Cell strain for producing biosimilar drug of ustekinumab and production method therefor

Abstract

Provided are a cell strain for producing a biosimilar drug of Ustekinumab and a production method therefor. Specifically, provided is a Chinese hamster ovary cell S cell strain, which is deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2020232. The cell strain expresses a fully human monoclonal antibody directed against P40 subunit shared by human IL-12 and human IL-23. The fully human monoclonal antibody directed against P40 subunit shared by human IL-12 and human IL-23 is a biosimilar drug of Ustekinumab, which not only exhibits high consistency with Ustekinumab in pre-clinical research, but also passes pharmacokinetic bioequivalence and safety similarity evaluation in clinical research. The biosimilar drug of Ustekinumab is the first one that has entered clinical trials in China, is the only one that has completed the I stage clinical trial, and is also one of the biosimilar drugs of Ustekinumab, which has the fastest progress in new drug application in the world.

Description

Cell line and production method for producing ustekinumab biosimilars Technical Field

[0001] The present invention relates to the field of antibody drugs. Specifically, the present invention relates to a method for producing Ustekinumab (Ustekinumab, trade name ) and a method for producing a biosimilar to ustekinumab using the CHO-S cell line. Background Art

[0002] Ustekinumab (trade name: ) was developed by Johnson & Johnson and is a fully human monoclonal antibody targeting the p40 subunit shared by human IL-12 and IL-23. IL-12 and IL-23 are heterodimeric cytokines expressed by activated antigen-presenting cells. IL-12 can activate NK cells and promote CD4 + T cells differentiate into helper T cells 1 (Th1), and IL-23 can activate the helper T cell 17 (Th17) pathway. Dysregulation of IL-12 and IL-23 is closely associated with immune-related diseases. Ustekinumab inhibits IL-12 and IL-23-mediated signaling and cytokine cascades by blocking the binding of the p40 subunit shared by IL-12 and IL-23 to the IL-12Rβ1 receptor protein on the surface of target cells, thereby exerting therapeutic effects in patients with autoimmune diseases such as psoriasis and inflammatory bowel disease. It was launched in Canada in 2008, and subsequently in Europe, the United States, Japan and other places. Its approved clinical indications include psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, etc.

[0003] Johnson & Johnson uses SP2 / 0 cells as host cells for expression of ustekinumab. Antibodies expressed by these SP2 / 0 cells have a high proportion of sialic acid modifications, posing a certain risk of immunogenicity when administered to humans. In theory, switching host cells for ustekinumab expression could reduce its immunogenicity risk. However, generally speaking, even if the amino acid sequence is identical, the same monoclonal antibody expressed by different host cell types can exhibit structural differences due to factors such as differing glycosylation patterns. This can easily lead to differences in physicochemical properties, biological activity, and in vivo metabolic behavior, resulting in low similarity between biosimilars and the original drug, making them unable to pass similarity assessments. This is a major challenge in biosimilar development and a key reason why, despite the impending expiration of ustekinumab's patent protection, there is so little information available about its biosimilars.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a cell line that can be used to produce a biosimilar drug that is highly similar to ustekinumab in key physicochemical parameters and biological functions, as well as a method for producing a biosimilar drug to ustekinumab.

[0006] Specifically, the present invention includes:

[0007] 1. A Chinese Hamster Ovary S (CHO-S) cell line, deposited with the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2020232; the cell line expresses a fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23, the heavy chain amino acid sequence of the fully human monoclonal antibody being shown in SEQ ID NO: 1, and the light chain amino acid sequence of the fully human monoclonal antibody being shown in SEQ ID NO: 3; the cell line comprises an expression plasmid for expressing the fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23, the expression plasmid comprising a nucleotide sequence encoding the heavy chain amino acid sequence of the fully human monoclonal antibody as shown in SEQ ID NO: 2, and a nucleotide sequence encoding the light chain amino acid sequence of the fully human monoclonal antibody as shown in SEQ ID NO: 4.

[0008] 2. The CHO-S cell line according to item 1, wherein the fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23 expressed by the cell line does not substantially contain sialylated components.

[0009] 3. The CHO-S cell line according to item 1, wherein the fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23 expressed by the cell line is a biosimilar to Ustekinumab.

[0010] 4. A fully human monoclonal antibody against the p40 subunit common to human IL-12 and human IL-23, expressed by the CHO-S cell line according to any one of items 1 to 3, which is a biosimilar to ustekinumab.

[0011] 5. The fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23 according to item 5, which is substantially free of sialylated components.

[0012] 6. A method for producing a biosimilar of ustekinumab, comprising culturing the CHO-S cell line according to any one of items 1 to 3, thereby producing the fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23 according to item 4 or 5.

[0013] 7. An expression vector comprising a DNA having a nucleotide sequence as shown in SEQ ID NO: 2 and a DNA having a nucleotide sequence as shown in SEQ ID NO: 4.

[0014] 8. A host cell comprising the expression vector according to item 7.

[0015] Effects of the Invention

[0016] The Chinese Hamster Ovary (CHO) S cell line was named CHO-S-4 and deposited with the China Center for Type Culture Collection (CCTCC) under the accession number CCTCC C2020232. This CHO-S cell line can be cultured to express a fully human monoclonal antibody targeting the p40 subunit shared by human IL-12 and IL-23, thereby producing a biosimilar to ustekinumab. This biosimilar is highly similar to the original ustekinumab in key physicochemical parameters and biological functions. The ustekinumab biosimilar (designated QX001S) expressed by this CHO-S cell line not only demonstrated high similarity to ustekinumab in preclinical studies but also passed pharmacokinetic bioequivalence and safety similarity evaluations in clinical studies. This makes it the first ustekinumab biosimilar to enter clinical trials in China and the only one to complete Phase I clinical trials. To our knowledge, it is also one of the fastest-progressing ustekinumab biosimilars in new drug applications internationally. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the heavy chain amino acid sequence and light chain amino acid sequence of ustekinumab.

[0018] Figure 2 shows the main pharmacokinetic parameters and blood concentration-time changes of QX001S and ustekinumab in crab-eating monkeys after a single subcutaneous injection of 1 mg / kg.

[0019] Figure 3 shows the main pharmacokinetic parameters and blood concentration-time variation patterns of QX001S and ustekinumab after a single subcutaneous injection in humans, i.e., C max , AUC 0-t and AUC 0-inf 90% confidence interval (CI) for the ratio of geometric means.

[0020] FIG4 shows the codon-optimized heavy chain gene sequence of QX001S.

[0021] FIG5 is the codon-optimized light chain gene sequence of QX001S. DETAILED DESCRIPTION

[0022] definition

[0023] As used herein, the term "cell line" refers to cultured cells with specific properties or hallmarks obtained from a primary culture or cell line by selection or cloning. The specific properties or hallmarks of a cell line must persist throughout the entire culture period. The genetic material of the cell line must remain unchanged.

[0024] The term "DNA" used in this article refers to a macromolecular polymer composed of deoxynucleotides. Deoxynucleotides are composed of bases, deoxyribose and phosphate. There are four types of bases: adenine (A), guanine (G), thymine (T) and cytosine (C). In the molecular structure, two polydeoxynucleotide chains are coiled around a common central axis to form a double helix structure. The deoxyribose-phosphate chain is on the outside of the helical structure, and the bases are facing inside. The two deoxynucleotide chains are reverse complementary and are connected by base pairing formed by hydrogen bonds between the bases, forming a fairly stable combination. DNA carries the genetic information necessary for the synthesis of RNA and proteins, and is an essential biological macromolecule for the development and normal functioning of organisms.

[0025] The term "expression vector" as used herein refers to a vector that adds expression elements (such as promoters, ribosome binding sites, terminators, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.

[0026] As used herein, the term "monoclonal antibody" refers to a highly homogeneous antibody produced by a single B cell clone that targets a specific antigenic epitope. These antibodies are typically produced using hybridoma technology, which is based on cell fusion technology, fusing primed B cells capable of secreting specific antibodies with myeloma cells, which have unlimited reproductive capacity, to form a B cell hybridoma. By culturing a single hybridoma cell with these properties into a cell population, a specific antibody against a single antigenic epitope, known as a monoclonal antibody, can be produced.

[0027] The term "humanized antibody" as used herein, also known as complementarity determining region transplanted antibody, refers to an antibody in which the variable region portion or the entire antibody is encoded by human antibody genes.

[0028] The term "humanized monoclonal antibody" as used in this article refers to an antibody produced by utilizing the numerous existing and well-analyzed mouse antibodies, taking the antibody fragment that directly contacts the antigen (complementarity determining region, CDR) and grafting it onto the human antibody framework. After affinity remodeling, the antibody can maintain its specificity and most of its affinity while almost eliminating immunogenicity and toxic side effects.

[0029] The term "fully humanized antibody" as used herein refers to the transfer of all human antibody-encoding genes into genetically engineered antibody-deficient animals through transgenic or transchromosomal techniques, allowing the animals to express human antibodies and achieve the goal of fully humanized antibodies. Various methods have been established to produce fully humanized antibodies, including phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0030] The present invention relates to a Chinese hamster ovary cell S (CHO-S) cell line, which is named CHO-S-4, and is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: C2020232 and a deposit date of December 3, 2020.

[0031] In a specific embodiment, the Chinese Hamster Ovary S (CHO-S) cell line expresses a fully human monoclonal antibody against the human IL-12 / IL-23p40 subunit, the heavy chain amino acid sequence of the fully human monoclonal antibody is the heavy chain amino acid sequence of ustekinumab, and the light chain amino acid sequence of the fully human monoclonal antibody is the light chain amino acid sequence of ustekinumab.

[0032] In a specific embodiment, the Chinese hamster ovary S (CHO-S) cell line expresses a fully human monoclonal antibody against the human IL-12 / IL-23p40 subunit, the heavy chain amino acid sequence of the fully human monoclonal antibody is shown in SEQ ID NO: 1, and the light chain amino acid sequence of the fully human monoclonal antibody is shown in SEQ ID NO: 3.

[0033] SEQ ID NO: 1

[0034]

[0035] SEQ ID NO: 3

[0036]

[0037] In a specific embodiment, the Chinese hamster ovary S (CHO-S) cell line contains an expression plasmid for expressing the fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23, and the expression plasmid contains a nucleotide sequence encoding the heavy chain amino acid sequence of the fully human monoclonal antibody as shown in SEQ ID NO: 2, and a nucleotide sequence encoding the light chain amino acid sequence of the fully human monoclonal antibody as shown in SEQ ID NO: 4.

[0038] SEQ ID NO: 2

[0039]

[0040]

[0041] SEQ ID NO: 4

[0042]

[0043] In a specific embodiment, the Chinese Hamster Ovary S (CHO-S) cell line expresses a fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23, wherein the fully human monoclonal antibody is substantially free of sialylated components.

[0044] The present invention also relates to a fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23, expressed by the above-mentioned CHO-S cell line, which is a biosimilar to Ustekinumab (named QX001S) and is highly similar to the original drug Ustekinumab.

[0045] Specifically, the primary, secondary and higher-order structures of QX001S are similar to those of ustekinumab; the glycosylation modification sites are similar; the analytical data of charge heterogeneity, such as the deglycosylated isoelectric point, acidic peak, and basic peak are similar; the data of aggregate and degradation product analysis are similar; the thermal stability is similar; the analytical data of in vitro biological activity, such as antigen IL-12 binding activity, antigen IL-12 affinity, antigen IL-23 binding activity, antigen IL-23 affinity, IL-12 receptor antagonist activity, IL-23 receptor antagonist activity, NK-92 MI cell activity, mouse spleen cell activity, Fc-related receptor affinity, species specificity analysis, and consistency analysis of antigen recognition epitopes are similar.

[0046] Pharmacodynamic studies in an IL-23-induced mouse ear epidermal hyperplasia model demonstrated highly similar pharmacodynamic effects between the QX001S and ustekinumab groups. Pharmacokinetic studies in cynomolgus monkeys demonstrated similar pharmacokinetic parameters and plasma concentration-time patterns for ustekinumab and QX001S. In vivo pharmacokinetic studies in humans demonstrated pharmacokinetic bioequivalence and similar safety profiles between QX001S injection and ustekinumab injection.

[0047] In addition, the present invention also relates to a method for producing a biosimilar of Ustekinumab, which comprises culturing the CHO-S cell line to produce a fully human monoclonal antibody against the p40 subunit shared by human IL-12 and human IL-23.

[0048] Example

[0049] Hereinafter, the present invention will be described in more detail by way of examples, but it should be understood that the present invention is not limited to these examples.

[0050] Example 1: Vector construction

[0051] The heavy chain amino acid sequence (SEQ ID NO: 1) and light chain amino acid sequence (SEQ ID NO: 3) of ustekinumab were determined through public information query and peptide analysis (see Figure 1).

[0052] The heavy chain amino acid sequence and light chain amino acid sequence of QX001S, a biosimilar to ustekinumab, are exactly the same as those of ustekinumab.

[0053] Using the heavy and light chain amino acid sequences of ustekinumab as templates, the heavy and light chain gene sequences of QX001S were designed, and expression plasmids were constructed.

[0054] The codon-optimized heavy chain gene sequence of QX001S is SEQ ID NO: 2 (as shown in FIG4 ).

[0055] The codon-optimized light chain gene sequence of QX001S is SEQ ID NO: 4 (as shown in FIG5 ).

[0056] The expression plasmid system consists of codon-optimized QX001S heavy and light chain genes constructed using known molecular biological methods into their respective blank expression vectors. These genes are then linked to upstream and downstream expression regulatory elements. The heavy chain and its expression regulatory elements are then enzymatically cleaved and ligated to the light chain expression vector to form the heavy- and light-chain linked plasmid, which is the final expression plasmid used for transfection and construction of engineered cells. This final expression plasmid also contains the glutamine synthetase (GS) gene selection marker, a widely used selection marker in the construction of engineered cell lines using CHO cells, which can be used for subsequent screening of stably transfected cells.

[0057] The expression plasmid obtained above was transiently expressed, and then the protein sequence of the expressed QX001S was identified and the activity was analyzed to confirm that the gene sequence of the constructed QX001S expression vector and the expressed protein were correct.

[0058] Example 2: Screening of cell lines

[0059] The gene sequence of the constructed QX001S expression vector and the expressed protein were confirmed to be correct, and a stable expression cell line of QX001S was constructed using CHO-S as the host cell. CHO-S host cells were purchased from ThermoFisher (formerly Invitrogen, product number A13696-01), and CHO-S was revived and cultured. TMAfter the linearized expression vector is electroporated into the host cell, it is randomly integrated into the host cell genome through the homology arms on the vector.

[0060] Using glutamine synthetase as a screening marker, a CHO-S cell line that stably and highly expresses QX001S, whose physicochemical properties are consistent with ustekinumab, was obtained through methionine methylene sulfone (MSX) pressure screening.

[0061] 1. Through step-by-step amplification, ELISA testing and other operations, a high-yield cell pool (mixed cell clone) was initially screened from 40 cell clones cultured in 96-well plates, and 14 high-yield cell pools were selected as candidate pools for the first round of subcloning.

[0062] 2. The 14 cell pools obtained were not monoclonal, and these cells needed to be monocloned to obtain a single stable cell line. The 14 high-yield cell pools were subcloned using the limiting dilution method, and high-expressing monoclones were screened by step-by-step amplification and ELISA detection. A total of 694 cell clones were amplified, and finally the 33 clones with the highest OD values ​​were selected and expanded to T25 (8 ml of CD CHO medium containing 100 μmol / L MSX) for suspension culture. After 8 days of cell culture in T25, the cell supernatant was harvested, the IgG content was detected by Fortebio, and QX001S was purified with Protein A for quality analysis.

[0063] 3. Considering that the quality attributes of the expressed QX001S must be consistent with those of ustekinumab, clones 4, 9, 25, and 27 were selected from the 33 monoclonal clones described above, whose secreted QX001S expressed a simple and low-content basic component after carboxypeptidase treatment. Furthermore, peptide mapping analysis comparing the QX001S expressed by clones 4, 25, and 27 with ustekinumab revealed essentially identical peptide maps, with no significant increase or decrease in peptide fragments. This indicates that the amino acid sequence of the QX001S secreted by these three clones is correct.

[0064] 4. To obtain a uniform, stable, and high-yielding QX001S production cell line, a second round of subcloning screening was performed on the four candidate clones. Clone 25 did not meet the cell status requirements during the second round of subcloning screening; clones obtained from clone 27 after the second round of subcloning had low yields; and clone 9 did not meet the requirements in the initial stability assessment. All three clones were eliminated.

[0065] That is, among the 33 clones that entered the quality analysis, only one clone, clone 4, met the requirements as a QX001S production cell line.

[0066] The CHO-S cell line of clone 4 was named Chinese Hamster Ovary Cell CHO-S-4 and deposited with the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2020232 on December 3, 2020. The fully human monoclonal antibody against human IL-12 / IL-23p40 subunit expressed by the CHO-S cell line of clone 4 was named QX001S.

[0067] Example 3: Pharmaceutical Similarity Study

[0068] The pharmaceutical similarity between QX001S expressed in the CHO-S cell line clone 4 and ustekinumab was investigated. A comprehensive comparison of the primary, secondary, and higher-order structures, post-translational modifications, charge heterogeneity, aggregates and degradation products, in vitro biological activity, and accelerated stability of QX001S and ustekinumab revealed a high degree of pharmaceutical similarity. QX001S expressed in this CHO-S cell line demonstrated substantial similarity to ustekinumab in 71 of the 78 parameters listed in Table 1.

[0069] The main indicators of difference between the two are in post-translational modification (5 indicators are different). In particular, it was determined that the proportion of sialylated components of ustekinumab was between 11.87% and 25.56%, while QX001S expressed by the CHO-S cell line basically did not contain sialylated components, suggesting that QX001S may have lower immunogenicity than ustekinumab.

[0070] Table 1. Summary of indicators for pharmaceutical similarity studies

[0071]

[0072]

[0073]

[0074] Example 4: Pharmacodynamic Study in Mice (Preclinical Animal Level Efficacy Evaluation)

[0075] In this example, the effect of QX001S on otic epidermal hyperplasia in a mouse IL-23-induced otic epidermal hyperplasia model was evaluated, and the pharmacodynamics of QX001S and the original drug ustekinumab (named QX001S-DZY in this study, EU marketed product: batch number FJS1W25) in this model were compared. The study showed that both ustekinumab and QX001S showed significant inhibition of rhIL-23-induced otic epidermal hyperplasia, as demonstrated by reduced ear thickness and reduced pathological comprehensive scores. The QX001S group and the ustekinumab group showed highly similar pharmacodynamic consistency.

[0076] Forty-eight C57BL / 6 mice were randomly divided into six groups, with eight mice in each group. Mice were intradermally injected with blank vehicle or rhIL-23 on the back of their right ears for eight consecutive days. QX001S or QX001S-DZY was intraperitoneally injected 1 hour before rhIL-23 injection. Ear thickness and ear thickness increment were measured on days 0, 2, 4, 6, and 8. On day 8, the right ears of all 48 mice were excised for paraffin section preparation and stained with HE. The stained sections were scored for degree of keratinization, epidermal thickness, dermal thickness, and lymphocyte infiltration (0-4 points for each item). The scores of these four components were added together to form a comprehensive pathological score (0-16 points).

[0077] The experimental results showed that there was no significant difference in right ear thickness, increased right ear thickness, and comprehensive pathology score between the QX001S 10 mg / kg and QX001S-DZY 10 mg / kg groups (see Tables 2 and 3 for specific data).

[0078] Table 2. Thickness of the right ear and the thickness increase of the right ear

[0079]

[0080] Data are expressed as mean ± SD.

[0081] # p<0.05, ## p < 0.01, compared with control; * p<0.05, ** p < 0.01, comparison model;

[0082] △ p<0.05, △△ p < 0.01, QX001S 3 mg / kg vs. QX001S-DZY 3 mg / kg;

[0083] ☆ p<0.05, ☆☆p<0.01, QX001S 10 mg / kg vs. QX001S-DZY 10 mg / kg.

[0084] Table 3. Pathological scores of right ear tissue sections after HE staining

[0085]

[0086]

[0087] Data are expressed as mean ± SD.

[0088] # p<0.05, ## p < 0.01, compared with control; * p<0.05, ** p < 0.01, comparison model;

[0089] △ p<0.05, △△ p < 0.01, QX001S 3 mg / kg vs. QX001S-DZY 3 mg / kg;

[0090] ☆ p<0.05, ☆☆ p<0.01, QX001S 10 mg / kg vs. QX001S-DZY 10 mg / kg.

[0091] Example 5: Pharmacokinetic Study in Cynomolgus Monkeys (Preclinical Animal-Level Pharmacokinetic Evaluation)

[0092] In this example, the pharmacokinetic characteristics of QX001S in cynomolgus monkeys were evaluated, and the pharmacokinetic parameters of QX001S and the original drug ustekinumab (designated QX001S-DZY in this study) were compared in cynomolgus monkeys. The study demonstrated that ustekinumab and QX001S exhibited similar pharmacokinetic parameters and plasma concentration-time patterns in cynomolgus monkeys.

[0093] In this example, 36 cynomolgus macaques were randomly divided into six groups of six animals each. Three groups received a single subcutaneous injection of QX001S (0.35, 1, and 3 mg / kg), one group received a single intravenous bolus of QX001S (1 mg / kg), one group received a single subcutaneous injection of QX001S-DZY (1 mg / kg), and one group received repeated subcutaneous injections of QX001S (1 mg / kg). Serum was collected intravenously from all animals at various time points before and after administration. Serum concentrations of QX001S and QX001S-DZY were measured using a validated ELISA method, and relevant pharmacokinetic parameters were calculated using a non-compartmental model.

[0094] The results of pharmacokinetic analysis showed that there were no significant differences in the main pharmacokinetic parameters between the QX001S and QX001S-DZY groups after a single subcutaneous injection of 1 mg / kg (p>0.05). The pharmacokinetic parameters and blood concentration-time patterns of the two drugs in crab-eating monkeys were similar (as shown in Figure 2).

[0095] The immunogenicity results showed that 83% (5 / 6) of individuals in the QX001S and QX001S-DZY groups received a single subcutaneous injection of 1 mg / kg and developed anti-drug antibodies, and the immunogenicity of the two drugs in crab-eating macaques was similar.

[0096] Example 6 Pharmacokinetic Study in Humans (Phase I Clinical Study)

[0097] QX001S injection is the world's first ustekinumab injection (Ustekinumab, trade name ) biosimilar, the Phase I clinical study, "A randomized, double-blind, single-dose, parallel comparison of the pharmacokinetic profile of QX001S injection and ustekinumab injection (marketed in the EU: manufacturing batch number GDS3IME) in healthy Chinese male volunteers," has been successfully completed, with the study results meeting all pre-specified primary and secondary endpoints (National Drug Clinical Trials Registration and Information Disclosure Platform registration number CTR20181658). Based on the positive results of the Phase I clinical study, we are fully preparing to initiate a Phase III clinical study of QX001S for the indication of psoriasis. Key results from the Phase I clinical study include:

[0098] 1. Pharmacokinetic bioequivalence

[0099] The 90% CIs of the geometric mean ratios of Cmax, AUC0-t, and AUC0-inf of QX001S and ustekinumab were 100.90% to 118.68%, 98.71% to 115.26%, and 98.49% to 115.81%, respectively, all falling within the prescribed equivalence range of 80.00% to 125.00%, indicating that QX001S injection and ustekinumab injection are pharmacokinetic bioequivalent (as shown in Figure 3).

[0100] 2. Similar security

[0101] Of the 177 subjects included in the safety analysis population, 98 experienced 228 TEAEs. No subjects experienced SAEs, and no withdrawals were due to AEs. The incidence of adverse drug reactions was 42.7% in the QX001S and 42.0% in the ustekinumab groups, demonstrating similar safety profiles.

[0102] In addition, among the 177 subjects who entered the immunogenicity analysis population in this study, there were 11 positive subjects in the QX001S group, with a positive rate of 12.4%, and 21 positive subjects in the ustekinumab group, with a positive rate of 23.9%.

[0103] It should also be noted that the original drug ustekinumab uses SP2 / 0 cells as the host cell for recombinant expression, while the biosimilar QX001S uses CHO-S cells as the host cell for recombinant expression. Generally speaking, even if the amino acid sequence of the same monoclonal antibody is the same, different host cells may express different structures due to factors such as different glycosylation patterns. This can easily lead to differences in physicochemical properties, biological activity, and in vivo metabolic behavior, resulting in inconsistencies between the biosimilar and the original drug, and failure to pass the consistency evaluation. This is a major difficulty in biosimilar development and a key reason why, despite the impending expiration of ustekinumab's patent protection, there is so little information available about its biosimilars. In contrast, the ustekinumab biosimilar QX001S, expressed by a specific CHO-S cell line obtained through screening in the present invention, not only demonstrated a high degree of consistency with ustekinumab in preclinical studies, but also passed the pharmacokinetic bioequivalence and safety similarity evaluations in clinical studies. It has become the first ustekinumab biosimilar to enter clinical trials in China and the only one to complete Phase I clinical trials. To our knowledge, it is also one of the ustekinumab biosimilars with the fastest progress in new drug application internationally.

[0104] While the embodiments of the present invention have been described above through specific examples, those skilled in the art will readily appreciate the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different perspectives and applications without departing from the spirit of the present invention.

Claims

1. A Chinese hamster ovary cell S (CHO-S) cell line, which is deposited with the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2020232; it expresses a fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23. The amino acid sequence of the heavy chain of the fully human monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the fully human monoclonal antibody is shown in SEQ ID NO: 3; it contains an expression plasmid expressing the fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23. The expression plasmid contains a nucleotide sequence encoding the amino acid sequence of the heavy chain of the fully human monoclonal antibody shown in SEQ ID NO: 2, and a nucleotide sequence encoding the amino acid sequence of the light chain of the fully human monoclonal antibody shown in SEQ ID NO:

4.

2. The CHO-S cell line according to claim 1, wherein the fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23 expressed by it is substantially free of sialylated components.

3. The CHO-S cell line according to claim 1, wherein the fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23 expressed by it is a biosimilar of ustekinumab.

4. A fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23 expressed by the CHO-S cell line according to any one of claims 1 to 3, which is a biosimilar of ustekinumab.

5. The fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23 according to claim 5, which is substantially free of sialylated components.

6. A method for producing a biosimilar of ustekinumab, which comprises culturing the CHO-S cell line according to any one of claims 1 to 3 to produce the fully human monoclonal antibody against the common p40 subunit of human IL-12 and human IL-23 according to claim 4 or 5.

7. An expression vector, which contains DNA with a nucleotide sequence shown in SEQ ID NO: 2 and DNA with a nucleotide sequence shown in SEQ ID NO:

4.

8. A host cell, which contains the expression vector according to claim 7.