Novel Fc-effect-eliminated framework flow antibody as well as preparation method and application of novel Fc-effect-eliminated framework flow antibody

Abstract

The invention discloses a novel Fc effect eliminated framework flow antibody, and belongs to the technical field of biological medicine. The antibody comprises a mutation combination of L234A, L235A, P238S and D265A 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 type of the antibody can be selected from multiple forms of scFv-Fc, (scFv) 2-Fc, scFv / scFv-Fc, Fab / scFv-Fc and the like. The invention also provides a nucleic acid sequence for coding the antibody and a preparation method of the antibody. The preparation method comprises the following steps: introducing specific mutation into a CH2 region of recombinant rabbit IgG, and expressing and purifying in a mammal expression system to obtain the antibody. When the antibody is applied to a flow cytometry detection reagent, a non-specific background signal caused by Fc receptor combination can be effectively eliminated, and the detection specificity and accuracy are improved.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering, specifically to a novel Fc-effect-eliminating backbone flow cytometry antibody, its preparation method, and its applications. Background Technology

[0002] Flow cytometry is an important technique for cell analysis, widely used in fields such as immunology, oncology, and cell biology. In flow cytometry, antibodies are key reagents for recognizing specific cell surface markers. Rabbit antibodies, due to their high affinity and specificity, have become a commonly used detection tool in flow cytometry. However, rabbit IgG antibodies face the problem of Fc-mediated non-specific binding in flow cytometry applications, severely limiting their detection performance and application scope.

[0003] Currently, the industry has a certain understanding of the Fc effector mechanism of rabbit-derived antibodies. Studies have shown that rabbit IgG and human IgG have significant structural differences in the CH2 region. Their unique amino acid sequences and spatial conformations result in a strong binding ability to human Fcγ receptors (such as FcγRI, FcγRII, and FcγRIII). This binding can easily lead to non-specific adsorption with cells expressing FcγR (such as monocytes, macrophages, and B cells) in flow cytometry, causing increased background signal and decreased signal-to-noise ratio. This effect is particularly pronounced in the detection of low-abundance antigens or the analysis of complex samples (such as whole blood and spleen cells).

[0004] To overcome Fc-mediated nonspecific binding, existing technologies mainly employ two strategies: First, replacing the complete IgG with the F(ab′)2 fragment. This fragment lacks the Fc region, thus avoiding FcγR binding. However, its enzymatic digestion process is complex, resulting in low yield, poor stability, and susceptibility to polymerization or degradation, limiting its standardization and large-scale application. Second, reducing effector function through site-directed mutations in the Fc region. For example, mutations such as "LALA" (L234A / L235A), widely used in human IgG, effectively weaken binding to FcγR. However, due to significant differences in the CH2 region sequence and structure between rabbit IgG and human IgG, 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] Existing patents, such as CN113348177A, provide an antibody design that modifies the constant region of the heavy chain to reduce effector function, but it focuses on human or murine antibodies and does not address the structural specificity of rabbit IgG. CN108299557A modifies the binding characteristics of the antibody to FcRn through mutation to regulate the half-life, but it also fails to solve the problem of non-specific binding of rabbit IgG in flow cytometry. Overall, there is currently a lack of mutation schemes that target the characteristics of the rabbit IgG Fc region, systematically eliminate its binding to FcγR, and do not affect antigen-binding activity.

[0006] Therefore, the technical problem to be solved by this invention is to provide a novel rabbit IgG backbone antibody that can effectively eliminate the Fc effect while maintaining high stability and antigen binding ability, addressing the problems of strong non-specific binding, high detection background, and low signal-to-noise ratio caused by the Fc effect in existing rabbit-derived flow cytometry antibodies, and to establish its reliable preparation method and application in flow cytometry detection. Summary of the Invention

[0007] The technical problem this invention aims to solve is to address the shortcomings of traditional rabbit IgG antibodies in flow cytometry, including: the strong Fc receptor binding ability of rabbit IgG leads to non-specific binding with myeloid cells (such as monocytes / macrophages) expressing FcγR; high background noise affects the detection of weakly expressed targets; existing technologies use the F(ab')2 fragment but suffer from low yield and poor stability; and while the known "LALA" mutation (L234A / L235A) in human IgG can reduce effector function, the human Fc elimination mutation is ineffective in rabbit IgG due to the unique binding epitope of the rabbit IgG CH2 structure.

[0008] The first technical solution provided by this invention is a novel Fc-effect-eliminating backbone flow cytometry rabbit antibody. The heavy chain CH2 region of the rabbit IgG parent contains the following mutation combinations: leucine at position 234 is replaced with alanine (L234A), leucine at position 235 is replaced with alanine (L235A), proline at position 238 is replaced with serine (P238S), and aspartic acid at position 265 is replaced with alanine (D265A). The amino acid sequence of the constant region of the heavy chain 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 comprises the following combination of mutations: serine at position 267 is replaced with glutamic acid (S267E) and leucine at position 328 is replaced with phenylalanine (L328F).

[0010] In some embodiments, the antibody that eliminates the Fc effect is selected from any of the following: scFv-Fc, (scFv)2-Fc, scFv / scFv-Fc, Fab / scFv-Fc, Fab / (scFv)2-Fc, Fab / Fab scFv Fc, Fab / Fab cross-FabFc, Fab / cross-Fab-Fc, IgG scFv, and IgG (scFv)2.

[0011] The present invention also provides a second technical solution, which is to encode the gene of the novel backbone flow cytometry rabbit antibody mutant described in the first technical solution.

[0012] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

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

[0014] The fourth technical solution provided by the present invention is a recombinant cell expressing the novel backbone flow cytometry rabbit antibody described in the first technical solution, or containing the gene described in the second technical solution, or transformed or transfected with the recombinant vector described in the third technical solution.

[0015] In some embodiments, the recombinant cells are hosted by Escherichia coli TG1, BL21(DE3) or mammalian HEK293F cells.

[0016] The fifth technical solution provided by the present invention is a method for preparing a novel flow cytometry rabbit antibody with Fc effect elimination, wherein the method involves culturing the recombinant cells described in the fourth technical solution to obtain a culture containing the novel flow cytometry rabbit antibody with the novel backbone described in the first technical solution.

[0017] The present invention provides six technical solutions for a method to eliminate the Fc effect of rabbit IgG antibodies. The method involves making the following mutation combinations in the CH2 region of the heavy chain of the rabbit IgG parent: replacing leucine at position 234 with alanine (L234A), leucine at position 235 with alanine (L235A), proline at position 238 with serine (P238S), aspartic acid at position 265 with alanine (D265A), serine at position 267 with glutamic acid (S267E), and leucine at position 328 with phenylalanine (L328F).

[0018] The seventh technical solution provided by this invention is the application of the novel backbone flow cytometry rabbit antibody described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution in flow cytometry detection or in the preparation of flow cytometry detection products.

[0019] Compared with the prior art, the present invention has the following beneficial effects: By introducing specific amino acid mutations, particularly the L234A / L235A / P238S / D265A / S267E / L328F mutation combination, into the CH2 region of rabbit IgG antibodies, the binding affinity of rabbit IgG antibodies to activating FcγR (FcγRI, FcγRIIa, FcγRIIIa) is significantly reduced, typically by several orders of magnitude; antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP) are almost completely eliminated; and binding to C1q is significantly weakened, thereby effectively inhibiting complement-dependent cytotoxicity (CDC). Compared with existing technologies, this invention solves the problem of non-specific binding of rabbit IgG antibodies in flow cytometry, reduces background noise, and improves the detection effect of weakly expressed targets; compared with the F(ab')2 fragment of existing technologies, the mutant rabbit IgG of this invention has higher yield and better stability, and in the application of flow cytometry detection reagents, it eliminates the non-specific background signal caused by Fc receptor binding compared with non-mutated antibodies. Attached Figure Description

[0020] Figure 1 For the stability assay of the novel CD3 backbone rabbit antibody by flow cytometry: A, SEC-HPLC purity analysis and SDS-PAGE purity analysis after storage at 37℃ for 0, 15, and 30 days; B, affinity assay after storage at 37℃ for 0, 15, and 30 days; C, flow cytometry experiment after storage at 37℃ for 0, 15, and 30 days.

[0021] Figure 2 Non-specific background signal comparison analysis for flow cytometry experiments of human PBMC samples with wild-type antibody, tetramutant antibody, and hexamutant antibody. Detailed Implementation

[0022] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0023] Raw materials used in the examples:

[0024] Experimental materials used in the examples: Human FcγRI, FcγRII, and FcγRIII receptor proteins were purchased from Wuhan Huamei Biotechnology Co., Ltd., with catalog numbers CSB-YP008537HU1, CSB-MP008540HU1, and CSB-AP005111HU, respectively. Human peripheral blood mononuclear cells (PBMCs) and human bone marrow mononuclear cells were purchased from Wuhan Pronosei Life Sciences Co., Ltd., with catalog numbers CP-H182 and CP-H185, respectively.

[0025] Example 1: Preparation of tetramutant antibody

[0026] This embodiment provides a novel Fc-effect-eliminating flow cytometry rabbit antibody with the following mutation combination in the CH2 region of the heavy chain: leucine at position 234 is replaced with alanine (L234A), leucine at position 235 is replaced with alanine (L235A), proline at position 238 is replaced with serine (P238S), and aspartic acid at position 265 is replaced with alanine (D265A). The specific steps include the following: I. Design recombinant rabbit IgG containing the above four mutation sites on CH2 against human CD3, CD14, CD33, and CD56, and add human interleukin-2-α receptor signal peptide to its N-terminus to induce antibody exocrine expression.

[0027] To obtain efficiently expressed antibody genes, this embodiment optimized the nucleic acid sequence encoding mutant antibodies. Specifically, based on the codon usage preferences of mammalian cells (especially CHO cells), the antibody gene sequence was codon-optimized to improve antibody expression levels in the expression system.

[0028] In this embodiment, the nucleic acid sequence of the four mutant antibodies (L234A / L235A / P238S / D265A) includes the following parts: 1. Signal peptide sequence: The nucleic acid sequence encoding the secretion signal peptide, used to guide the secretion of antibody molecules extracellularly. A highly efficient human interleukin-2-α receptor signal peptide sequence, approximately 63 nucleotides in length, was selected.

[0029] 2. Constant Region Sequence: The nucleic acid sequence encoding the antibody's constant region, including the CH2 region sequence containing four mutation sites. The specific nucleotide changes corresponding to these mutation sites are as follows: - L234A: CTG / CTC / CTA / CTT / TTA / TTG → GCC / GCT / GCA / GCG - L235A: CTG / CTC / CTA / CTT / TTA / TTG → GCC / GCT / GCA / GCG - P238S: CCT / CCC / CCA / CCG → TCT / TCC / TCA / TCG - D265A: GAT / GAC → GCT / GCC / GCA / GCG The amino acid sequence of the constant region of the four mutant Fc is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.12.

[0030] 3. Variable Region Sequence: The amino acid sequence encoding the variable regions of the antibody heavy and light chains determines the antigen specificity of the antibody. The variable region sequence varies depending on the different antigen targets. The amino acid sequence of the CD3 antibody heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the CD14 antibody heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7; the amino acid sequence of the CD33 antibody heavy chain variable region is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9; the amino acid sequence of the CD56 antibody heavy chain variable region 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.

[0031] 4. Stop codon and polyadenylate signal sequence: used to terminate translation and guide mRNA tailing.

[0032] The desired amino acid mutation was introduced into the CH2 region of recombinant rabbit IgG using site-directed mutagenesis. First, primers containing the target mutation site were designed based on the wild-type rabbit IgG sequence. Then, the mutation was introduced using overlap extension PCR or a commercially available site-directed mutagenesis kit. Finally, the correctness of the mutation was verified by DNA sequencing.

[0033] For the four-mutant antibody (L234A / L235A / P238S / D265A), all four mutations can be introduced at once or in stages. This embodiment adopts a staged strategy: first, the L234A and L235A mutations are introduced, then the P238S mutation is introduced on this basis, and finally the D265A mutation is introduced.

[0034] 2. Xba I and Age I restriction sites were designed at both ends of the above gene sequence, cloned into pcNDA3.4, and then the mutant antibody was expressed in a mammalian expression system.

[0035] The expression vector containing the mutant sequence was transfected into a mammalian expression system to express the mutant antibody. In this example, CHO cell lines (such as CHO-K1, CHO-S, or CHO-DG44) were mainly used as the expression host, but HEK293 cell line can also be used as an alternative.

[0036] Transfection methods can include liposome transfection (such as Lipofectamine 3000), electroporation, or viral transduction. This example uses liposome transfection, with the following specific steps: Mix 1-5 μg of expression vector DNA with 10-15 μL of transfection reagent, incubate at room temperature for 15-20 minutes, and then add to CHO cells with 70-80% confluence. 24 hours after transfection, add a selective antibiotic (such as puromycin, at a concentration of 30-50 μg / mL) to screen for stably transfected cells. After 2-3 weeks of screening, a stable expression cell pool is obtained.

[0037] For high-yield requirements, this embodiment also included monoclonal screening and amplification. Specifically, stable expression cell pools were seeded into 96-well plates using a limit dilution method (0.5-1 cells per well on average). After culturing for 2-3 weeks, the monoclonal cell supernatant was collected, and antibody expression levels were detected using ELISA. High-expression clones were selected for amplification and culture.

[0038] 3. The target antibody was obtained by purification through protein A affinity chromatography.

[0039] High-purity tetramutant antibodies L234A / L235A / P238S / D265A were obtained through a multi-step chromatography process. The specific purification procedure is as follows: 1. Collect cell culture supernatant: When the cell culture reaches the optimal harvest time (usually 7-14 days after transfection, with cell viability >80%), collect the culture supernatant and remove cells and debris by centrifugation (5000 g, 20 minutes) and filtration (0.22 μm filter membrane).

[0040] 2. Affinity chromatography: The antibody was purified using a protein A or protein G affinity column (such as GE Healthcare's MabSelect SuRe or Thermo Fisher's Protein A Sepharose). The specific procedure was as follows: column equilibration (PBS, pH 7.4), loading (cell culture supernatant), washing (PBS, pH 7.4), elution (0.1 M glycine-HCl, pH 2.7-3.0), and neutralization (1 M Tris-HCl, pH 8.0).

[0041] 3. Ion exchange chromatography: The antibody is further purified using an anion exchange column (such as GE Healthcare's Q Sepharose) to remove impurities such as aggregates, host cell proteins, and DNA. The specific procedure is as follows: column equilibration (20 mM Tris-HCl, pH 8.0), sample loading (protein A purification product), washing (20 mM Tris-HCl, pH 8.0), and gradient elution (20 mM Tris-HCl, pH 8.0, containing 0-500 mM NaCl).

[0042] 4. Concentration and formulation adjustment: Concentrate the purified antibody to the desired concentration (usually 1-10 mg / mL) using ultrafiltration centrifuge tubes (such as Millipore's Amicon Ultra) and adjust to the final formulation (such as PBS, pH 7.4, containing 0.05% Tween 20 and 5% glycerol) by buffer exchange.

[0043] Furthermore, the mutant antibody in this embodiment also exhibits good stability and manufacturability. For example, the anti-human CD3 mutant antibody, after being stored at 37°C for 30 days, remains stable with a single component and high biological activity. Figure 1 The expression level in mammalian cell expression systems can reach 1-2 g / L, meeting the needs of large-scale production.

[0044] Example 2: Preparation of six mutant antibodies

[0045] Compared to Example 1, this example introduces two additional mutation sites in the CH2 region of the heavy chain of recombinant rabbit IgG containing anti-human CD3, CD14, CD33, and CD56: serine at position 267 is replaced with glutamic acid (S267E) and leucine at position 328 is replaced with phenylalanine (L328F). These two additional mutation sites further enhance the Fc effector elimination ability of the antibody, reducing the antibody's binding affinity to the Fc receptor to an even lower level.

[0046] Specifically, replacing serine at position 267 with glutamate (S267E) mainly affects the interaction between the antibody and FcγRIIa and FcγRIIb receptors, while replacing leucine at position 328 with phenylalanine (L328F) mainly affects the interaction between the antibody and FcγRIII receptor.

[0047] The six-mutant antibody (L234A / L235A / P238S / D265A / S267E / L328F) described in this embodiment has a nucleic acid sequence that, based on the four-mutant antibody sequence mentioned above, also adopts a stepwise strategy, first introducing the S267E mutation, then introducing the L328F mutation, and additionally includes nucleotide changes at the following two mutation sites: - S267E:TCT / TCC / TCA / TCG → GAA / GAG - L328F: CTG / CTC / CTA / CTT / TTA / TTG → TTT / TTC The amino acid sequence of the Fc constant region of the six mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.13.

[0048] Following the method described in Example 1, recombinant expression and purification were performed to prepare the six-mutant antibody.

[0049] Comparative Example 1

[0050] Compared to Examples 1 and 2, this example uses recombinant rabbit IgG against human CD3, CD14, CD33, and CD56 as parental antibodies, and constructs a quadrupole A: L234A / L235A / P238S / S267E using the molecular biology methods described in Examples 1 and 2.

[0051] Comparative Example 2

[0052] Compared to Examples 1 and 2, this example uses recombinant rabbit IgG against human CD3, CD14, CD33, and CD56 as parental antibodies, and constructs a quadrupole B: L234A / L235A / D265A / S267E using the molecular biology methods described in Examples 1 and 2.

[0053] Comparative Example 3

[0054] Compared to Examples 1 and 2, this example uses recombinant rabbit IgG against human CD3, CD14, CD33, and CD56 as parental antibodies, and constructs a quadruple mutant C: L234A / L235A / P238S / L328F using the molecular biology methods described in Examples 1 and 2.

[0055] Comparative Example 4

[0056] Compared to Examples 1 and 2, this example uses recombinant rabbit IgG against human CD3, CD14, CD33, and CD56 as parental antibodies, and constructs a quadruple mutant D: L234A / L235A / D265A / L328F using the molecular biology methods described in Examples 1 and 2.

[0057] Comparative Example 5

[0058] Compared to Examples 1 and 2, this example uses recombinant rabbit IgG against human CD3, CD14, CD33, and CD56 as parental antibodies, and constructs a five-mutant A: L234A / L235A / P238S / D265A / S267E using the molecular biology methods described in Examples 1 and 2.

[0059] Comparative Example 6

[0060] Compared to Examples 1 and 2, this example uses recombinant rabbit IgG against human CD3, CD14, CD33, and CD56 as parental antibodies, and constructs a five-mutant B: L234A / L235A / D265A / S267E / L328F using the molecular biology methods described in Examples 1 and 2.

[0061] All mutants in Comparative Examples 1-6 were cloned into the pcDNA3.4 vector according to the method described in Example 1, transiently transfected and expressed in HEK293F cells, and purified by Protein A affinity chromatography to obtain high-purity antibodies for subsequent testing.

[0062] Test case

[0063] I. This embodiment verifies the application effect of the four mutants of Example 1, the six mutants of Example 2, and the other four and five mutants of Comparative Examples 1 to 6 in the flow cytometry detection reagent, especially their advantages in eliminating non-specific background signals caused by Fc receptor binding.

[0064] 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 used to recognize and label specific cell surface or intracellular molecules. However, the Fc region of conventional antibodies may bind to Fc receptors on the cell surface, generating non-specific background signals and affecting the accuracy of the detection results.

[0065] This test case selected several typical samples containing cells with high levels of Fc receptor expression for testing, including: 1. Human peripheral blood mononuclear cells (PBMCs): These are cells that express various Fc receptors, such as monocytes, macrophages, B cells, and NK cells.

[0066] 2. Human bone marrow sample: contains various hematopoietic progenitor cells and mature blood cells, with some cells expressing high levels of Fc receptors.

[0067] The testing method is as follows: The cell samples described above were incubated with wild-type antibody, the tetramutated antibody of Example 1, and the hexamutated antibody of Example 2, respectively. These antibodies were all labeled with the same fluorescent dye (such as PE or APC) and targeted the same antigen. The incubation conditions were 4°C for 30 minutes. After incubation, unbound antibodies were washed to remove them, and the fluorescence signal of the cells was finally analyzed by flow cytometry.

[0068] The results showed that in human PBMC samples ( Figure 2 Compared to wild-type antibodies, the non-specific background signal of the L234A / L235A / P238S / D265A tetramutant antibody was reduced by approximately 75-85%, and its performance was significantly better than other tetramutants. The non-specific background signal of the L234A / L235A / P238S / D265A / S267E / L328F hexamutant antibody was reduced by approximately 97-99%, and its performance was significantly better than other mutants. In particular, the reduction in background signal was most significant in CD14+ monocytes and CD56+ NK cells, which is consistent with the high level of Fc receptor expression in these cells.

[0069] In human bone marrow samples, the L234A / L235A / P238S / D265A tetramutant antibody and the L234A / L235A / P238S / D265A / S267E / L328F hexamutant antibody reduced nonspecific background signal by approximately 55-80% and 95-99%, respectively. The reduction in background signal was most significant in CD33+ bone marrow cells and CD14+ monocyte populations.

[0070] II. The binding affinity of the mutant antibody to various Fc receptors was determined using surface plasmon resonance (SPR) technology. Different Fc receptors were coupled to a CM5 chip, and binding kinetic curves were measured using mutant and wild-type antibodies at different concentrations (3.125-100 nM) as analytes. The results showed that, compared with the wild-type antibody (rabbit IgG), the binding affinity of the six-mutant antibody in Example 2 to FcγRI, FcγRII, and FcγRIII receptors was reduced by approximately 100-fold, 50-fold, and 20-fold, respectively, while the background signal-to-noise ratio was improved by approximately 4.14-fold, as shown in Table 1. The data indicate that the elimination efficacy of the Fc effect is not determined by the number of mutations, but rather by a specific combination of sites. The performance of some five-mutants is inferior to that of the four-mutants of this invention, and the significant differences in effects among different four-mutants highlight the unpredictable synergistic effect produced by the specific combination L234A / L235A / P238S / D265A / S267E / L328F.

[0071] III. This embodiment tested the thermal stability of the mutant antibodies in Examples 1 and 2 and Comparative Examples 1-6. Differential scanning calorimetry (DSC) was used to determine the thermal stability of the mutant and wild-type antibodies. Each antibody sample and PBS buffer were injected into the sample and reference cells, respectively, and the temperature was increased from 20°C to 100°C at a rate of 1°C / min. The change in heat capacity was monitored in real time, and the data were normalized by subtracting the buffer baseline and concentration. The DSC results showed that the melting temperature (Tm) of the six-mutant antibody was 72.8°C, which was similar to that of the wild-type antibody (Tm = 73.6°C), as shown in Table 1.

[0072] Table 1 Comparison of performance parameters of each mutant mutant FcγRI binds to KD (nM) FcγRIIa binds to KD (nM) FcγRIIIa binds to KD (nM) Streaming 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 The four mutants of this invention 250±30 1800±200 4500±400 5.8±0.4 72.8±0.4 The six mutants of this invention 1050±120 5200±300 11500±800 8.7±0.5 72.8±0.4 Comparative Example 1 (quadrigenetic mutant A) 95±10 650±70 2200±250 3.5±0.3 72.5±0.5 Comparative Example 2 (Tetramutant B) 180±20 1200±150 3000±350 4.2±0.3 72.3±0.6 Comparative Example 3 (quadrivariate C) 120±15 1100±120 3800±350 5.0±0.4 72.4±0.6 Comparative Example 4 (quadrigenetic 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 (Pentamutant B) 230±23 1400±110 3700±380 4.5±0.4 72.8±0.4 Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A novel rabbit antibody with a backbone for flow cytometry, characterized in that, The heavy chain CH2 region of the rabbit IgG parent contains the following mutation combinations: leucine at position 234 is replaced with alanine, leucine at position 235 is replaced with alanine, proline at position 238 is replaced with serine, and aspartic acid at position 265 is replaced with alanine. The amino acid sequence of the constant region of the heavy chain of the rabbit IgG parent is shown in SEQ ID NO.

1.

2. The novel backbone flow cytometry rabbit antibody according to claim 1, characterized in that, The heavy chain CH2 region also contains the following mutation combination: serine at position 267 is replaced with glutamic acid and leucine at position 328 is replaced with phenylalanine.

3. The gene encoding the novel backbone flow cytometry rabbit antibody of claim 1 or 2.

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

5. The recombinant vector according to claim 4, characterized in that, The recombinant vectors are expressed using the phage display vector pDAN5, the prokaryotic expression vector pET28a, or the eukaryotic expression vector pcDNA3.

4.

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

7. The recombinant cell according to claim 6, characterized in that, The recombinant cells used Escherichia coli TG1, BL21(DE3) or mammalian HEK293F cells as hosts.

8. A method for preparing a novel flow cytometry rabbit antibody with an Fc effect-eliminating backbone, characterized in that, The method involves culturing the recombinant cells as described in claim 6 or 7 to obtain a culture containing the novel backbone rabbit antibody as described in claim 1 or 2.

9. A method for eliminating the Fc effect of rabbit IgG antibodies, characterized in that, The method involves the following mutation combination in the CH2 region of the heavy chain of the rabbit IgG parent: leucine at position 234 is replaced with alanine, leucine at position 235 is replaced with alanine, proline at position 238 is replaced with serine, aspartic acid at position 265 is replaced with alanine, serine at position 267 is replaced with glutamic acid, and leucine at position 328 is replaced with phenylalanine.

10. The novel backbone rabbit antibody of claim 1 or 2, or the gene of claim 3, or the recombinant vector of claim 4 or 5, or the recombinant cell of claim 6 or 7, or the method of claim 8, or the method of claim 9, in flow cytometry detection or in the preparation of flow cytometry detection products.

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