and mixed so that the final concentration of the sample was 1.0mg/mL; centrifuging at 3000rpm at room temperature for 30sec, and incubating at 70 deg.C for 15min; after the incubation is finished, cooling to room temperature, and centrifuging at 12000rpm for 5min; and respectively taking 75 mu l of sample solution out of the sample tubes to sample injection bottles to avoid bubbles, and detecting by using a capillary electrophoresis apparatus (PA 800PLUS, SCIEX). The antibody sample purity calculation method comprises the following steps: antibody protein purity (%) = (antibody heavy chain peak area + antibody light chain peak area)/total peak area.
Detection of antigen binding Activity of antibodies by ELISA method
After 1.0. Mu.g/mL of the MFc-tagged human ACE-2 (manufactured by June Suzhou, lot: 20210203) was coated using a microplate reader from Thermo Scientific, 2% Mill (Anchor) was blocked, and the antibody to be tested was added in a gradient (4-fold gradient dilution was performed with 4.0. Mu.g/mL as the starting concentration for a total of 12 concentrations); anti-human IgG (Fc-specific) -peroxidase antibody (Sigma-Aldrich, A0170) was diluted 5000-fold and detected as a detection antibody, followed by development with 0.1mg/ml TMB (Sigma, T2885), and finally the reaction was stopped with 2M hydrochloric acid solution, and the plate was read at 450nm/620 nm. The activity calculation method comprises the following steps: fitting the antibody protein concentration and the color rendering value by utilizing a log (aginst) vs. response-Variable slope light squares fit nonlinear fitting model in Graphpad software to obtain an EC50 value. ACE2 binding activity (%) = different time points EC 50/starting time points EC50.
The instrument and column/cartridge information used for each test method is shown in table 4 below.
TABLE 4 information on the instruments used for the assay and the column/cartridge
Figure BDA0003765028910000101
Figure BDA0003765028910000111
The results of stability tests of m11B11 and H11B11 monoclonal antibodies after being left at 40 ℃ for 4 weeks are shown in Table 5 below.
TABLE 5 stability of the m11B11 and H11B11 monoclonal antibodies at a high temperature of 40 ℃ for 4 weeks
Figure BDA0003765028910000112
Note: "-" represents map abnormalities and no data is reported.
As can be seen from Table 5, after the humanized monoclonal antibody H11B11 was left at a high temperature of 40 ℃ for 4 weeks, the proportion of the reduced and non-reduced CE-SDS main peak samples showed a decreasing trend with time, and the biological activity was significantly decreased; these results suggest that: the humanized monoclonal antibody H11B11 may have stability problems.
Example 4: construction and expression of H11B11 mutant antibody molecules
In order to improve the stability of the humanized monoclonal antibody H11B11, in this example, a series of mutations and screening were performed on the variable region of the H11B11 antibody, and two mutant antibody molecules H11B11-mut1 and H11B11-mut2 were finally obtained; relative to the humanized monoclonal antibody H11B11 before the modification, the heavy chain variable region HCDR3 of the mutant antibody molecules H11B11-mut1 and H11B11-mut2 each comprises an amino acid mutation; the HCDR1, HCDR2 and HCDR3 sequences of antibodies H11B11, H11B11-mut1 and H11B11-mut2 are shown in Table 6 below.
TABLE 6 sequences of HCDRs 1 to 3 of H11B11, H11B11-mut1 and H11B11-mut2
Figure BDA0003765028910000113
Figure BDA0003765028910000121
As can be seen from Table 6, the remaining segments of the two mutant antibody molecules are identical to the antibody H11B11, except for the HCDR3 difference.
The heavy chain sequence of H11B11-mut1 is shown as SEQ ID NO. 11, and the light chain sequence is shown as SEQ ID NO. 12; the heavy chain sequence of H11B11-mut2 is shown as SEQ ID NO 13, and the light chain sequence is shown as SEQ ID NO 12; according to the sequence information, carrying out whole gene synthesis of light chains and heavy chains of two mutant antibodies; then, according to the method described in example 1, respectively constructing mutant antibody H11B11-mut1, H11B11-mut1 complete light, heavy chain expression plasmid, and according to the method described in example 2 for antibody expression and determination of its expression level; the light and heavy chain compatibility and expression level of the monoclonal antibodies H11B11, H11B11-mut1 and H11B11-mut2 are shown in Table 7 below.
TABLE 7 light and heavy chain compatibility and expression level of H11B11, H11B11-mut1 and H11B11-mut2
Figure BDA0003765028910000122
As is clear from Table 7, the expression levels of the mutant antibodies H11B11-mut1 and H11B11-mut1 were comparable to that of H11B11, and they were all expressed at high efficiency.
Example 5: antigen binding activity of mutant antibodies H11B11-mut1 and H11B11-mut2 (i.e., binding to human ACE 2)
In this example, the antigen binding activity (EC 50 value) of the antibody H11B11 and the mutant antibodies H11B11-mut1 and H11B11-mut1 was measured by ELISA.
Specifically, after 1.0. Mu.g/mL of an mFc-labeled human ACE2 (manufactured by Suzhou Jun Seiki, lot: 20210203) was coated and 2% milk (Anchor) was blocked using a microplate reader from Thermo Scientific, a gradient-diluted antibody to be tested (4-fold gradient dilution was performed with 4.0. Mu.g/mL as the starting concentration for 12 concentrations) was added; anti-human IgG (Fc-specific) -peroxidase antibody (Sigma-Aldrich, A0170) was diluted 5000-fold and detected as a detection antibody, followed by development with 0.1mg/ml TMB (Sigma, T2885), and finally the reaction was stopped with 2M hydrochloric acid solution, and the plate was read at 450nm/620 nm.
Four parameter logistic regression (4 PL) model fitting was performed using software GraphPad Prism to obtain EC50 (ng/mL), which can reflect the antigen binding activity of each antibody; the results are shown in table 8 below.
TABLE 8 antigen binding Activity (EC 50 values) of H11B11, H11B11-mut1 and H11B11-mut2 antibodies
Sample name EC50(ng/mL)
H11B11 4.1
H11B11-mut1 3.5
H11B11-mut2 2.7
As can be seen from Table 8, the antigen binding activity of the mutant antibodies H11B11-mut1 and H11B11-mut1 is significantly higher than that of the humanized monoclonal antibody H11B11, i.e., the mutant antibodies H11B11-mut1 and H11B11-mut1 have significantly higher antigen binding activity than the original antibody H11B 11.
Example 6: high temperature stability of mutant antibodies H11B11-mut1 and H11B11-mut2
In this example, the stability of the mutant antibodies H11B11-mut1 and H11B11-mut1 after standing at a high temperature of 40 ℃ for 4 weeks was determined according to the detection method described in example 3; the results are shown in table 9 below.
TABLE 9 high temperature stability of H11B11-mut1 and H11B11-mut2
Figure BDA0003765028910000131
Comparing the stability data of the two mutant antibodies in table 9 with the stability data of H11B11 in table 3 (both under the same test conditions), it can be seen that the high temperature stability of the mutant antibodies H11B11-mut1 and H11B11-mut1 is significantly improved compared with the original antibody H11B11, and thus the pharmaceutical properties are significantly improved.
The above examples show that the mutant antibodies H11B11-mut1 and H11B11-mut1 can specifically bind to human ACE2 with high binding activity, and have high stability and good drug-forming property, so that the mutant antibodies are expected to be therapeutic antibody drugs for infection of coronavirus (including SARS-CoV-2) with ACE2 as a receptor.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
The sequences used herein are as follows:
1, SEQ ID NO: HCDR1 of antibody H11B11-mut1 of the invention
DYYMN；
2, SEQ ID NO: HCDR2 of antibody H11B11-mut1 of the invention
FIRNKANDYTTEYST；
3, SEQ ID NO: HCDR3 of antibody H11B11-mut1 of the invention
HMYDHGFDF；
4, SEQ ID NO: LCDR1 of antibody H11B11-mut 1or H11B11-mut2 of the inventionRASSSVRYMH；
5, SEQ ID NO: LCDR2 of the antibodies H11B11-mut 1or H11B11-mut2 of the invention
DTSKLAS；
6 of SEQ ID NO: LCDR3 of the antibodies H11B11-mut 1or H11B11-mut2 of the inventionQQWSYNPLT；
7, SEQ ID NO: HCDR3 of antibody H11B11-mut2 of the invention
HMYDNGFDF；
8, SEQ ID NO: heavy chain variable region of antibody H11B11-mut1 of the invention
EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDHGFDFWGQGTLVTVSS；
9 of SEQ ID NO: variable region of light chain of antibody H11B11-mut 1or H11B11-mut2 of the present invention
DIQMTQSPSSLSASVGDRVTITCRASSSVRYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSYNPLTFGQGTKLEIK；
10, SEQ ID NO: heavy chain variable region of antibody H11B11-mut2 of the invention
EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDNGFDFWGQGTLVTVSS；
11, SEQ ID NO: heavy chain of antibody H11B11-mut1 of the invention
EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDHGFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；
12, SEQ ID NO: light chain of an antibody H11B11-mut 1or H11B11-mut2 of the invention
DIQMTQSPSSLSASVGDRVTITCRASSSVRYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSYNPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
13 in SEQ ID NO: heavy chain of antibody H11B11-mut2 of the invention
EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDNGFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；
14, SEQ ID NO: heavy chain variable region of murine monoclonal antibody m11B11
EVKLVESGGGLVQPGGSLRLSCTTSGFTFIDYYMNWVRQPPGKALEWLGFIRNKANDYTTEYSTSVKGRFTISRDNSQSILYLQLNNLRAEDSGTYYCASHMYDDGFDFWGQGTTVTVSS；
15, SEQ ID NO: light chain variable region of murine monoclonal antibody m11B11
DNVLTQSPAIMSAFPGEKVTMTCNASSSVRYMHWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSYNPLTFGAGTKLEIK；
16 in SEQ ID NO: heavy chain variable region of humanized monoclonal antibody H11B11
EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDDGFDFWGQGTLVTVSS；
17 in SEQ ID NO: human IgG1 mutant antibody constant regions
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；
18, SEQ ID NO: human kappa light chain constant region
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
19, SEQ ID NO: heavy chain of murine monoclonal antibody m11B11
EVKLVESGGGLVQPGGSLRLSCTTSGFTFIDYYMNWVRQPPGKALEWLGFIRNKANDYTTEYSTSVKGRFTISRDNSQSILYLQLNNLRAEDSGTYYCASHMYDDGFDFWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK；
20, SEQ ID NO: light chain of murine monoclonal antibody m11B11
DNVLTQSPAIMSAFPGEKVTMTCNASSSVRYMHWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSYNPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC；
21, SEQ ID NO: heavy chain of humanized monoclonal antibody H11B11
EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDDGFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。
Figure IDA0003765028970000011
Figure IDA0003765028970000021
Figure IDA0003765028970000031
Figure IDA0003765028970000041
Figure IDA0003765028970000051
Figure IDA0003765028970000061
Figure IDA0003765028970000071
Figure IDA0003765028970000081

Claims (15)

1. A humanized monoclonal antibody or antigen binding fragment thereof that specifically binds human ACE2, characterized in that the humanized monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region and a set of light chain variable regions selected from the group consisting of:
(1) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively; or
(2) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.
2. The humanized monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising a set of heavy chain variable regions and light chain variable regions selected from the group consisting of:
(1) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8; and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9;
(2) A heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.
3. The humanized monoclonal antibody or antigen-binding fragment thereof of claim 2, comprising a set of heavy chain variable regions and light chain variable regions selected from the group consisting of:
(1) A heavy chain variable region having an amino acid sequence set forth in SEQ ID NO 8; and a light chain variable region having an amino acid sequence set forth in SEQ ID NO 9;
(2) A heavy chain variable region having an amino acid sequence set forth in SEQ ID NO 10; and a light chain variable region having an amino acid sequence set forth in SEQ ID NO 9.
4. The humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain set selected from the group consisting of:
(1) A heavy chain having the amino acid sequence shown as SEQ ID NO. 11, or an amino acid sequence having at least 95%,96%,97%,98%, or 99% sequence identity to the amino acid sequence shown as SEQ ID NO. 11; and a light chain having an amino acid sequence set forth in SEQ ID NO. 12, or an amino acid sequence having at least 95%,96%,97%,98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12;
(2) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 13, or an amino acid sequence having at least 95%,96%,97%,98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13; and a light chain having an amino acid sequence as set forth in SEQ ID NO. 12, or an amino acid sequence having at least 95%,96%,97%,98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12.
5. The humanized monoclonal antibody or antigen-binding fragment thereof of claim 4, comprising heavy and light chain sets selected from the group consisting of:
(1) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 11; and a light chain having an amino acid sequence as set forth in SEQ ID NO 12;
(2) A heavy chain having an amino acid sequence as set forth in SEQ ID NO 13; and a light chain having an amino acid sequence set forth in SEQ ID NO 12.
6. The humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the humanized monoclonal antibody or antigen-binding fragment thereof is humanized;
preferably, the monoclonal antibody is an IgG 1-type monoclonal antibody;
preferably, the antigen binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, diabodies.
7. A bispecific antibody comprising the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6.
8. A polynucleotide encoding the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, or encoding the bispecific antibody of claim 7.
9. A nucleic acid construct comprising the polynucleotide of claim 8, and optionally, at least one expression control element operably linked to the polynucleotide.
10. An expression vector comprising the nucleic acid construct of claim 9; preferably, the expression vector is a eukaryotic expression vector.
11. A transformed host cell comprising the polynucleotide of claim 8, the nucleic acid construct of claim 9, or the expression vector of claim 10;
preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
12. A method of making the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising:
1) Culturing the transformed host cell of claim 12 to express the humanized monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the humanized monoclonal antibody or antigen-binding fragment thereof;
2) Recovering the expressed humanized monoclonal antibody or antigen-binding fragment thereof from the host cell or culture thereof.
13. A drug conjugate comprising the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6 or the bispecific antibody of claim 7, and an effector molecule conjugated directly or indirectly via a spacer to the humanized monoclonal antibody or antigen-binding fragment thereof;
preferably, the effector molecule is a detectable label, toxin, and/or chemotherapeutic agent;
further preferably, the detectable label is an enzyme label, a fluorescein label, an isotope label, a biotin label, a chemiluminescent group, or a metal particle.
14. A pharmaceutical composition comprising the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, the bispecific antibody of claim 7, the polynucleotide of claim 8, the nucleic acid construct of claim 9, the expression vector of claim 10, the transformed host cell of claim 11, and/or the drug conjugate of claim 13, and a pharmaceutically acceptable carrier and/or excipient.
15. Use of the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, the bispecific antibody of claim 7, the polynucleotide of claim 8, the nucleic acid construct of claim 9, the expression vector of claim 10, the transformed host cell of claim 11, the drug conjugate of claim 13, and/or the pharmaceutical composition of claim 14 in the manufacture of a medicament for the prevention and/or treatment of an ACE 2-receptor coronavirus infection;
preferably, the ACE 2-receptor coronavirus is selected from the group consisting of: SARS-CoV and SARS-CoV-2;
further preferably, the SARS-CoV-2 is a SARS-CoV-2 prototype strain and/or a variant strain thereof.
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AU2021333886A1 (en) * 2020-08-26 2023-05-04 The University Of Queensland Modified polypeptides with improved properties
CN115925934A (en) * 2022-07-26 2023-04-07 北京昌平实验室 Humanized monoclonal antibody with improved stability and application thereof

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WO2024021839A1 (en) * 2022-07-26 2024-02-01 北京昌平实验室 Humanized monoclonal antibody having improved stability, and use thereof

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