Double site labeled antibodies of unnatural amino acids and methods of making the same

Abstract

The present application provides a method for synchronously incorporating two unnatural amino acids at specific sites of an antibody, comprising: (i) constructing a vector 1 containing an aminoacyl tRNA synthetase / tRNA orthogonal pair sequence capable of recognizing a TAG codon to encode an unnatural amino acid; (ii) constructing a vector 2 containing a tRNA synthetase / tRNA orthogonal pair sequence capable of recognizing a TAA codon to encode an unnatural amino acid; (iii) constructing a vector 3 containing a nucleic acid sequence in which the amino acid codons at specific sites of the antibody are replaced with TAG and TAA respectively; (iv) co-transfecting the vector 1 in step (i), the vector 2 in step (ii) and the vector 3 in step (iii) into a mammalian cell, and adding an unnatural amino acid inducer, thereby obtaining an antibody in which two unnatural amino acids are simultaneously incorporated at specific sites. The present application also provides an aminoacyl tRNA synthetase / tRNA orthogonal pair for recognizing an unnatural amino acid, an antibody prepared by the foregoing method, and the use of the antibody in preparing an antibody-drug conjugate (ADC).

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a method for incorporating non-natural amino acids at specific sites on an antibody, antibodies prepared according to this method, and the application of antibodies incorporating non-natural amino acids at specific sites in the preparation of antibody-drug conjugates (ADCs). Background Technology

[0002] Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies that target tumor-specific antigens or tumor-associated antigens, linked to varying numbers of small-molecule cytotoxic payloads via linkers. Combining the high targeting specificity of monoclonal antibodies with the high activity of cytotoxic drugs in tumor tissues, ADCs are one of the fastest-growing drug classes in the field of targeted cancer therapy in recent years.

[0003] Among natural amino acids, only lysine and cysteine ​​are typically used for conjugation. However, the preparation of antibody-drug conjugates using these two amino acids often suffers from poor batch-to-batch uniformity and low stability, which greatly affects efficacy and therapeutic window.

[0004] Site-specific incorporation of non-natural amino acids provides a new technical approach for the development of antibody-drug conjugates (ADCs). Non-natural amino acids enable site-specific antibody-drug conjugation. Compared to traditional ADCs, antibodies incorporating non-natural amino acids as linkers can achieve site-specific and quantitative conjugation, resulting in ADCs with higher efficacy, better stability, higher safety, and more uniform drug-to-antibody ratio (DAR).

[0005] Based on site-specific labeling of non-natural amino acids, highly homogeneous dual-load and multi-load ADC drugs can be prepared on a site-specific and quantitative basis.

[0006] Dual-load ADCs offer several advantages over single-load ADCs: 1. Improved efficacy and selectivity: Dual-load ADCs can simultaneously carry two different payloads, potentially enhancing their ability to kill tumor cells, especially in heterogeneous tumor microenvironments. This design can target different tumor cell subpopulations, improving therapeutic efficacy. 2. Reduced drug resistance: Tumor cells may develop resistance to single chemotherapeutic drugs. Dual-load ADCs, by carrying two payloads with different mechanisms, can generate a synergistic effect within cells, reducing the occurrence of drug resistance. 3. Expanded indications: The innovative strategy of dual-load ADCs can expand the range of drug indications, fully leveraging the advantages of dual-load delivery.

[0007] There are currently no reported methods to simultaneously encode non-natural amino acids at two or more sites in the same antibody protein.

[0008] Brief Description of the Invention

[0009] This invention aims to provide a method for simultaneously incorporating two non-natural amino acids at specific sites on an antibody, and to synthesize the antibody in living cells using synthetic biology techniques. Based on this, the invention uses herceptin antibody as a model protein, selecting sites A121 and N297 on the heavy chain and T109 and V110 on the light chain for site-specific labeling of the non-natural amino acids. Orthogonal non-natural amino acids pAcF, pAzF, and AzK are selected for site-specific labeling of the antibody protein.

[0010] Therefore, a first aspect of the present invention provides a method for incorporating non-natural amino acids at specific sites on an antibody, the method comprising:

[0011] (i) Construct a vector containing an aminoacyl-tRNA synthetase / tRNA orthogonal pair sequence encoding a non-natural amino acid that can recognize the TAG codon;

[0012] (ii) Construct vector 2 containing an aminoacyl-tRNA synthetase / tRNA orthogonal pair sequence that can recognize the TAA codon encoding non-natural amino acids;

[0013] (iii) Constructing a vector containing antibody nucleic acid sequences with amino acid codons at specific sites replaced by TAG and TAA, respectively;

[0014] (iv) Transfect mammalian cells with vector 1 from step (i), vector 2 from step (ii), and recombinant vector 3 from step (iii), and induce them with non-natural amino acids, thereby simultaneously incorporating two non-natural amino acids at two specific sites on the antibody.

[0015] Furthermore, the non-natural amino acid is selected from, but not limited to, one or more of pAcF, pAzF, or AzK.

[0016] Furthermore, the aminoacyl-tRNA synthetase / tRNA orthogonal pair that recognizes the TAG codon encoding the non-natural amino acid pAcF is pAcFRS / Tyr tRNA. CUA Further, pAcFRS has the nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2; further, Tyr tRNA CUA It has the nucleotide sequence shown in SEQ ID NO:3.

[0017] Furthermore, the aminoacyl-tRNA synthetase / tRNA orthogonal pair that recognizes the TAA codon encoding the non-natural amino acid pAzF is Mm pAzFRS / Pyl tRNA. UUAFurther, Mm pAzFRS has the nucleotide sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 7; further, Pyl tRNA UUA It has a nucleotide sequence as shown in SEQ ID NO: 8.

[0018] Furthermore, the aminoacyl-tRNA synthetase that recognizes the TAA codon encoding the non-natural amino acid AzK is MmAzKRS / Pyl-tRNA. UUA Further, Mm AzKRS has a nucleotide sequence as shown in SEQ ID NO: 36 or an amino acid sequence as shown in SEQ ID NO: 37; further, Pyl tRNA UUA It has a nucleotide sequence as shown in SEQ ID NO: 8.

[0019] Furthermore, the specific sites of antibodies incorporating non-natural amino acids can be one, two, or more.

[0020] Furthermore, the aforementioned antibody is selected from murine antibodies, human antibodies, humanized antibodies, antigen-binding domains of antibodies, or single-chain Fv of antibodies. In one specific embodiment, the aforementioned antibody is herceptin; in one specific embodiment, the herceptin antibody has a heavy chain as shown in SEQ ID NO. 27 and a light chain as shown in SEQ ID NO. 28.

[0021] A second aspect of the present invention provides a method for preparing antibody-drug conjugates (ADCs), wherein the method further involves conjugating toxin molecules to an incorporated non-natural amino acid via a linker, based on the aforementioned method.

[0022] Furthermore, when the incorporated non-natural amino acid is pAZF and / or AzK, the linker can be a molecule with alkyne, DBCO, or BCN groups; when the incorporated non-natural amino acid is pAcF, the linker can be a molecule with hydroxylamine groups.

[0023] A third aspect of the present invention provides an antibody incorporating a non-natural amino acid at a specific site, wherein the antibody is prepared by the aforementioned method.

[0024] Furthermore, the antibodies of the present invention incorporating non-natural amino acids at specific sites have a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 28; or have a heavy chain as shown in SEQ ID NO: 24 and a light chain as shown in SEQ ID NO: 28; or have a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 25; or have a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 26; or have a heavy chain as shown in SEQ ID NO: 29 and a light chain as shown in SEQ ID NO: 28; or have a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 28; or have a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 31; or have a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 32; or have a heavy chain as shown in SEQ ID NO: 33 and a light chain as shown in SEQ ID NO: 28; or have a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 34.

[0025] A fourth aspect of the invention provides a nucleotide sequence encoding an antibody incorporating a non-natural amino acid at a specific site.

[0026] Furthermore, the nucleotide sequences of antibodies incorporating non-natural amino acids at specific sites are as follows: having a heavy chain as shown in SEQ ID NO: 11 and a light chain as shown in SEQ ID NO: 16; or having a heavy chain as shown in SEQ ID NO: 12 and a light chain as shown in SEQ ID NO: 16; or having a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 13; or having a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 14; or having a heavy chain as shown in SEQ ID NO: 17 and a light chain as shown in SEQ ID NO: 16; or having a heavy chain as shown in SEQ ID NO: 18 and a light chain as shown in SEQ ID NO: 16; or having a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 19; or having a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20; or having a heavy chain as shown in SEQ ID NO: 21 and a light chain as shown in SEQ ID NO: 16; or having a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 16. The light chain shown in IDNO: 22;

[0027] A fifth aspect of the invention provides a vector comprising the aforementioned encoded nucleic acid.

[0028] A sixth aspect of the invention provides the use of antibodies incorporating non-natural amino acids at specific sites in the preparation of antibody-drug conjugates (ADCs). Attached Figure Description

[0029] The accompanying drawings constituting this application are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention. In the drawings:

[0030] Figure 1 The chemical structure of the non-natural amino acids used in the embodiments of this invention.

[0031] Figure 2 This is an SDS-PAGE of the antibodies, where lane 1 contains Herceptin, lane 2 contains Herceptin A121pAcF, and lane 3 contains Herceptin A121AzK.

[0032] Figure 3 SDS-PAGE image of herceptin A121pAcF+N297pAcF

[0033] Figure 4 SDA-PAGE image of Herceptin T109pAzK+V110AzK

[0034] Figure 5 SDS-PAGE image of Herceptin A121pAcF+N297pAcF / T109AzK+V110AzK

[0035] Figure 6 SDS-PAGE gel images of wild-type and non-natural amino acid-incorporated Herceptin antibody proteins.

[0036] Figure 7 shows the proteomic profiles of wild-type and non-natural amino acid-incorporated Herceptin antibodies: A) WT protein; B) A121pAcF protein; C) N297AzK protein; D) A12lpAcF+N297AzK protein.

[0037] Figure 8 The antitumor toxin molecules used in this application are DBCO-PEG4-GGFG-Dxd (A) and Auristatin-PEG2-hydroxylamine (B).

[0038] Figure 9SDS-PAGE of Herceptin antibody conjugate with antitumor toxin molecule

[0039] Figure 10 Mass spectra of the conjugated products of non-natural amino acid-incorporated Herceptin antibodies and antitumor toxin molecules: A) is the cross-linked product of A121pAcF+N297AzK protein and DBCO-PEG4-GGFG-Dxd molecule; B) is the cross-linked product of A121pAcF+N297AzK-Dxd protein and Auristatin-PEG2-hydroxylamine.

[0040] Figure 11 Assay for the binding affinity of Herceptin antibody-antitumor toxin conjugates to Her2 antigen protein.

[0041] Figure 12 To test the cytotoxic activity of Herceptin antibody-drug conjugates against Her2-positive and Her2-negative tumor cells, we tested: A) the in vitro cytotoxic activity of the ADC molecules against MDA-MB-435Her2+ cells; and B) the in vitro cytotoxic activity of the ADC molecules against MDA-MB-435Her2- cells.

[0042] Detailed Description of the Invention

[0043] This invention is described in detail herein by reference to the definitions and embodiments described below. All patents and publications mentioned herein, including all sequences disclosed in such patents and publications, are expressly incorporated herein by reference.

[0044] In this invention, a "bioorthogonal pair" refers to a pair composed of an engineered orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA, wherein aaRS can specifically aminoacylate homologous tRNA with specific non-natural amino acids, dedicated to decoding specific codons (usually the stop codon UAG), and subsequently recognize tRNA via cellular ribosomes. CUA This allows for the specific incorporation of the UAA-corresponding codon site into the synthetic peptide during translation. In this invention, aaRS / tRNA includes pAcFRS / Tyr tRNA that recognizes the coupled non-natural amino acid pAcF. CUA Orthogonal pairs of Mm pAzFRS and Pyl tRNA that can recognize the coupled non-natural amino acid pAzF UUA Orthogonal pairs and Mm AzKRS and Pyl tRNA that can recognize the coupled non-natural amino acid AzK UUA Orthogonal pair. Example

[0045] It should be noted that, unless otherwise specified, the embodiments in this application are merely illustrative and are not intended to limit the invention in any way.

[0046] Prepared dual non-natural amino acid-labeled antibody protein based on gene codon expansion technology

[0047] To achieve the simultaneous encoding of two different types of non-natural amino acids in a protein, it is necessary to address the issues of codon assignment and orthogonality between codons and non-natural amino acids. Here, we combine pAcFRS / Tyr tRNA. CUA Orthogonal pairs, Mm pAzFRS and Pyl tRNA UUA And Mm AzKRS and Pyl tRNA UUA The orthogonal pair decodes the TAG and TAA codons orthogonally within the cell to translate and produce antibody proteins that simultaneously label two non-natural amino acids at specific sites.

[0048] Orthogonally coupled non-natural amino acids pAcF, pAzF, and AzK were selected for site-specific labeling of antibody proteins. The side chain of pAcF amino acid can undergo orthogonal coupling reactions with molecules containing hydroxylamine groups under slightly acidic conditions. pAzF and AzK can undergo cycloaddition coupling reactions with alkynes under copper catalysis, or be used for strain-promoted cycloaddition of alkyne azides (SPAAC) in copper-free click chemistry.

[0049] E. coli TyrRS / Tyr tRNA CUA The orthogonal pair was modified to recognize the TAG codon encoding the non-natural amino acid pAcF, and the E. coli TyrRS protein was modified to contain the following site mutations (Y37I, D182G, F183M, L186A) (nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2), named pAcFRS. The corresponding orthogonal Tyr tRNA... CUA See SEQ ID NO: 3.

[0050] pAcFRS with Tyr tRNA CUA The sequences were cloned into the pCDNA3.1 vector plasmid, and pAcFRS expression was driven by the CMV promoter (SEQ ID NO: 4) and Tyr tRNA expression was driven by the U6 promoter (SEQ ID NO: 5). CUA Transcription, plasmid named pCDNA3.1-pAcFRS-Tyr tRNA CUA .

[0051] Methanosarcina mazei(Mm)PylRS / Pyl tRNA CUAThe orthogonal pair was modified to recognize the TAA codon encoding the non-natural amino acids pAzF and Azk. The Mm PylRS protein was then modified to contain the following site mutations (N346A, C348A) (nucleotide sequence SEQ ID NO: 6, amino acid sequence SEQ ID NO: 7), and named pAzFRS. The corresponding orthogonal PyltRNA... UUA See SEQ ID NO: 8. The Mm PylRS protein was modified to contain the following mutation (Y384F) (nucleotide sequence SEQ ID NO: 36, amino acid sequence SEQ ID NO: 37), named AzKRS, and the corresponding orthogonal Pyl tRNA was... UUA See SEQ ID NO: 8.

[0052] Mm pAzFRS or Pyl tRNA UUA The sequences were cloned into the pFuse vector plasmid, and pAzFRS expression was driven by the HTLV promoter (SEQ ID NO: 9) and Pyl tRNA expression was driven by the h7SK promoter (SEQ ID NO: 10). UUA Transcription, plasmid named pFuse-pAzFRS-Pyl tRNA UUA Mm AzKRS or Pyl tRNA UUA The sequences were cloned into the pFuse vector plasmid, and MmAzKRS expression was driven by the HTLV promoter (SEQ ID NO: 9), while Pyl tRNA expression was driven by the h7SK promoter (SEQ ID NO: 10). UUA Transcription, plasmid named pFuse-AzKRS-Pyl tRNA UUA .

[0053] Herceptin protein was used as the model antibody molecule. The A121 and N297 sites of the heavy chain (HC) and the T109 and V110 sites of the light chain (LC) were selected for site-directed labeling of non-natural amino acids. The codons at the A121 and N297 sites of the heavy chain and the T109 and V110 sites of the light chain were modified to TAG codons (the nucleic acid sequences correspond to SEQ ID NO: 11-14, respectively). The codons at the A121 and N297 sites of the heavy chain and the T109 and V110 sites of the light chain were modified to TAA codons (the nucleic acid sequences correspond to SEQ ID NO: 17-20, respectively). The nucleic acid sequence of the wild-type heavy chain (named HC-wt) of the antibody Herceptin is SEQ ID NO: 15, and the nucleic acid sequence of the wild-type light chain (named LC-wt) is SEQ ID NO: 16. Construct the combined mutant sequence of heavy chain (HC)A121TAG+N297TAA (nucleic acid sequence SEQ ID NO: 21) and the combined mutant sequence of heavy chain (HC)A121TAA+N297TAG (nucleic acid sequence SEQ ID NO: 20).

[0054] Different plasmid architectures were arranged according to heavy chain / light chain / pCDNA3.1-pAcFRS-Tyr tRNA CUA / pFuse-pAzFRS-Pyl tRNA UUA HEK 293F cells were transfected at a ratio of 3:3:1:1. After 6 hours, the corresponding non-natural amino acids were added to the culture medium, and the cells were cultured until the cell viability dropped below 70%. The culture supernatant was harvested by centrifugation, and antibody affinity purification was performed using Protein A resin. The protein was then concentrated using a 30kDa ultrafiltration concentrator and replaced with DPBS buffer. SDS-PAGE electrophoresis was performed on the concentrated protein samples. SDS-PAGE electrophoresis results ( Figure 2-5 The results showed that the exogenous addition of non-natural amino acids to the culture medium could successfully induce mammalian cells to express antibodies incorporating coupled non-natural amino acids.

[0055] Table 1. Gene and amino acid sequence list

[0056] E.coliMj TyrRS protein >SEQ ID NO: 2 <![CDATA[Tyr tRNA CUA ]]> Nucleotides >SEQ ID NO: 3 CMV promoter Nucleotides >SEQ ID NO: 4 U6 Starter Nucleotides >SEQ ID NO: 5 Mm PylRS Nucleotides >SEQ ID NO: 6 Mm PylpAzFRS protein >SEQ ID NO: 7 <![CDATA[Pyl tRNA UUA ]]> Nucleotides >SEQ ID NO: 8 HTLV promoter Nucleotides >SEQ ID NO: 9 h7SK promoter Nucleotides >SEQ ID NO: 10 HC-A121TAG Nucleotides >SEQ ID NO: 11 HC-N297TAG Nucleotides >SEQ ID NO: 12 LC-T109TAG Nucleotides >SEQ ID NO: 13 LC-V110TAG Nucleotides >SEQ ID NO: 14 Herceptin HC Nucleotides >SEQ ID NO: 15 Herceptin LC Nucleotides >SEQ ID NO: 16 HC-A121TAA Nucleotides >SEQ ID NO: 17 HC-N297TAA Nucleotides >SEQ ID NO: 18 LC-T109TAA Nucleotides >SEQ ID NO: 19 LC-V110TAA Nucleotides >SEQ ID NO: 20 A121TAG+N297TAA Nucleotides >SEQ ID NO: 21 HC A121TAA+N297TAG Nucleotides >SEQ ID NO: 22 HC-A121pAcF protein >SEQ ID NO: 23 HC-N297pAcF amino acids >SEQ ID NO: 24 LC-T109pAcF amino acids >SEQ ID NO.25 LC-V110pAcF amino acids >SEQ ID NO.26 HC amino acids >SEQ ID NO: 27 LC amino acids >SEQ ID NO: 28 HC A121AzK amino acids >SEQ ID NO: 29 HC N297AzK amino acids >SEQ ID NO: 30 LC-T109AzK amino acids >SEQ ID NO.31 LC-V110AzK amino acids >SEQ ID NO.32 HC A121pAcF+N297AzF amino acids >SEQ ID NO: 33 LC T109pAzF+V110pAcF amino acids >SEQ ID NO.34 HC A121pAcF+N297AzK protein >SEQ ID NO: 35 Mm AzKRS Nucleotides >SEQ ID NO: 36 Mm AzKRS amino acids >SEQ ID NO: 37

[0057] Table 2 Non-natural amino acid-labeled antibodies

[0058]

[0059] Plasmids containing the nucleic acid sequence SEQ ID NO: 15 and SEQ ID NO: 16 were mixed at a 1:1 ratio and transfected into HEK 293F cells using PEI transfection reagent. After 6 hours, 2-5 mM of non-natural amino acid pAcF was added to the culture medium, and the cells were cultured until the cell viability dropped below 70%. The culture supernatant was harvested by centrifugation, and antibody affinity purification was performed using Protein A resin. The protein was concentrated using a 30 kDa ultrafiltration concentrator and then transferred to DPBS buffer. SDS-PAGE electrophoresis was performed on the concentrated protein samples. SDS-PAGE electrophoresis results (…) Figure 6 The results showed that the exogenous addition of non-natural amino acids to the culture medium successfully induced the expression of wild-type full-length antibodies in mammalian cells. Mass spectrometry analysis was performed after PNGase deglycosylation and treatment with 10 mM DTT. Figure 7A As can be seen, the molecular weights of both the light and heavy chains of the antibody are consistent with the theoretical values.

[0060] Plasmid containing the nucleic acid sequence SEQ ID NO: 11, plasmid containing the nucleic acid sequence SEQ ID NO: 16, and pCDNA3.1-pAcFRS-Tyr tRNA were prepared. CUA Plasmids were mixed in a 3:1:1 ratio and transfected into HEK 293F cells using PEI transfection reagent. After 6 hours, 2-5 mM of non-natural amino acid pAcF was added to the culture medium, and the cells were cultured until cell viability dropped below 70%. The culture supernatant was harvested by centrifugation, and antibody affinity purification was performed using Protein A resin. The protein was concentrated using a 30 kDa ultrafiltration tube and then transferred to DPBS buffer. SDS-PAGE electrophoresis was performed on the concentrated protein samples. SDS-PAGE electrophoresis results (…) Figure 6 The results showed that the exogenous addition of non-natural amino acids to the culture medium successfully induced mammalian cells to express the full-length Herceptin A121pAcF antibody with a single-site incorporation of pAcF amino acids. Mass spectrometry analysis was performed after PNGase deglycosylation and treatment with 10 mM DTT. Figure 7B As can be seen, the molecular weights of both the light and heavy chains of the antibody are consistent with the theoretical values.

[0061] Plasmid containing the nucleic acid sequence SEQ ID NO: 18, plasmid containing the nucleic acid sequence SEQ ID NO: 16, and pFuse-pAzFRS-Pyl tRNA were used. UUAPlasmids were mixed in a 3:1:1 ratio and transfected into HEK 293F cells using PEI transfection reagent. After 6 hours, 2-5 mM of the non-natural amino acid AzK was added to the culture medium, and the cells were cultured until cell viability dropped below 70%. The culture supernatant was harvested by centrifugation, and antibody affinity purification was performed using Protein A resin. The protein was concentrated using a 30 kDa ultrafiltration tube and then transferred to DPBS buffer. SDS-PAGE electrophoresis was performed on the concentrated protein samples. SDS-PAGE electrophoresis results (…) Figure 6 The results showed that the exogenous addition of non-natural amino acids to the culture medium successfully induced the expression of the full-length Herceptin N297AzK antibody with a single-site AzK amino acid incorporation in mammalian cells. Mass spectrometry analysis was performed after PNGase deglycosylation and treatment with 10 mM DTT. Figure 7C As can be seen, the molecular weights of both the light and heavy chains of the antibody are consistent with the theoretical values.

[0062] Prepared dual non-natural amino acid-labeled antibody protein based on gene codon expansion technology

[0063] Plasmid containing the nucleic acid sequence SEQ ID NO: 21, plasmid containing the nucleic acid sequence SEQ ID NO: 16, and pCDNA3.1-pAcFRS-Tyr tRNA were used. CUA Plasmids and pFuse-pAzFRS-Pyl tRNA UUA Plasmids were mixed in a 3:1:1:1 ratio and transfected into HEK 293F cells using PEI transfection reagent. After 6 hours, 2-5 mM of non-natural amino acids pAcF and AzK were added to the culture medium, and the cells were cultured until cell viability dropped below 70%. The culture supernatant was harvested by centrifugation, and antibody affinity purification was performed using Protein A resin. The protein was concentrated using a 30 kDa ultrafiltration tube and then transferred to DPBS buffer. SDS-PAGE electrophoresis was performed on the concentrated protein samples. SDS-PAGE electrophoresis results (…) Figure 6 The results showed that the addition of exogenous non-natural amino acids to the culture medium successfully induced mammalian cells to express the full-length Herceptin A121pAcF+N297AzK antibody with pAcF and AzK amino acids incorporated at two sites. Mass spectrometry was performed after PNGase deglycosylation and treatment with 10 mM DTT. Figure 7D As can be seen, the molecular weights of both the light and heavy chains of the antibody are consistent with the theoretical values.

[0064] Orthogonal coupling of dual non-natural amino acid-labeled antibodies with antitumor toxin small molecules

[0065] The dual non-natural amino acid site-directed antibody Herceptin A121pAcF+N297AzK protein was replaced in DPBS pH 7.4 buffer and adjusted to a concentration of 5-10 mg / mL. Then, 10 molar equivalents of DBCO-PEG4-GGFG-Dxd molecules (molecular structure as shown) were added. Figure 8 As shown in Figure A), after thorough mixing, the mixture was incubated at 4°C for 12 hours for orthogonal coupling reaction. The reaction product was purified using Protein A resin for antibody affinity, and the protein was concentrated using a 30kDa ultrafiltration tube and then transferred to DPBS buffer. The concentrated protein samples were then analyzed by SDS-PAGE electrophoresis. Figure 9 Mass spectrometry was performed after PNGase deglycosylation and 10 mM DTT treatment. Figure 10 A) As can be seen, the molecular weights of the antibody light chain are consistent with the theoretical values, and the molecular weights of the heavy chain and the conjugate products are consistent. The heavy chain protein reacts completely with the DBCO-PEG4-GGFG-Dxd molecule. The conjugate product is labeled HerceptinA121pAcF+N297AzK+Dxd.

[0066] The Herceptin A121pAcF+N297pAzK+Dxd conjugate was replaced with 25 mM sodium acetate pH 4.5 buffer and adjusted to a concentration of 5-10 mg / mL. Then, 10 molar equivalents of Auristatin-PEG2-hydroxylamine molecules (molecular structure as shown) were added. Figure 8 (As shown in B), after thorough mixing, the mixture was incubated at 20°C for 48 hours for orthogonal coupling reaction. The reaction product was purified using Protein A resin for antibody affinity, and the protein was concentrated using a 30kDa ultrafiltration tube and then transferred to DPBS buffer. The concentrated protein samples were then subjected to SDS-PAGE electrophoresis for detection. Figure 9 Mass spectrometry was performed after PNGase deglycosylation and 10 mM DTT treatment. Figure 10 B) It can be seen that the molecular weight of the antibody light chain is consistent with the theoretical value, and the molecular weight of the heavy chain is consistent with the molecular weight of the conjugate product. The conjugate product is labeled as Herceptin A121pAcF+N297AzK+Dxd+Auristatin.

[0067] Antigen-binding activity assay of dual non-natural amino acid-labeled antibodies and their antitumor toxin small molecule conjugates

[0068] Her2 ECD protein was diluted to 1 μg / mL with DPBS buffer, and 100 μL / well was used to coat Maxisorp ELISA plates overnight at 4°C. The next day, the supernatant was discarded, and the plates were washed twice with DPBS + 0.05% Tween-20 buffer, followed by blocking with DPBS + 2% BSA for 1 hour. The supernatant was discarded again, and the plates were washed twice with DPBS + 0.05% Tween-20 buffer. Herceptin WT, A121pAcF+N297pAzK, and antibody conjugates Herceptin A121pAcF+N297AzK+Dxd and Herceptin A121pAcF+N297AzK+Dxd+Auristatin were serially diluted 10x (to an initial concentration of 10 μg / mL) with DPBS + 2% BSA, and 100 μL was added to each well of the ELISA plate and incubated at room temperature for 1 hour. After discarding the supernatant, the plates were washed three times with DPBS + 0.05% Tween-20 buffer. After incubation with mouse anti-human IgG Fc HRP antibody (SouthernBiotech, cat#9040-05), the plates were washed three times with DPBS + 0.05% Tween-20 buffer. A colorimetric reaction was then performed using TMB chromogenic buffer, and the OD570nm value was measured using a microplate reader. The results are shown below. Figure 11 Compared with wild-type Herceptin antibodies, the incorporation of non-natural amino acids at the A12lpAcF+N297AzK dual site and further single-toxin and dual-toxin conjugation did not affect the binding activity of the antibody molecules to the Her2 antigen protein.

[0069] Tumor cell killing activity assay of dual non-natural amino acid-labeled antibody and antitumor toxin small molecule conjugate

[0070] MDA-MB-435Her2+ cells are stable cell lines obtained from MDA-MB-435Her2- cells after lentiviral infection. Both MDA-MB-435Her2+ and MDA-MB-435Her2- cells were passaged in DMEM medium containing 10% (vol / vol) FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin. After trypsin digestion, both MDA-MB-435Her2+ and MDA-MB-435Her2- cells were seeded into 96-well plates at approximately 1000 cells / 90 μL / well and incubated overnight at 37°C in a CO2 incubator. The following day, Herceptin WT, A21pAcF+N297AzK, and antibody conjugates Herceptin A121pAcF+N297AzK+Dxd and Herceptin A121pAcF+N297AzK+Dxd+Auristatin were serially diluted in culture medium and added to each cell well at 10 μL / well. A no-drug group (100% viability control) and a 5 μM taxol-induced cell killing group (0% viability control) were set up. The plates were incubated at 37°C in a CO2 incubator for 72 hours. Then, 80 μL of CellTiter-Glo was added to each well, and the plates were incubated at room temperature in the dark for 15 minutes. The chemiluminescence values ​​were then read using a microplate reader, and the cell viability of each well was calculated. Results are as follows: Figure 12 As shown, the antibodies Herceptin WT and A121pAcF+N297AzK themselves had no killing activity against either Her2+ or Her2- tumor cells. However, the conjugates Herceptin A121pAcF+N297AzK+Dxd and Herceptin A121pAcF+N297AzK+Dxd+Auristatin both exhibited dose-dependent killing activity against Her2+ tumor cells. The antitumor activity of Herceptin A121pAcF+N297AzK+Dxd+Auristatin, conjugated with both toxin molecules, was significantly higher than that of Herceptin A121pAcF+N297AzK+Dxd, conjugated with a single toxin molecule.

[0071] In summary, this invention successfully utilizes synthetic biology techniques to encode and synthesize antibody molecules within mammalian cells that simultaneously incorporate two orthogonal non-natural amino acids at two specific sites. These antibody molecules can achieve highly efficient orthogonal coupling reactions with different chemical mechanisms. In this invention, this technology is applied to the design and preparation of dual-loaded antibody-drug conjugates, which exhibit significantly higher antitumor activity than single-loaded antibody-drug conjugates. Furthermore, the method of this invention can also be widely applied to the homogenization preparation of various types and multiple-loaded conjugates, such as antibody-toxin, antibody-isotope, and antibody-immunoagonist conjugates.

Claims

1. A method for incorporating non-natural amino acids at specific sites on an antibody, the method comprising: (i) Construct a vector containing an aminoacyl-tRNA synthetase / tRNA orthogonal pair sequence that encodes non-natural amino acids using a recognizable TAG codon; (ii) Construct a vector 2 containing an aminoacyl-tRNA synthetase / tRNA orthogonal pair sequence that encodes non-natural amino acids using a recognizable TAA codon; (iii) Construct a vector containing nucleic acid sequences in which the amino acid codons at specific sites of the antibody are replaced by TAG and TAA, respectively; (iV) The vector 1 in step (i), the vector 2 in step (ii), and the recombinant vector 3 in step (iii) are co-transfected into mammalian cells, and non-natural amino acids are added to induce the simultaneous incorporation of two non-natural amino acids at two specific sites on the antibody.

2. The method of claim 1, wherein the non-natural amino acid includes, but is not limited to, pAcF, pAzF, or AzK.

3. The method of claim 1 or 2, wherein the aminoacyl-tRNA synthetase / tRNA orthogonal pair encoding the non-natural amino acid pAcF by recognizing the TAG codon is pAcFRS / Tyr tRNA. CUA Preferably, the pAcFRS has a nucleotide sequence as shown in SEQ ID NO: 1 or an amino acid sequence as shown in SEQ ID NO: 2, and the Tyr tRNA CUA It has the nucleotide sequence shown in SEQ ID NO:

3.

4. The method of claim 1 or 2, wherein the aminoacyl-tRNA synthetase / tRNA orthogonal pair that recognizes the TAA codon encoding the non-natural amino acid pAzF is Mm pAzFRS / Pyl tRNA UUA Preferably, the Mm pAzFRS has a nucleotide sequence as shown in SEQ ID NO: 6 or an amino acid sequence as shown in SEQ ID NO: 7, and the Pyl tRNA UUA It has a nucleotide sequence as shown in SEQ ID NO:

8.

5. The method of claim 1 or 2, wherein the aminoacyl-tRNA synthetase / tRNA orthogonal pair encoding the non-natural amino acid AzK by recognizing the TAA codon is MmAzKRS / Pyl-tRNA UUA Preferably, the Mm AzKRS has a nucleotide sequence as shown in SEQ ID NO: 36 or an amino acid sequence as shown in SEQ ID NO: 37, and the Pyl tRNA UUA It has a nucleotide sequence as shown in SEQ ID NO:

8.

6. The method according to any one of claims 1-5, wherein the antibody is selected from murine antibodies, human antibodies, humanized antibodies, antigen-binding domains of antibodies, or single-chain Fv of antibodies, etc.

7. A method for preparing an antibody-drug conjugate (ADC), wherein the method comprises: using a linker to conjugate a toxin molecule to a non-natural amino acid of the antibody of any one of claims 1-6, in accordance with the method of any one of claims 1-6.

8. An aminoacyl-tRNA synthetase / tRNA orthogonal pair, characterized in that, The orthogonal pair can identify non-natural amino acids encoded by the TAG or TAA codons; preferably, the non-natural amino acids are selected from, but not limited to, pAcF, pAzF, or AzK.

9. The orthogonal pair as described in claim 8, characterized in that: The orthogonal pair of the TAG codon encoding the non-natural amino acid pAcF is pAcFRS / Tyr tRNA. CUA Preferably, the pAcFRS has a nucleotide sequence as shown in SEQ ID NO: 1 or an amino acid sequence as shown in SEQ ID NO: 2, and the Tyr tRNA CUA It has the nucleotide sequence shown in SEQ ID NO: 3; The orthogonal pair of the TAA codon encoding the non-natural amino acid pAzF is Mm pAzFRS / PyI tRNA. UUA Preferably, the Mm pAzFRS has a nucleotide sequence as shown in SEQ ID NO: 6 or an amino acid sequence as shown in SEQ ID NO: 7, and the PyItRNA UUA It has the nucleotide sequence shown in SEQ ID NO: 8; The orthogonal pair of the TAA codon encoding the non-natural amino acid AzK is MmAzKRS / PyI tRNA. UUA Preferably, the Mm AzKRS has a nucleotide sequence as shown in SEQ ID NO: 36 or an amino acid sequence as shown in SEQ ID NO: 37, and the PyItRNA UUA It has a nucleotide sequence as shown in SEQ ID NO:

8.

10. An antibody incorporating a non-natural amino acid at a specific site, said antibody being obtained by the method of any one of claims 1-6; preferably; the antibody has a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 24 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 25; or has a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 26; or has a heavy chain as shown in SEQ ID NO: 29 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 31; or has a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 32; or has a heavy chain as shown in SEQ ID NO: 33 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 29 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 29 and a light chain as shown in SEQ ID NO: 28; or has a heavy chain as shown in SEQ ID NO: 32 and a light chain as shown in SEQ ID NO: 33; or has a heavy chain The heavy chain shown in NO:27 and the light chain shown in SEQ ID NO:

34.

11. A nucleic acid encoding the antibody as described in claim 10.

12. A vector comprising the nucleic acid of claim 11.

13. The use of the antibody as described in claim 10 in the preparation of antibody-drug conjugates (ADCs).

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