Fusion proteins useful for site-directed conjugation of antibodies and uses thereof

Abstract

The application provides a fusion protein for antibody site-specific coupling and application thereof. The fusion protein of the application is mainly fused by an antibody Fc affinity protein binding monomer and an avidin protein, realizes direct coupling of the antibody Fc affinity protein and a biotin-labeled molecule, and no longer uses a click chemistry scheme, so that the production and use difficulty of the antibody Fc affinity protein Protein G is greatly simplified, the Protein G protein no longer needs to be activated through the click chemistry in the production, the transportation and storage conditions no longer need to be strictly controlled, and even one-step antibody molecule coupling can be realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fusion protein that can be used for site-specific antibody conjugation, as well as methods and applications for site-specific antibody conjugation. Background Technology

[0002] Antibody conjugation refers to the modification of antibody molecules with chemical substances (such as small molecule drugs, nucleic acid molecules, fluorescent groups, etc.). Antibody conjugation has a wide range of applications, including antibody-drug conjugation, flow cytometry antibody-fluorescence conjugation, single-cell labeled antibody-nucleic acid conjugation, and antigen detection antibody conjugation.

[0003] Antibody conjugation technology is mainly divided into two categories: non-directional conjugation and site-directed conjugation. Non-directional antibody conjugation generally employs click chemistry to link functional groups (small molecules, nucleic acids, proteins, etc.) to specific amino acid residues on the antibody, such as NH2, COOH, and SH. Because antibodies typically have numerous amino acid residue sites such as NH2, COOH, and SH, the conjugation site is often uncertain.

[0004] Currently, the main types of site-directed antibody conjugation technologies are as follows:

[0005] a) Introducing non-natural amino acids into the antibody sequence as site-directed active groups allows for specific conjugation of the antibody to the functional group site. This approach requires targeted modification of the antibody sequence and is suitable for the development of ADC drugs with a limited number of antibody development projects. However, it is not suitable for site-directed conjugation of large-scale, shelf-stable antibody products.

[0006] b) Enzymatic coupling: This method introduces additional sequences using specialized enzymes to achieve targeted coupling sites. These enzymes include glutamine transferase, sorting enzyme StrA, formylglycine synthase, and isoprenoid transferase. However, this approach is limited for large-scale shelf-sold antibodies due to several reasons, such as the need to modify antibody sequences or low efficiency.

[0007] c) Affinity conjugation: This method uses molecules of targeted affinity antibodies, such as Protein A, Protein G, and aptamers, to anchor to the Fc fragment of the antibody, providing and achieving a targeted conjugation site. This type of approach can be used with large quantities of shelf-sold antibodies and is relatively flexible.

[0008] Currently, commercially available affinity conjugation technologies, such as AlphaThera Inc.'s oYo-link antibody conjugation technology, primarily utilize engineered Protein G proteins, introducing click-chemically active groups onto the Protein G protein. The oYo antibody conjugation process involves first, the engineered Protein G binding directionally to the antibody Fc fragment. One Fc fragment can bind to one or two Protein G proteins. Then, activated functional groups (such as nucleic acids or fuels with azide) react with click-chemically active groups on the Protein G (such as DBCO), thereby achieving site-specific conjugation.

[0009] The oYo-link antibody conjugation technology requires modification of the Protein G protein sequence to introduce click chemically active groups via enzymatic or chemical methods. Therefore, the concentration ratio of click chemical substances needs to be optimized and controlled during the conjugation reaction. Interference from solution reagents needs to be controlled during the conjugation reaction. The transport, storage and use of activated Protein G must be strictly controlled under storage conditions and usage time; otherwise, the DBCO group on the Protein G protein will be rapidly oxidized and degraded when exposed to air.

[0010] In addition, JPWO2008130053A1 provides a fusion scheme for protein G and avidin, where the core protein, protein G, is wild-type protein G, and the monomeric avidin-binding protein is a monomeric form of streptavidin. To reduce non-specific binding of protein G, JPWO2008130053A1 also used the protein G protein subdomain region without implementing affinity-enhancing mutations, resulting in a significant reduction in the Fc affinity of protein G. The monomeric streptavidin used in JPWO2008130053A1 also suffers from decreased biotin affinity, with the Kd value dropping to the nM level. DeMonte, in his paper "Structure based engineering of streptavidin monomer with a reduced biotin dissociation rate," published in 2013 that the Kd value of the engineered monomeric streptavidin ranged from 0.53 to 0.78 nM. Therefore, it is necessary to further explore fusion protein schemes that can improve application performance for antibody site-specific conjugation. Summary of the Invention

[0011] Therefore, it is necessary to provide a fusion protein that can be used for antibody site-directed conjugation, as well as a method and application for antibody site-directed conjugation, which adopts a non-click chemistry scheme, and the transportation and storage conditions are no longer strictly controlled, making it quick and convenient to use.

[0012] The present invention adopts the following technical solution:

[0013] This invention provides a fusion protein that can be used for site-specific antibody conjugation. The fusion protein is mainly composed of an antibody Fc affinity protein sequence and a biotinylate affinity protein monomer sequence, and the antibody Fc affinity protein sequence is as follows:

[0014] MTFKLIINGKTLKGEITIEAVDAAEAEKIDKQYANDYGIDGEWTYDDATKTFTVTE.

[0015] The biotin affinity protein monomer sequence is selected from the avidin monomer sequence, and the sequence is as follows:

[0016] EFGPAIWQGQDTFQYVPTTEGSFDASNFKDFSSIASASSSWQNQHGSTMIIQVDSFGNVSGQYVNRAEGTGCQNSPYPLTGRVNGTFIDFSVKWNNSTENCNSNTQWTGYAQVNGNNTEIVTRWNLKYE.

[0017] Preferably, the fusion protein further includes a fusion protein linker sequence. The fusion protein consists of an antibody Fc affinity protein sequence, a monomeric avidin sequence, a linker, and a His-tag purified tag sequence, as shown in SEQ ID NO: 1.

[0018] The present invention can also provide an antibody fusion protein labeled complex, which is prepared by reacting the above-mentioned fusion protein with an antibody and a biotin-labeled molecule.

[0019] The present invention may also provide a kit for detecting target antigens, comprising the above-mentioned fusion protein or the above-mentioned antibody fusion protein labeled complex.

[0020] The present invention can also provide a method for site-specific antibody conjugation, which involves reacting the above-mentioned fusion protein with an antibody and a biotin-labeled molecule. Preferably, the molar ratio of the fusion protein, antibody, and biotin-labeled molecule is 1.5:1:2.

[0021] The present invention may also provide a gene encoding the above-mentioned fusion protein, and an expression vector containing the above-mentioned gene.

[0022] The present invention also provides a method for preparing the above-mentioned fusion protein, comprising the following steps: synthesizing a gene encoding the above-mentioned fusion protein and cloning it into a vector; transforming Escherichia coli, inducing expression, and collecting bacterial cells; disrupting the bacterial cells, centrifuging, and collecting inclusion body precipitate; transferring the inclusion body precipitate into a denaturing solution for treatment, followed by renaturation treatment to obtain a renatured protein solution; passing the renatured protein solution through a Ni column and an ion exchange column to remove impurities, obtaining a crude fusion protein; dialyzing the crude fusion protein into PBS buffer containing a stabilizer, and freezing to obtain the final product.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] Compared to existing antibody-site conjugation methods relying on click chemistry, the fusion protein of this invention is mainly composed of a modified antibody Fc affinity protein sequence and a biotin affinity protein monomer sequence. This invention achieves site-specific conjugation by leveraging the high affinity of the modified antibody Fc and the high affinity of the monomeric avidin and the biotin-labeled molecule, resulting in an affinity of the fusion protein at the nM level or higher. Furthermore, the fusion protein of this invention can be expressed efficiently and stably, is quick and convenient for client-side transportation, storage, and use, and offers high cost-effectiveness.

[0025] In particular, pfGA (protein fusion of protein G and monomeric Avidin) fusion protein can meet the conjugation needs of most off-the-shelf antibody products, and the operation is very convenient. Customers only need to prepare or purchase off-the-shelf antibodies, pfGA protein, biomarker molecules, etc., and add them in sequence to achieve targeted conjugation of antibody and molecule to obtain antibody-pfGA-labeled molecule complex. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the antibody-pfGA-labeled molecule complex.

[0027] Figure 2 This is a statistical graph showing the dissociation rate of antibody-fusion protein (pfGA)-biotinlyted DNA in Experiment Example 3.

[0028] Figure 3 This is a diagram showing the site-specific conjugation of antibodies for single-cell surface antigen labeling and classification in Experiment Example 4. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0030] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0031] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0032] Experimental Example 1

[0033] The antibody-site conjugation protocol consists of three parts: antibody, fusion protein, and biotin-labeled molecule. The three parts form an antibody-fusion protein-biotin-labeled molecule complex to achieve the purpose of antibody-site conjugation.

[0034] The design of the antibody-conjugated fusion protein in this experiment mainly considered two components: one is a modified antibody Fc affinity protein; the other is a monomeric biotinylate affinity protein, such as moneric streptavidin or monomericrhizavidin.

[0035] Streptavidin is a homotetrameric protein with a relatively large molecular weight. Each subunit can bind one molecule of biotin, with binding capacity at the fM level. If steptavidin is to be used for site-directed coupling, the biotin-binding capacity of three subunits needs to be inactivated, while retaining one biotin-binding subunit. Furthermore, through genetic engineering techniques, it has been found that the biotin-binding capacity of the monomeric form of streptavidin is reduced to the nM level. This is evident in DeMonte's 2013 publication of an engineered monomeric streptavidin with a Kd value of 0.53–0.78 nM (Structure based engineering of streptavidin monomer with a reduced biotin dissociation rate).

[0036] Rhizavidin is a homodimeric protein, with each subunit capable of binding one molecule of biotin. Through structural analysis and genetic engineering, the monomeric form of rhizavidin retains a strong biotin-binding capacity, reaching the pM level, while maintaining a relatively small molecular weight. Lee reported in 2016 an engineered monomeric rhizavidin protein with a Kd value of 37 pM at 37°C (A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates). Therefore, the monomeric form of rhizavidin is considered a candidate protein for site-specific antibody conjugation.

[0037] In addition, high-affinity proteins for antibody Fc fragments generally include Protein A and Protein G. Protein G or Protein A consists of multiple repeating domains, which can bind to the antibody's Fc or Fab fragments, enabling multi-site antibody binding or indirect antibody conjugation. If Protein G is used for site-specific antibody conjugation, its Fab binding ability needs to be inactivated, and one domain needs to be retained to achieve site-specific binding to the Fc fragment.

[0038] To further reduce the size of the Protein G protein molecule, the inventors extracted a single protein subdomain with a molecular weight of approximately 8 kDa and performed site-directed mutagenesis.

[0039] N37Y eliminates its Fab bonding capability;

[0040] F30D can significantly increase its Fc affinity by about 5 times.

[0041] Based on extensive research, the inventors' team constructed several fusion proteins with different fusion forms, mainly considering the N-terminal or C-terminal positions of the monomer avidin and the modified Protein G protein (antibody Fc affinity protein sequence), and the His-tag purification label at the N-terminal or C-terminal position.

[0042] The fusion scheme involving the modified Protein G protein at the C-terminus and the monomeric avidin significantly improved protein expression and solubility, while the fusion scheme involving the modified Protein G protein at the C-terminus and the monomeric avidin at the N-terminus significantly reduced protein expression and solubility. The GS linker did not affect protein expression or solubility, but it was used for the fusion scheme to ensure protein structural flexibility. Because the fusion scheme involving the modified Protein G protein at the N-terminus and the monomeric avidin at the C-terminus performed better, the purification tag could only be placed at the C-terminus of the fusion protein, thus not affecting the affinity activity of the modified Protein G protein.

[0043] The final decision was made to fuse the modified Protein G protein with the monomeric avidin, tentatively named protein fusion of protein G and monomeric avidin (pfGA).

[0044] The modified Protein G can specifically bind to the antibody Fc fragment, and the monomer avidin has a biotin-binding site that can bind to biotin-labeled molecules. Therefore, the pfGA fusion protein has the ability to bind both antibody Fc fragments and biotin-labeled molecules.

[0045] The preferred pfGA fusion expression design is as follows: the modified Protein G protein is located at the N-terminus, the avidin protein is located at the C-terminus, the His-tag purification tag is located at the C-terminus, and the avidin protein is linked to the GS linker.

[0046] The amino acid sequence of the pfGA fusion protein is as follows:

[0047]

[0048] In the amino acid sequence of the pfGA fusion protein, the underlined segment corresponds to the modified antibody Fc affinity protein sequence, and the wavy line corresponds to the biotinylate affinity protein monomer sequence.

[0049] The gene sequence encoding the pfGA fusion protein is as follows:

[0050] (SEQ ID NO:2)

[0051] Experimental Example 2

[0052] This experimental example provides a method for the expression and purification of pfGA fusion protein, including the following steps:

[0053] S1, synthesize the pfGA gene as shown in SEQ ID NO:2 and clone it into the pET28a vector.

[0054] S2, the pET28a vector was transformed into E. coli BL(DE3) and cultured at 37°C until OD. 600nm Add IPTG to a final concentration of 1 mM, bring to 1.0, and continue culturing at 37°C for 4 hours. Centrifuge and collect the bacterial cells.

[0055] S3, the collected bacterial cells were lysed and broken by high pressure in 1×PBST solution, centrifuged and the supernatant was discarded, and the precipitate was retained; the precipitate was rinsed with 1×PBST 3 times, 30 min each time, centrifuged and the supernatant was discarded, and the precipitate inclusion bodies were retained.

[0056] S4. Dissolve inclusion bodies in denaturing solution (50 mM Tris-HCl, pH 8.0, 6 M GdnHCl, 5 mM eta-Me, 250 mM NaCl), centrifuge to collect the supernatant, and discard the precipitate. Adjust the concentration of denatured protein in the supernatant to approximately 1 mg / ml. Add the denatured protein solution to 80 volumes of pre-cooled 1×PBS using a rapid dilution method, incubate at 4°C with slow stirring overnight to obtain the renatured protein solution.

[0057] S5. Equilibrate the Ni-NTA column with solution A (50mM Tris-HCl, pH 8.0, 250mM NaCl) containing 10mM imidazole. Add the refolded protein solution to the Ni column. Wash the Ni column with solution A containing 50mM imidazole until the UV baseline is stable. Then elute the Ni column with solution A containing 500mM imidazole.

[0058] S6. Purification was continued using a HiTrap Q FF column ion exchange column with linear elution (0–100% B). Uniform and symmetrical elution peaks were collected to ensure uniform protein folding conformation, and finally the pfGA fusion protein was obtained.

[0059] S7. Dialyze the crude pfGA fusion protein into a 1×PBS buffer solution containing 50% glycerol and freeze at -20°C for later use.

[0060] Experimental Example 3

[0061] This experimental example provides a method for the binding and dissociation assay of pfGA fusion protein with antibodies and biotin-labeled molecules, including the following steps:

[0062] Taq antibody, pfGA fusion protein prepared in Example 2, and biotin-labeled DNA molecule (FAM 5'-GGTTTTTTATGCATGCTTTTTT-3'BIOTIN) were added to a 1×PBS buffer reaction system, with final concentrations of 1 μM, 0.7 μM, and 0.5 μM, respectively, and incubated at 25°C for 30 min.

[0063] The reaction products were stored at 37°C, and samples were taken every hour and stored at 4°C for later use, for a total of 10 samplings. Then, the sampled proteins were analyzed 10 times by native-PAGE. During the electrophoresis process, the electrophoresis tank was placed in an ice bath to keep the electrophoresis buffer pre-cooled.

[0064] Electrophoretic gel analysis was performed using a fluorescence imaging device to calculate the ratio of DNA molecule binding to detachment; the dissociation rate was 2.6 × 10⁻⁶. -5 s -1 .

[0065] In fact, this experimental example provides an antibody fusion protein labeled complex and a method for site-specific antibody conjugation, which involves reacting the fusion protein with an antibody and biotin-labeled fluorescent DNA molecules. Preferably, the molar ratio of the fusion protein, antibody, and biotin-labeled fluorescent DNA molecules is 1.5:1:2, which ensures that all antibody molecules are labeled with biotin molecules.

[0066] Test Example 4

[0067] This experimental example provides a method for single-cell molecular marker sequencing, including the following steps:

[0068] S1, nine antibodies (CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD45, CD56) and their corresponding biotin-labeled DNA molecules (see Table 1 below for the labeled nucleic acid sequences of the CD antibodies) were conjugated with the pfGA fusion protein prepared in Experiment Example 2. Specifically, the antibodies, pfGA fusion protein, and their corresponding biotin-labeled DNA molecules were added to a 1×PBS buffer reaction system to a final concentration of 1 μM, 0.7 μM, and 0.5 μM, respectively. The mixture was incubated at 25°C for 30 min to obtain the antibody-nucleic acid conjugate product (AOC), which was then placed on ice for later use.

[0069] Table 1. Labeled nucleic acid sequences of CD antibodies

[0070]

[0071]

[0072] S2. Mix the nine antibody-nucleic acid conjugate products (AOC) in equal volume ratios to form an antibody-nucleic acid conjugate mixture (AOCMix), and place it on ice for later use.

[0073] S3, resuspend PMBC single cells in 1×PBS buffer. Mix 1 μL of AOCMix with 1 million single-cell suspension and incubate gently upside down at 4°C for 30 min. Centrifuge at 600g for 3 min and gently aspirate the supernatant. Wash the cells once more with 1 mL of wash buffer, centrifuge at 600g for 3 min, and gently aspirate the supernatant. Then, perform library construction and sequencing according to the 10Xgenomic single-cell sequencing protocol.

[0074] The test results for antibody-conjugated antibodies used for single-cell surface antigen labeling and classification are shown in [the original text]. Figure 3 .

[0075] Depend on Figure 3 It can be seen that antibody-nucleic acid conjugate mixture (AOCMix) can be used for single-cell surface protein labeling, cell type clustering, and detection of abundant surface antigens.

[0076] In addition, the present invention can also provide a kit for detecting target antigens, comprising the above-mentioned fusion protein or the above-mentioned antibody fusion protein labeled complex, for rapid detection of target antigens.

[0077] In fact, the pfGA fusion protein described above can meet the conjugation requirements of most off-the-shelf antibody products, exhibiting excellent affinity and being very easy to handle. Customers only need to prepare or purchase off-the-shelf antibodies, pfGA protein, biomarker molecules, etc., and add them in sequence to achieve targeted conjugation of the antibody and the molecule to obtain an antibody-pfGA-labeled molecule complex.

[0078] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fusion protein useful for site-directed conjugation of antibodies, characterized in that, The fusion protein is composed of an antibody Fc affinity protein sequence, a monomer avidin sequence, a linker and a His-tag purification tag sequence, and the sequence is shown as SEQ ID NO:

1.

2. The fusion protein useful for site-directed conjugation of antibodies according to claim 1, characterized in that, The antibody Fc affinity protein sequence is MTKLIINGKTLKGEITIEAVDAAEAEKIDKQYANDYGIDGEWTYDDATKTFTVTE.

3. A gene encoding the fusion protein of claim 1 or 2.

4. An expression vector comprising the gene of claim 3.

5. An antibody fusion protein marker complex, characterized in that, Prepared by reacting the fusion protein of claim 1 or 2 with an antibody and a biotin-labeled molecule.

6. A method of site-directed conjugation of an antibody, characterized in that, Reacting the fusion protein of claim 1 or 2 with an antibody and a biotin-labeled molecule.

7. The method of antibody site-directed conjugation according to claim 6, wherein, The molar ratio of the reaction of the fusion protein, the antibody and the biotin-labeled molecule is 1.5:1:

2.

8. A kit for detecting a target antigen, characterized by, The fusion protein of claim 1 or 2 or the antibody fusion protein-labeled complex of claim 5.

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