A novel backbone flow antibody with fc effector elimination and its preparation method and application

Abstract

The application discloses a novel Fc effector-eliminated scaffold flow antibody, and belongs to the technical field of biological medicine. The antibody comprises a L234A, L235A, P238S and D265A mutation combination in a heavy chain CH2 region, and can further comprise S267E and L328F mutations, so that the Fc receptor binding capacity is effectively reduced. The antibody type can be selected from a scFv-Fc, (scFv)2-Fc, scFv / scFv-Fc and Fab / scFv-Fc and the like. The application further provides a nucleic acid sequence for coding the antibody and a preparation method, which comprises introducing specific mutations in a CH2 region of recombinant rabbit IgG, expressing in a mammalian expression system and purifying to obtain the antibody. When the antibody is applied to a flow cytometry detection reagent, the non-specific background signal caused by Fc receptor binding can be effectively eliminated, and the specificity and accuracy of detection are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering, in particular to a novel framework flow antibody with Fc effector elimination and a preparation method and application thereof. BACKGROUND

[0002] Flow cytometry is an important technology for cell analysis, widely used in immunology, oncology and cell biology, etc. In flow cytometry, antibodies are the key reagents for recognizing specific cell surface markers. Rabbit-derived antibodies have become a common detection tool in flow cytometry due to their high affinity and specificity. However, rabbit IgG antibodies face the problem of Fc-mediated non-specific binding in flow cytometry applications, which seriously restricts their detection performance and application range.

[0003] Currently, the industry has some understanding of the Fc effect mechanism of rabbit-derived antibodies. Studies have shown that there are significant structural differences between rabbit IgG and human IgG in the CH2 region, and its unique amino acid sequence and spatial conformation lead to its strong binding ability to human Fcγ receptors (such as FcγRI, FcγRII and FcγRIII). This binding easily causes non-specific adsorption with cells expressing FcγR (such as monocytes, macrophages, B cells, etc.) in flow detection, resulting in an increase in background signal and a decrease in signal-to-noise ratio, especially in the detection of low-abundance antigens or the analysis of complex samples (such as whole blood, spleen cells, etc.).

[0004] To overcome the Fc-mediated non-specific binding, two main strategies are used in the prior art: one is to use F(ab')2 fragments instead of complete IgG, which lacks the Fc region and can avoid FcγR binding, but its enzyme cutting preparation process is complex, the yield is low, the stability is poor, and it is easy to aggregate or degrade, which limits its standardization and large-scale application; the second is to reduce the effector function by site-directed mutagenesis of the Fc region, for example, the "LALA" (L234A / L235A) mutation widely used in human IgG can effectively weaken the binding to FcγR. However, due to the large difference in sequence and structure between rabbit IgG and human IgG in the CH2 region, the mutation strategy of directly transplanting human antibodies cannot effectively eliminate the Fc effect of rabbit IgG, and may even lead to structural instability or loss of affinity.

[0005] The existing patent technology such as CN113348177A provides an antibody design of a modified heavy chain constant region for reducing effector function, but it focuses on human or murine antibodies and does not involve the structural specificity of rabbit IgG; CN108299557A adjusts the half-life by mutating to change the binding property of the antibody to FcRn, and also does not solve the problem of non-specific binding of rabbit IgG in flow detection. In general, there is currently a lack of mutation schemes for the characteristics of the Fc region of rabbit IgG, which can systematically eliminate its binding to FcγR and do not affect the antigen binding activity.

[0006] Therefore, the technical problem to be solved by the present application is to provide a new rabbit IgG framework antibody that can effectively eliminate Fc effect while maintaining high stability and antigen binding capacity, and to establish a reliable preparation method and application form in flow detection, in order to solve the problems of strong non-specific binding, high detection background and low signal-to-noise ratio caused by Fc effect of existing rabbit source flow antibodies. SUMMARY

[0007] The technical problem to be solved by the present application is to solve the defects of traditional rabbit IgG antibodies in flow cytometry, including: due to the strong Fc receptor binding ability of rabbit IgG, non-specific binding occurs with myeloid cells (such as mononuclear / macrophages) expressing FcγR; high background noise affects the detection of weakly expressed targets; the existing technology uses F(ab')2 fragments but has the defects of low yield and poor stability; and the known "LALA" mutation (L234A / L235A) of human IgG can reduce the effector function, but due to the unique binding epitope of the CH2 structure of rabbit IgG, the human Fc elimination mutation is invalid in rabbit IgG.

[0008] The first technical solution provided by the present application is a new framework flow rabbit antibody with Fc effect elimination, wherein the heavy chain CH2 region of the rabbit IgG parent contains the following mutation combinations: the 234th leucine is replaced by alanine (L234A), the 235th leucine is replaced by alanine (L235A), the 238th proline is replaced by serine (P238S), and the 265th aspartic acid is replaced by alanine (D265A), and the amino acid sequence of the heavy chain constant region of the rabbit IgG parent is shown in SEQ ID NO. 1.

[0009] In some embodiments, the heavy chain CH2 region of the rabbit antibody further contains the following mutation combinations: the 267th serine is replaced by glutamic acid (S267E), and the 328th leucine is replaced by phenylalanine (L328F).

[0010] In some embodiments, the Fc effector-eliminated antibody is selected from the group consisting of scFv-Fc, (scFv)2-Fc, scFv / scFv-Fc, Fab / scFv-Fc, Fab / (scFv)2-Fc, Fab / Fab scFv Fc, Fab / Fab crossFabFc, Fab / crossFab-Fc, IgG scFv, and IgG (scFv)2.

[0011] The present application also provides a second technical solution, which is a gene encoding the novel framework flow rabbit antibody mutant of the first technical solution.

[0012] The present application provides a third technical solution, which is a recombinant vector carrying the gene of the second technical solution.

[0013] In some embodiments, the recombinant vector is expressed in a phage display vector pDAN5, a prokaryotic expression vector pET28a, or a eukaryotic expression vector pcDNA3.4 expression vector.

[0014] The present application provides a fourth technical solution, which is a recombinant cell expressing the novel framework flow rabbit antibody of the first technical solution, or containing the gene of the second technical solution, or transformed or transfected with the recombinant vector of the third technical solution.

[0015] In some embodiments, the recombinant cell is used as a host for E. coli TG1, BL21(DE3), or mammalian HEK293F cells.

[0016] The present application also provides a fifth technical solution, which is a method for preparing the Fc effector-eliminated novel framework flow rabbit antibody, the method being culturing the recombinant cell of the fourth technical solution to obtain a culture containing the novel framework flow rabbit antibody of the first technical solution.

[0017] The present application provides a sixth technical solution, which is a method for eliminating the Fc effect of rabbit IgG antibody, the method being mutating the heavy chain CH2 region of the rabbit IgG parent as follows: replacing the 234th leucine with alanine (L234A), replacing the 235th leucine with alanine (L235A), replacing the 238th proline with serine (P238S), replacing the 265th aspartic acid with alanine (D265A), replacing the 267th serine with glutamic acid (S267E), and replacing the 328th leucine with phenylalanine (L328F).

[0018] The seventh technical solution of the present application provides the application of the novel framework flow rabbit antibody of the first technical solution, or the gene of the second technical solution, or the recombinant vector of the third technical solution, or the recombinant cell of the fourth technical solution, or the method of the fifth technical solution, or the method of the sixth technical solution in flow cytometry or in the preparation of flow cytometry products.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] By introducing specific amino acid mutations in the CH2 region of rabbit IgG antibody, especially the L234A / L235A / P238S / D265A / S267E / L328F mutation combination, the binding affinity of rabbit IgG antibody to activated FcγR (FcγRI, FcγRIIa, FcγRIIIa) is significantly reduced, usually by several orders of magnitude; almost completely eliminates antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP); significantly reduces the binding to C1q, thereby effectively inhibiting complement-dependent cellular cytotoxicity (CDC). Compared with the prior art, the present application solves the problem of non-specific binding of rabbit IgG antibody in flow cytometry, reduces the background noise, and improves the detection effect of weakly expressed targets; compared with the F(ab')2 fragment of the prior art, the mutant rabbit IgG of the present application has higher yield and better stability, and in the application of flow cytometry reagents, compared with non-mutant antibodies, the non-specific background signal caused by Fc receptor binding is eliminated. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For CD3 novel framework flow rabbit antibody stability detection: A, SEC-HPLC purity analysis, SDS-PAGE purity analysis of 0 days, 15 days, 30 days storage at 37℃; B, affinity determination of 0 days, 15 days, 30 days storage at 37℃; C, flow cytometry experiment of 0 days, 15 days, 30 days storage at 37℃.

[0022] Figure 2 For wild-type antibody, four-mutation antibody and six-mutation antibody human PBMC sample flow cytometry experiment non-specific background signal comparison analysis. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explanation of the present application and are not used to limit the present application.

[0024] Raw materials used in the examples:

[0025] Experimental materials in the examples:

[0026] Human FcγRI, FcγRII and FcγRIII receptor proteins were purchased from Wuhan Huamei Bioengineering Co., Ltd., with item numbers CSB-YP008537HU1, CSB-MP008540HU1 and CSB-AP005111HU, respectively. Human peripheral blood mononuclear cells (PBMC) and human bone marrow mononuclear cells were purchased from Wuhan Punsai Life Science and Technology Co., Ltd., with item numbers CP-H182 and CP-H185, respectively.

[0027] Example 1. Preparation of four-mutant antibody

[0028] This example provides a new type of Fc effector-eliminated flow rabbit antibody, which comprises the following combination of mutations in the CH2 region of the heavy chain: substitution of leucine at position 234 to alanine (L234A), substitution of leucine at position 235 to alanine (L235A), substitution of proline at position 238 to serine (P238S), and substitution of aspartic acid at position 265 to alanine (D265A), and specifically comprises the following steps:

[0029] I. Design of anti-human CD3, CD14, CD33 and CD56 recombinant rabbit IgG with CH2 containing the above four mutation sites, and addition of human interleukin-2-alpha receptor signal peptide at the N-terminus to enable extracellular secretion of the antibody.

[0030] In order to obtain an antibody gene with high expression, the nucleic acid sequence encoding the mutant antibody was designed and optimized in this example. Specifically, according to the codon usage bias of mammalian cells (particularly CHO cells), the antibody gene sequence was codon-optimized to improve the expression level of the antibody in the expression system.

[0031] In this example, the nucleic acid sequence of the four-mutant antibody (L234A / L235A / P238S / D265A) comprises the following parts:

[0032] 1. Signal peptide sequence: a nucleic acid sequence encoding a secretion signal peptide, which is used to guide the secretion of the antibody molecule outside the cell. A highly efficient human interleukin-2-alpha receptor signal peptide sequence was selected, with a length of about 63 nucleotides.

[0033] 2. Constant region sequence: a nucleic acid sequence encoding the constant region of the antibody, including the sequence of the CH2 region containing the four mutation sites. The corresponding nucleotide changes of the specific mutation sites are as follows:

[0034] - L234A: CTG / CTC / CTA / CTT / TTA / TTG → GCC / GCT / GCA / GCG

[0035] - L235A: CTG / CTC / CTA / CTT / TTA / TTG -> GCC / GCT / GCA / GCG

[0036] - P238S: CCT / CCC / CCA / CCG -> TCT / TCC / TCA / TCG

[0037] - D265A: GAT / GAC -> GCT / GCC / GCA / GCG

[0038] The amino acid sequence of the four-mutant Fc constant region is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 12.

[0039] 3. Variable region sequence: the amino acid sequence encoding the variable region of the heavy and light chains of the antibody, which determines the antigen specificity of the antibody. The variable region sequence is different according to different antigen targets. The amino acid sequence of the heavy chain variable region of the CD3 antibody is shown in SEQ ID NO. 4, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 5, the amino acid sequence of the heavy chain variable region of the CD14 antibody is shown in SEQ ID NO. 6, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7, the amino acid sequence of the heavy chain variable region of the CD33 antibody is shown in SEQ ID NO. 8, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 9, the amino acid sequence of the heavy chain variable region of the CD56 antibody is shown in SEQ ID NO. 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 11.

[0040] 4. Stop codon and polyadenylation signal sequence: used to terminate translation and guide mRNA tailing.

[0041] The desired amino acid mutations are introduced into the CH2 region of the recombinant rabbit IgG by site-directed mutagenesis technology. First, primers containing the target mutation site are designed based on the wild-type rabbit IgG sequence; then, the mutations are introduced using the overlap extension PCR method or commercial site-directed mutagenesis kit; finally, the correctness of the mutations is verified by DNA sequencing.

[0042] For the four-mutant antibody (L234A / L235A / P238S / D265A), all four mutations can be introduced at once, or they can be introduced step by step. This embodiment uses a step-by-step strategy: first, introduce the L234A and L235A mutations, then introduce the P238S mutation based on this, and finally introduce the D265A mutation.

[0043] II. The Xba I and Age I restriction enzyme sites are designed at both ends of the above-mentioned gene sequence, which is cloned into pcNDA3.4, and then the mutant antibody is expressed in a mammalian expression system.

[0044] The expression vector containing the mutant sequence is transfected into a mammalian expression system to express the mutant antibody. In this embodiment, a CHO cell line (such as CHO-K1, CHO-S or CHO-DG44) is mainly used as the expression host, and a HEK293 cell line can also be used as an alternative.

[0045] The transfection method can be selected from lipofection (such as Lipofectamine 3000), electroporation or viral transduction. In this embodiment, the lipofection method is used, and the specific operation is as follows: 1-5 μg of expression vector DNA is mixed with 10-15 μL of transfection reagent, incubated at room temperature for 15-20 minutes, and then added to CHO cells with a confluence of 70-80%; 24 hours after transfection, a selective antibiotic (such as puromycin, at a concentration of 30-50 μg / mL) is added to screen the stably transfected cells; after 2-3 weeks of screening, a stable expression cell pool is obtained.

[0046] For high yield requirements, monoclonal screening and expansion are also carried out in this embodiment. The specific method is as follows: the stable expression cell pool is inoculated into a 96-well plate by the limiting dilution method (an average of 0.5-1 cells per well), after 2-3 weeks of culture, the monoclonal cell supernatant is collected, the antibody expression amount is detected by ELISA method, and the high expression clone is selected for expansion culture.

[0047] III. The target antibody is purified by protein A affinity chromatography.

[0048] High-purity four-mutant antibody L234A / L235A / P238S / D265A is obtained by multi-step chromatography purification, and the specific purification process is as follows:

[0049] 1. Collecting cell culture supernatant: when the cell culture reaches the optimal harvest time point (usually 7-14 days after transfection, cell viability > 80%), collect the culture supernatant, remove cells and debris by centrifugation (5000 g, 20 minutes) and filtration (0.22 μm filter membrane).

[0050] 2. Affinity chromatography: protein A or protein G affinity column (such as GE Healthcare's MabSelect SuRe or Thermo Fisher's Protein A Sepharose) is used to purify the antibody. The specific operation is as follows: equilibrate the column (PBS, pH 7.4), sample (cell culture supernatant), wash (PBS, pH 7.4), elute (0.1 M glycine-HCl, pH 2.7-3.0), and neutralize (1 M Tris-HCl, pH 8.0).

[0051] 3. Ion exchange chromatography: The antibody is further purified by using an anion exchange column (such as Q Sepharose of GE Healthcare) to remove impurities such as aggregates, host cell proteins and DNA. The specific operation is as follows: equilibration of the column (20 mM Tris-HCl, pH 8.0), sample loading (protein A purification product), washing (20 mM Tris-HCl, pH 8.0), gradient elution (20 mM Tris-HCl, pH 8.0 containing 0-500 mM NaCl).

[0052] 4. Concentration and formulation adjustment: The purified antibody is concentrated to the required concentration (usually 1-10 mg / mL) by using an ultrafiltration centrifuge tube (such as Amicon Ultra of Millipore), and adjusted to the final formulation (such as PBS, pH 7.4, containing 0.05% Tween 20 and 5% glycerol) by buffer exchange.

[0053] In addition, the mutant antibodies in this embodiment also exhibit good stability and producibility. For example, the anti-human CD3 mutant antibody is still single and stable after being stored at 37°C for 30 days, and has high biological activity (EC50= 0.1 ng / mL). Figure 1 The expression amount in the mammalian cell expression system can reach 1-2 g / L, meeting the needs of large-scale production.

[0054] Preparation of six-mutant antibody

[0055] Compared with Example 1, this embodiment additionally introduces two mutation sites in the CH2 region of the anti-human CD3, CD14, CD33 and CD56 recombinant rabbit IgG heavy chain: the 267th serine is replaced by glutamic acid (S267E) and the 328th leucine is replaced by phenylalanine (L328F). These two additional mutation sites further enhance the Fc effector elimination ability of the antibody, reducing the binding ability of the antibody to the Fc receptor to a lower level.

[0056] Specifically, the 267th serine replaced by glutamic acid (S267E) mainly affects the interaction of the antibody with FcyRIIa and FcyRIIb receptors, while the 328th leucine replaced by phenylalanine (L328F) mainly affects the interaction of the antibody with FcyRIII receptor.

[0057] The six-mutant antibody (L234A / L235A / P238S / D265A / S267E / L328F) described in this embodiment, on the basis of the nucleic acid sequence of the four-mutant antibody described above, also uses a step-by-step strategy, first introducing the S267E mutation, and then introducing the L328F mutation, and additionally containing the following two mutation sites of nucleotide changes:

[0058] - S267E: TCT / TCC / TCA / TCG → GAA / GAG

[0059] - L328F: CTG / CTC / CTA / CTT / TTA / TTG → TTT / TTC

[0060] The amino acid sequence of the six mutant Fc constant region is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 13.

[0061] The recombinant expression and purification preparation of the six mutant antibody were carried out according to the method of Reference Example 1.

[0062] Comparative Example 1

[0063] In comparison with Example 1 and Example 2, the four mutant A: L234A / L235A / P238S / S267E was constructed by the molecular biology method described in Example 1 and 2, using the anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG as the parent antibody.

[0064] Comparative Example 2

[0065] In comparison with Example 1 and Example 2, the four mutant B: L234A / L235A / D265A / S267E was constructed by the molecular biology method described in Example 1 and 2, using the anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG as the parent antibody.

[0066] Comparative Example 3

[0067] In comparison with Example 1 and Example 2, the four mutant C: L234A / L235A / P238S / L328F was constructed by the molecular biology method described in Example 1 and 2, using the anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG as the parent antibody.

[0068] Comparative Example 4

[0069] In comparison with Example 1 and Example 2, the four mutant D: L234A / L235A / D265A / L328F was constructed by the molecular biology method described in Example 1 and 2, using the anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG as the parent antibody.

[0070] Comparative Example 5

[0071] Compared with Example 1 and Example 2, this embodiment takes anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG as the parent antibody, and constructs five mutant A: L234A / L235A / P238S / D265A / S267E through the molecular biology method described in Examples 1 and 2.

[0072] Comparative Example 6

[0073] Compared with Example 1 and Example 2, this embodiment takes anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG as the parent antibody, and constructs five mutant B: L234A / L235A / D265A / S267E / L328F through the molecular biology method described in Examples 1 and 2.

[0074] All the mutants in Comparative Examples 1-6 are cloned into the pcDNA3.4 vector according to the method described in Example 1, expressed in HEK293F cells through transient transfection, and purified through Protein A affinity chromatography to obtain high-purity antibodies for subsequent testing.

[0075] Test Example

[0076] First, this embodiment verifies the application effect of the four mutants in Example 1, the six mutants in Example 2, and the other four mutants and five mutants in Comparative Examples 1-6 in the anti-human CD3, CD14, CD33, CD56 recombinant rabbit IgG in flow cytometry reagents, especially their advantages in eliminating non-specific background signals caused by Fc receptor binding.

[0077] Flow cytometry is a powerful cell analysis technique widely used in immunology, oncology, and cell biology research. In flow cytometry, fluorescently labeled antibodies are key reagents for identifying and labeling specific cell surface or intracellular molecules. However, the Fc region of traditional antibodies may bind to Fc receptors on the cell surface, producing non-specific background signals and affecting the accuracy of the detection results.

[0078] This test example selects several typical samples containing high levels of Fc receptor-expressing cells for testing, including:

[0079] 1. Human peripheral blood mononuclear cells (PBMCs): containing various Fc receptor-expressing cells such as monocytes, macrophages, B cells, and NK cells, etc.

[0080] 2. Human bone marrow samples: containing various hematopoietic precursor cells and mature blood cells, some of which express high levels of Fc receptors.

[0081] The test method is as follows: the above cell samples are respectively incubated with wild type antibody, four-mutation antibody of Example 1 and six-mutation antibody of Example 2, which are all labeled with the same fluorescent dye (such as PE or APC) and are directed against the same antigen target. The incubation condition is 4℃ for 30 minutes, then the unbound antibody is removed by washing, and finally the fluorescence signal of the cells is analyzed by flow cytometry.

[0082] The results show that in human PBMC samples, Figure 2 compared with the wild type antibody, the non-specific background signal of the L234A / L235A / P238S / D265A four-mutation antibody is reduced by about 75-85%, which is significantly better than other four-mutation antibodies, and the non-specific background signal of the L234A / L235A / P238S / D265A / S267E / L328F six-mutation antibody is reduced by about 97-99%, which is significantly better than other mutations, especially in CD14+ monocyte and CD56+ NK cell populations, the reduction of background signal is the most significant, which is consistent with the expression of high level Fc receptor by these cells.

[0083] In human bone marrow samples, the non-specific background signal of the L234A / L235A / P238S / D265A four-mutation antibody and the L234A / L235A / P238S / D265A / S267E / L328F six-mutation antibody is reduced by about 55-80% and 95-99%, respectively. The reduction of background signal is the most significant in CD33+ bone marrow cells and CD14+ monocyte populations.

[0084] II. The binding affinity of mutant antibodies to various Fc receptors is determined by surface plasmon resonance (SPR) technology. Different Fc receptors are coupled to a CM5 chip, and different concentrations (3.125-100 nM) of mutant antibodies and wild type antibodies are used as analytes to determine the binding kinetic curve. The results show that compared with the wild type antibody (rabbit IgG), the binding affinity of the six-mutation antibody of Example 2 to FcγRI, FcγRII and FcγRIII receptors is reduced by about 100 times, 50 times and 20 times, respectively, and the background signal-to-noise ratio is increased by about 4.14 times, as shown in Table 1. The data show that the elimination of Fc effect is not determined by the number of mutations, but depends on the specific combination of sites. The performance of some five-mutation antibodies is not as good as the four-mutation antibodies of the present application, and the significant difference in effect between different four-mutation antibodies highlights the unpredictable synergistic effect of the specific combination of L234A / L235A / P238S / D265A / S267E / L328F.

[0085] Third, the thermal stability of the mutant antibodies in Examples 1, 2 and Comparative Examples 1-6 was detected. Differential scanning calorimetry (DSC) was used to determine the thermal stability of the mutant antibodies and wild-type antibodies. Each antibody sample and PBS buffer were injected into the sample cell and reference cell, respectively, and the temperature was raised from 20°C to 100°C at a rate of 1°C / min. The heat capacity change was monitored in real time, and the data was processed by subtracting the buffer baseline and normalizing the concentration. The differential scanning calorimetry (DSC) determination results showed that the melting temperature (Tm) of the six mutant antibodies was 72.8°C, which was similar to that of the wild-type antibody (Tm = 73.6°C), as shown in Table 1.

[0086] Table 1 Comparison of performance parameters of various mutants

[0087] Mutant FcγRI binding KD (nM) FcγRIIa binding KD (nM) FcγRIIIa binding KD (nM) Flow background signal to noise ratio (S / N) Tm value (°C) Wild type 10±2 100±15 500±50 2.1±0.3 73.6±0.5 Four mutant of the present invention 250±30 1800±200 4500±400 5.8±0.4 72.8±0.4 Six mutant of the present invention 1050±120 5200±300 11500±800 8.7±0.5 72.8±0.4 Comparative Example 1 (four mutant A) 95±10 650±70 2200±250 3.5±0.3 72.5±0.5 Comparative Example 2 (four mutant B) 180±20 1200±150 3000±350 4.2±0.3 72.3±0.6 Comparative Example 3 (four mutant C) 120±15 1100±120 3800±350 5.0±0.4 72.4±0.6 Comparative Example 4 (four mutant D) 175±12 1200±150 3400±300 4.5±0.4 72.2±0.6 Comparative Example 5 (five mutant A) 215±20 1250±120 3500±350 4.2±0.5 72.0±0.5 Comparative Example 6 (five mutant B) 230±23 1400±110 3700±380 4.5±0.4 72.8±0.4

[0088] Although the present application has been disclosed in the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application should be defined by the claims.

Claims

1. A novel scaffold flow rabbit antibody, characterized in that, The heavy chain CH2 region of the rabbit IgG parent is subjected to the following combinations of mutations: the 234th leucine is replaced by alanine, the 235th leucine is replaced by alanine, the 238th proline is replaced by serine, the 265th aspartic acid is replaced by alanine, the 267th serine is replaced by glutamic acid, and the 328th leucine is replaced by phenylalanine, the amino acid sequence of the heavy chain constant region of the rabbit IgG parent is shown as SEQ ID NO. 1, and the amino acid sequence of the mutated Fc constant region is shown as SEQ ID NO.

3.

2. A gene encoding the novel backbone flow rabbit antibody of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector of claim 3, wherein, The recombinant vector is expressed in a phage display vector pDAN5, a prokaryotic expression vector pET28a, or a eukaryotic expression vector pcDNA3.4 expression vector.

5. Recombinant cells expressing the novel backbone flow rabbit antibody of claim 1, or containing the gene of claim 2, or transformed or transfected with the recombinant vector of claim 3 or 4.

6. The recombinant cell of claim 5, wherein, The recombinant cells are expressed in E. coli TG1, BL21 (DE3), or mammalian HEK293F cells as hosts.

7. A method of making a novel scaffold flow rabbit antibody with Fc effectorless, characterized in that, The method is to culture the recombinant cells of claim 5 or 6 to obtain a culture containing the novel backbone flow rabbit antibody of claim 1.

8. Use of the novel backbone flow rabbit antibody of claim 1, or the gene of claim 2, or the recombinant vector of claim 3 or 4, or the recombinant cells of claim 5 or 6, or the method of claim 7 in the preparation of flow cytometry detection products.

Images