Herceptin antibody mutant and nk cell conjugated thereto

Abstract

The present invention provides a herceptin antibody mutant. The herceptin antibody mutant is a VH-R50G, VH-R50A, VH-R50V, VH-R50L, VH-R50I, VL-F53L, VL-R66G, VL-H91Q, VH-R59T, VH-G103R, VH-F104L, VH-R50G / G103R, VH-G103R / F104L, VH-R50G / R59T / G103R, or VL-H91Q / VH-R50G mutant based on a wild-type light chain variable region (VL) and heavy chain variable region (VH). The mutant has lower antigen affinity, and ensures that effector cells can promptly dissociate from a target cell antigen upon exerting the cytotoxic effect thereof, thereby reducing self-apoptosis. Thus, the mutant exhibits enhanced tumor inhibition and / or killing activity and a prolonged in vivo half-life.

Description

A Herceptin antibody mutant and its coupled NK cells Technical Field

[0001] This invention relates to the conjugation and application of antibody mutants with cells, and belongs to the fields of polypeptides and cell biology. Background Technology

[0002] Trastuzumab Deruxtecan (DS-8201) is an antibody-drug conjugate (ADC) targeting HER2, consisting of an antibody (Herceptin) and derlutecan (an irinotecan-like chemotherapy drug). It is used to treat unresectable or metastatic HER2-positive solid tumors and is widely recognized as the most significant breakthrough drug for HER2-positive tumors to date. Based on clinical data from DESTINY-PanTumor 02, DS-8201 demonstrated excellent efficacy against various HER2-positive solid tumors. In a study of 267 HER2 IHC 3+ (strongly positive) patients covering multiple tumor types including endometrial cancer, cervical cancer, ovarian cancer, bladder cancer, and bile duct cancer, the objective response rate (ORR) was 37.1%, and the median duration of response (mDOR) was 11.3 months. However, even among strongly HER2-positive patients, nearly 63% did not benefit from this treatment. Furthermore, the side effects of the drug cannot be ignored. 84.6% of patients experienced drug-related adverse events, with 40.8% experiencing grade 3 or higher adverse events, and 28.8% discontinuing or reducing their dosage as a result (J Clin Oncol, 2023, 42:47-58.). Therefore, the biopharmaceutical field has been searching for novel therapies targeting HER2. Among these, the combination of Herceptin and cellular immunotherapy has become a hot topic. Herceptin can induce NK cells to exert a killing effect in vivo through antibody-mediated cytotoxicity (ADCC) (Br J Cancer, 2006, 94: 0.). Based on this, the combination therapy of Herceptin and NK cell immunotherapy has been extensively studied by cell therapy companies both domestically and internationally. For example, the haNK cell (high-affinity Natural Killer cell) produced by NantKwest, when used in combination with Herceptin, has shown a more potent killing effect on HER2-positive tumor cell lines (Blood, 2020, 135: 399-410.). Therefore, NK cell-related immunotherapy has developed rapidly in the field of cancer treatment. Nearly 30% of the 30 NK-related companies in China have entered clinical trials, and there are over 2,000 NK cell clinical trials conducted or ongoing globally, a significant portion of which target HER2-positive tumors. However, because antibody molecules and NK cells have their own tissue distribution characteristics in the body, and NK cells themselves do not have targeting specific tissues, no significant clinical benefit has been observed when used in combination.To address the lack of targeting, constructing CAR-NK cells that target tumor antigens can enable NK cells to achieve a certain degree of targeting. Artiva Biotherapeutics' AB-102 and Catamaran Bio's CAT-179 are both CAR-NK products targeting HER2. In addition, well-known NK therapy companies such as NKArta and Fate Therapeutics have also developed multiple CAR-NK products targeting different targets. However, CAR-NK products currently generally suffer from low transfection efficiency, long preparation cycles, and high costs, and viral vectors also pose certain safety risks. Technical issues

[0003] Studies have shown that if NK cells fail to separate from target cells after cytotoxicity, it leads to a large number of NK cell deaths, with most NK cell death occurring when NK cells fail to detach from their target (J Cell Biol, 2018, 217: 0.). Furthermore, Misty R. Jenkins et al. found that prolonged contact time between NK cells and target cells can induce excessive inflammation (J Exp Med, 2015, 212: 0.), thereby inhibiting NK cell activity. These findings indicate that rapid separation of NK cells from target cells after exerting their cytotoxic effect is crucial for NK cell survival. NK cells that fail to separate from target cells in a timely manner are more likely to undergo apoptosis.

[0004] The purpose of this invention is to provide a low-affinity anti-HER2 antibody. This antibody can not only covalently couple to NK cells to form a cell antibody-drug conjugate, thereby endowing NK cells with targeting ability to enhance their killing ability against HER2-positive tumor cells, but also, through its low-affinity characteristic, make it easier for NK cells to separate from target cells after exerting their killing effect, thereby improving the survival ability of NK cells in the tumor microenvironment. Technical solutions

[0005] To achieve the above objectives, the present invention first provides a Herceptin antibody mutant, wherein the Herceptin antibody mutant is a light chain variable region F53L mutant (abbreviated as VL-F53L, Mutant1) based on the light chain variable region shown in amino acids 1-107 of SEQ ID NO.1 and the heavy chain variable region shown in amino acids 1-120 of SEQ ID NO.2, or

[0006] The light chain variable region R66G mutant (abbreviated as VL-R66G, Mutant2), or

[0007] The light chain variable region H91Q mutant (abbreviated as VL-H91Q, Mutant3), or

[0008] Heavy chain variable region R50G mutant (abbreviated as VH-R50G, Mutant4), or

[0009] Heavy chain variable region R50A mutant (abbreviated as VH-R50A, Mutant4-1), or

[0010] Heavy chain variable region R50V mutant (abbreviated as VH-R50V, Mutant4-2), or

[0011] Heavy chain variable region R50L mutant (abbreviated as VH-R50L, Mutant4-3), or

[0012] Heavy chain variable region R50I mutant (abbreviated as VH-R50I, Mutant4-4), or

[0013] Heavy chain variable region R59T mutant (abbreviated as VH-R59T, Mutant5), or

[0014] Heavy chain variable region G103R mutant (abbreviated as VH-G103R, Mutant7), or

[0015] Heavy chain variable region F104L mutant (abbreviated as VH-F104L, Mutant8), or

[0016] The light chain variable region H91Q and heavy chain variable region R50G mutants (abbreviated as VL-H91Q / VH-R50G, Mutant9), or

[0017] Heavy chain variable region R50G and G103R mutants (abbreviated as VH-R50G / G103R, Mutant12), or

[0018] Heavy chain variable region G103R and F104L mutants (abbreviated as VH-G103R / F104L, Mutant13), or

[0019] The heavy chain variable region mutants R50G, R59T and G103R (abbreviated as VH-R50G / R59T / G103R, Mutant15).

[0020] In a preferred embodiment, the amino acid sequence of the light chain constant region of the Herceptin antibody mutant is shown as amino acids 108-214 of SEQ ID NO.1, and the amino acid sequence of the heavy chain constant region is shown as amino acids 121-450 of SEQ ID NO.2.

[0021] Secondly, this invention provides a polynucleotide encoding the aforementioned Herceptin antibody mutant. Based on common knowledge of those skilled in the art, following the triplet codon rule of protein coding, the same amino acid can be encoded by different triplet nucleotides. Therefore, any amino acid sequence capable of encoding both the heavy chain variable region and the light chain variable region of the aforementioned Herceptin antibody mutant falls within the scope of the polynucleotide encoding the Herceptin antibody mutant defined by this invention.

[0022] In a preferred embodiment, the polynucleotide sequence encoding the R50G mutant (Mutant4) heavy chain variable region of the Herceptin antibody is shown in SEQ ID NO. 6, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, pp. 49-369.

[0023] The polynucleotide sequence encoding the R50A mutant heavy chain variable region of the Herceptin antibody (Mutant4-1) is shown in SEQ ID NO.7, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.3, digits 49-369.

[0024] The polynucleotide sequence encoding the R50V mutant (Mutant4-2) heavy chain variable region of the Herceptin antibody is shown in SEQ ID NO. 8, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, digits 49-369, or...

[0025] The polynucleotide sequence encoding the R50L mutant (Mutant4-3) heavy chain variable region of the Herceptin antibody is shown in SEQ ID NO. 9, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, digits 49-369, or...

[0026] The polynucleotide sequence encoding the heavy chain variable region of the Herceptin antibody R50I mutant (Mutant4-4) is shown in SEQ ID NO. 10, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, digits 49-369, or...

[0027] The polynucleotide sequences encoding the light chain variable region H91Q and the heavy chain variable region R50G mutant (Mutant9) of the Herceptin antibody are shown in SEQ ID NO. 11, and the polynucleotide sequences encoding the heavy chain variable region are shown in SEQ ID NO. 12, or...

[0028] The polynucleotide sequences encoding the heavy chain variable regions R50G and G103R mutants (Mutant12) of the Herceptin antibody are shown in SEQ ID NO.13, and the polynucleotide sequences encoding the light chain variable regions are shown in SEQ ID NO.3, pp. 49-369.

[0029] In another preferred embodiment, the polynucleotide sequence encoding the heavy chain constant region of the Herceptin antibody mutant is shown in positions 1690-2679 of SEQ ID NO.3, and the polynucleotide sequence encoding the light chain constant region of the Herceptin antibody mutant is shown in positions 370-690 of SEQ ID NO.3.

[0030] Third, the present invention provides a vector for expressing the Herceptin antibody mutant, the vector containing a polynucleotide encoding the Herceptin antibody mutant. In one specific embodiment of the present invention, the vector is a commercially available pTT5 vector; other conventional expression vectors in the field of genetic engineering can also be used in the present invention.

[0031] Fourth, the present invention provides a host cell for the Herceptin antibody mutant, wherein the host cell contains the vector expressing the Herceptin antibody mutant. In one specific embodiment of the present invention, the host cell is a HEK293 cell, but other conventional host cells in the field of genetic engineering can also be used in the present invention.

[0032] Fifth, this invention provides the application of the Herceptin antibody mutant in the preparation of drugs for treating tumor diseases. The tumor disease is caused by tumor cells expressing the HER2 antigen.

[0033] Sixth, the present invention provides an effector cell coupled with the Herceptin antibody mutant, which has tumor-inhibiting and / or tumor-killing activity.

[0034] In a preferred embodiment, the effector cells are NK cells. In a specific embodiment of the invention, the cells are NK92 cells.

[0035] Seventh, this invention provides the application of the effector cells described above in the preparation of drugs for treating tumor diseases. The tumor disease is a disease caused by tumor cells expressing the HER2 antigen.

[0036] Finally, this invention provides a method for preparing Herceptin antibody or Herceptin antibody mutant by conjugating it to effector cells, the method comprising the following steps:

[0037] (1) Modify the Herceptin antibody or its mutant to obtain Herceptin antibody or antibody mutant linked to TCO-PEG4;

[0038] (2) The Herceptin antibody or antibody mutant linked to TCO-PEG4 obtained in step (1) is reacted with GDP-Fucose-PEG4-Methyltetrazine (guanosine 5′-bisphosphate-fucose-triazole-ethylene glycol-methyltetraazine) to obtain Herceptin antibody or antibody mutant coupled with GDP-Fucose;

[0039] (3) Use fucosyltransferase to couple the Herceptin antibody or antibody mutant conjugated with GDP-Fucose obtained in step (2) to N-GlcNac on the surface of NK cell membrane. Beneficial effects

[0040] This invention obtained Herceptin antibody mutants with lower HER antigen affinity compared to Herceptin antibodies using an AICD mutation screening platform. Furthermore, using the method provided by this invention, the Herceptin antibody mutants were conjugated with NK cells. NK92 cells conjugated with the low-affinity Herceptin mutants exhibited higher killing efficiency than NK92 cells conjugated with Herceptin antibodies. The killing efficiency increased from 37.7% to 40.5% within 4 hours and from 53% to 60.6% within 16 hours. The low antigen affinity of the antibody ensured that the conjugated NK92 cells could promptly separate from the target cell antigen after exerting their killing effect, thereby reducing apoptosis. Flow cytometry results showed that the number of apoptotic cells in NK cells conjugated with low-affinity Herceptin was less than that in NK cells conjugated with wild-type Herceptin, approaching the number of apoptotic cells in fresh NK cells. In vivo tumor suppression experiments in mice showed that NK cells conjugated with low-affinity Herceptin mutants exhibited more sustained killing ability than NK cells conjugated with Herceptin antibodies. Furthermore, NK cells conjugated with low-affinity Herceptin mutants exhibit a longer half-life in mice. These results demonstrate the promising application of the low-affinity Herceptin mutants provided by this invention, as well as effector cells such as NK cells conjugated with said Herceptin antibody mutants, possessing inhibitory and / or cytotoxic activity, in the preparation of drugs for treating tumor diseases. Attached Figure Description

[0041] Figure 1 shows the in vitro killing results of wild-type Herceptin or its mutant in combination with NK92 cells in Example 4 of the present invention.

[0042] Figure 2 shows the in vitro killing results of NK92 cells coupled with wild-type Herceptin or its mutant in Example 5 of the present invention.

[0043] Figure 3 shows the activity detection results of NK92 cells coupled with wild-type Herceptin or its mutant after incubation with target cells for 4 hours in Example 5 of the present invention;

[0044] Figure 4 is a fluorescence imaging diagram of the anti-tumor test of the mouse tumor model in Example 6 of the present invention;

[0045] Figure 5 is a curve showing the change of bioluminescence intensity of tumor burden over time in Example 6 of the present invention;

[0046] Figure 6 shows the results of the half-life evaluation experiment of NK92 coupled with Herceptin mutant in mouse tumors in Example 7 of the present invention. Embodiments of the present invention

[0047] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0048] Example 1. Screening for Herceptin mutants

[0049] In humoral immunity, antibody diversity depends on somatic high-frequency mutations (SHM) and class-switching recombination (CSR) occurring in germinal centers. Activation-induced cytidine deaminase (AICD), the only enzyme to date capable of inducing mutations in human genome DNA, plays a crucial role in this process. AICD deaminates cytosine (C) in single-stranded DNA to uracil (U), resulting in a C:U mismatch. During subsequent DNA replication, a rich variety of point mutations or fragment deletions, insertions, and recombinations occur through mispairing repair (MMR), short-patch BER, or long-patch BER mechanisms. Based on the mutagenic properties of AICD on DNA, several teams have used AICD-mediated mutagenesis to screen antibodies in mammalian cells (Proc Natl Acad Sci USA, 2011, 108: 0.) and to direct the evolution of fluorescent proteins (Proc Natl Acad Sci USA, 2004, 101: 0.).

[0050] Herceptin is a humanized monoclonal antibody derived from the murine anti-HER2 monoclonal antibody 4D5. The humanization process was conducted by antibody engineer Paul Carter. Carter created eight humanized versions of the 4D5 antibody, among which the antibody named humAb4D5-8 became the precursor and basis of Herceptin due to its higher affinity for HER2 and better inhibitory effect on HER2-positive tumor cells (Proc Natl Acad Sci USA, 1992, 89: 0.). This invention uses the amino acid sequence of humAb4D5-8 (light chain GI: 1685823435; heavy chain GI: 442924) as wild-type Herceptin.

[0051] This invention first constructs a wild-type Herceptin expression plasmid, cloning the light and heavy chain polynucleotide sequences of wild-type Herceptin into the multiple cloning site of a pcDNA3.1 vector carrying the Zeocin gene. An intrinsic ribosomal entry site (IRES) is added between the light and heavy chain sequences to achieve simultaneous expression of the antibody light and heavy chains on a single vector. The light chain sequence is located before the 5' end of the IRES sequence, and the heavy chain sequence is located after the 3' end of the IRES sequence. Furthermore, the transmembrane region (TM) of PGDFR is fused to the Fc terminus of the heavy chain to enable the expressed antibody to be displayed on the eukaryotic cell membrane surface. The antibody described herein is named “Herceptin-wt-TM”, and the DNA coding sequence of Herceptin-wt-TM is shown in SEQ ID NO.3 (in SEQ ID NO.3, 1-48: CD33 signal peptide; 49-369: Herceptin-wt light chain variable region; 370-693: light chain constant region + stop codon; 694-1281: IRES sequence; 1282-1329: CD33 signal peptide; 1330-1689: Herceptin-wt heavy chain variable region; 1690-2679: heavy chain constant region; 2680-2826: PGDFR transmembrane region sequence).

[0052] Secondly, a human AICD expression plasmid was constructed, and the coding region DNA of human AICD was cloned into the multiple cloning site of pEE14.4 (Transient gene expression levels from multigene expression vectors. Biotechnol Prog. 2007 Mar-Apr;23(2):435-43.) (DNA coding sequence as shown in SEQ ID NO.4).

[0053] Third, this invention constructs a HER2 protein expression plasmid by cloning the extracellular DNA sequence of HER2 into the multiple cloning site of the pTT5 universal vector, and sequentially fusing an HIS×6 tag and a Twin-Strep tag to its C-terminus. The HER2 fusion protein is named "HER2-Twin-Strep" in this invention, and its DNA coding sequence is shown in SEQ ID NO. 5. AICD expression plasmid and Herceptin-wt-TM expression plasmid are simultaneously transfected into CHO cells. Herceptin-wt-TM expression on the cell membrane surface is detected by HER2-Twin-Strep, and cell populations with weakened affinity for HER2 are progressively sorted using flow cytometry. Mutants of Herceptin-wt-TM are identified by PCR and TA cloning sequencing.

[0054] In the specific implementation, pcDNA3.1-Herceptin-wt-TM was first introduced into CHO cells using an electroporator (Gene Pulser Xcell™, BIO-RAD), and selected for 2 weeks with a working concentration of 300 μg / mL Zeocin. Subsequently, pEE14.4-AICD plasmid DNA was introduced via electroporation. The pEE14.4 plasmid carries the glutamine synthase gene, and cells were selected for 2 weeks with a working concentration of 50 μg / mL L-Methionine-DL-sulfoximine (MSX) glutamine synthase inhibitor to obtain a cell bank simultaneously expressing Herceptin-wt-TM and AICD. AICD-mediated DNA mutations gradually accumulate with the proliferation of double-transfected cells.

[0055] Next, pTT5-HER2-Twin-Strep was transiently transfected into HEK293 cells for secretory expression. The cell culture supernatant was obtained and purified using an HIS purification column to prepare the HER2-Twin-Strep fusion protein. This fusion protein carries a Twin-Strep-Tag and can be detected using fluorescently labeled streptavidin (such as Streptavidin-PE).

[0056] The expression of Herceptin-wt-TM on the surface of CHO cell membranes was detected by anti-human IgG antibody staining and flow cytometry (NovoSampler Pro NS200, Agilent). The binding of the antibody to HER2 was detected using the HER2-Twin-Strep fusion protein for subsequent screening of Herceptin mutants. After double transfection of CHO cells, AICD began to be expressed in the cells and gradually introduced mutations into the Herceptin gene. These mutations were random mutations and had a chance of altering antibody affinity. After one week of culture, double-transfected CHO cells were stained with HER2-Twin-Strep fusion protein and Streptavidin-PE, and cells with weakened HER2 binding were screened using a flow cytometry system (CytoFLEX SRT cell sorter, Beckman Coulter) for continued culture until significant differences were observed.

[0057] Cells with weakened HER2 binding were isolated, precipitated, and chromosomal DNA was extracted. At this point, the Herceptin sequence had been stably integrated into the genome of the CHO cells. PCR amplification was performed using the following primers, yielding DNA products that may contain mutated Herceptin light and heavy chains.

[0058] F: 5'-AGGGAGACCCAAGCTGGCTAG-3';

[0059] R: 5'-CAGTGGGAGTGGCACCTTCCAGG-3';

[0060] PCR reaction volume: 10 μL;

[0061]

[0062] PCR reaction procedure: Step 1: 94℃, 5 min; Step 2: 94℃, 30 s; Step 3: 55℃, 30 s; Step 4: 72℃, 1 min; Step 5: 72℃, 5 min; Step 2 and Step 4 are repeated 30 times.

[0063] After PCR, the products were validated by agarose gel electrophoresis. After gel recovery, the PCR products were cloned and sequenced using the TA cloning strategy (Suzhou Hongxun Biotechnology Co., Ltd.). Based on the sequencing results, a total of 16 Herceptin mutants were screened. The sequence comparisons between each mutant and wild-type Herceptin are shown in Table 1. The amino acid sequence numbers were all obtained using the Kabat numbering system.

[0064] Table 1. List of mutation sites in 16 Herceptin mutants (VL indicates light chain variable region; VH indicates heavy chain variable region)

[0065]

[0066] Wild-type Herceptin and mutant sequences were cloned into the pTT5 vector. Mammalian cells (HEK293 or CHO) were used as host cells for secretory expression via transient transfection. The obtained cell supernatant was purified using Protein A affinity. The expression levels and purity of the Herceptin mutant are shown in Table 2.

[0067] Table 2. Transient expression levels and purity of Herceptin mutants in HEK293 cells.

[0068]

[0069] Example 2. Determination of the affinity of Herceptin and its mutants for HER2 using biolayer interferometry (BLI).

[0070] Wild-type Herceptin and Herceptin mutants used in the experiment were obtained by transient expression in mammalian cells HEK293. HER2 antigen (extracellular fragment) was produced by Shanghai Baiying Biotechnology Co., Ltd. The affinity of HER2 antigen for Herceptin mutants was tested using biolayer interferomeory (BLI) technology to further screen and validate Herceptin mutants with weakened HER2 affinity. Buffer formulation: 10 mM HEPES, 150 mM sodium chloride, 3 mM EDTA, 0.1% BSA, and 0.05% Tween 20; Octet® AHC2 biosensor (Sartorius, catalog number #18-5142); BLI device: Octet R8 manufactured by Sartorius; Data acquisition and analysis were performed using Data acquisition 12.0 and Data analysis 12.2 software, respectively. The process is briefly described below:

[0071] 1. Preparation of wild-type Herceptin and Herceptin mutant samples

[0072] Dilute wild-type Herceptin and Herceptin mutant with buffer to a concentration of 10 μg / ml, add to column 2 of a 96-well assay plate, and set the program to Loading for 600 s. Each row contains an independent Herceptin mutant, with wild-type Herceptin in row 1.

[0073] 2. HER2 antigen sample preparation

[0074] The HER2 antigen was diluted to 100 nM with buffer, and then serially diluted downwards to achieve HER2 analytical concentrations of 100, 20, 5, 2.5, 1.25 nM, and 0. These concentrations were then added to columns 4-9 of a 96-well plate, with the control program set to Association for 200 seconds. Buffer was added to columns 1, 3, 10, and 11 of the 96-well plate, and glycine (pH 1.7) was added to column 12. The sample and solution volume for all samples was 200 μL.

[0075] 3. Detect the affinity of the antibody for HER2.

[0076] Eight Octet® AHC2 biosensors (Sartorius, catalog number #18-5142) were placed in the AH slot of column 1 of the sensor holder. The detection conditions were set in Data Acquisition 12.0 software as follows: 1. Pre-humidification: Baseline, 60s, Position: Column 1. 2. Cyclic detection: Column 2: Loading, 600s; Column 3: Baseline 1, 60s; Samples: Columns 4-9: Association, 200s; Column 10: Dissociation, 600s; Column 11: Neutralization; Column 12: Regeneration.

[0077] 4. Data Analysis

[0078] Data was analyzed using Data Analysis 12.2 software. With 0 concentration as the control and background subtraction, KD values ​​were calculated using a Fitting curve.

[0079] The affinity of each Herceptin mutant for HER2 is shown in Table 3. The results show that the binding affinity of wild-type Herceptin to HER2 is 3.79 × 10⁻⁶. -9 Of the 16 mutants obtained, 3 mutants, Mutant4, Mutant9, and Mutant12, had a binding affinity reduced to 10. -7 M level; binding affinity of 8 mutants decreased to 10. -8 M~10 -9 M level; the binding affinity of other mutants is below the detection range and cannot be detected. This embodiment shows that the present invention obtains the expected low-affinity Herceptin mutant through the AICD mutation screening platform.

[0080] Table 3. List of Herceptin mutants' affinity for HER2 (NB no binding)

[0081]

[0082] Example 3. Preparation and expression optimization of Herceptin mutants

[0083] In this embodiment, the Herceptin mutant Mutant4 was selected for expression level optimization. The arginine R at the 50th amino acid position of the N-terminus of the heavy chain of Mutant4 was mutated to glycine G, reducing its affinity for HER2 to 3.9 × 10⁻⁶. -7 Mutant4 expressed a low level of only 8.9 mg / L, but at a low concentration. To obtain a low-affinity mutant with a high expression level without affecting antibody affinity, the 50th amino acid of the Mutant4 heavy chain, glycine (G), was sequentially mutated to alanine (A), valine (V), leucine (L), and isoleucine (I), all of which are non-electrolyte aliphatic amino acids with properties similar to glycine. These mutants were named Mutant4-1 (Herceptin-VH-R50A), Mutant4-2 (Herceptin-VH-R50V), Mutant4-3 (Herceptin-VH-R50L), and Mutant4-4 (Herceptin-VH-R50I), respectively. The protein production process is briefly described below:

[0084] 1. Suzhou Junji Biotechnology Co., Ltd. was commissioned to synthesize plasmid DNA. The light chain and heavy chain DNA sequences of the above-mentioned Herceptin antibody and its mutant were cloned into the pTT5 universal vector, and the plasmids were prepared according to the operation methods mentioned in the "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit". The sequences of wild-type Herceptin (Herceptin-wt) and its mutant (Mutant) are as follows: The nucleic acid sequence of wild-type Herceptin is shown in SEQ ID NO.3, lines 49-2679: 49-369: Herceptin-wt light chain variable region; 370-693: light chain constant region + stop codon; 1330-1689: Herceptin-wt heavy chain variable region; 1690-2679: heavy chain constant region; The nucleic acid sequence of the Mutant4 heavy chain variable region is shown in SEQ ID NO.6; the nucleic acid sequence of the Mutant4-1 heavy chain variable region is shown in SEQ ID NO.7; the nucleic acid sequence of the Mutant4-2 heavy chain variable region is shown in SEQ ID NO.8; the nucleic acid sequence of the Mutant4-3 heavy chain variable region is shown in SEQ ID NO.9; the nucleic acid sequence of the Mutant4-4 heavy chain variable region is shown in SEQ ID NO.10; the nucleic acid sequence of the Mutant9 light chain variable region is shown in SEQ ID NO. NO.11, the heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.12, and the Mutant12 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.13.

[0085] 2. Transient expression was performed in mammalian HEK293 cells. Cell density was adjusted to 2 × 10⁶ cells on the day of transfection. 6 For each cell / ml sample, 30 μg of plasmid (15 μg each of light and heavy chain plasmids) was added to 1.5 mL of Opti-MEM (Thermo Fisher, catalog number 11058021), vortexed, and incubated at room temperature for 5 min. Simultaneously, 150 μg of PEI (Thermo Fisher, catalog number 919012) was added to 1.5 mL of Opti-MEM and mixed. The two solutions were combined and mixed thoroughly, incubated at room temperature for 20 min, and then added to 30 mL of HEK293 cells. After 96 hours of transfection, the cells were centrifuged and the supernatant was collected.

[0086] 3. Protein purification: The antibody protein in the supernatant was purified by affinity using 1 mL of Ezfast AT protein A (Borglon).

[0087] The purified Herceptin mutant was tested for its affinity for HER2 using biolayer interferomeory (BLI). The procedure was the same as in Example 2.

[0088] The transient expression levels, purity, and HER2 binding affinity of each Herceptin mutant in this embodiment are shown in Table 4. The binding affinity of Mutant4-1 (Herceptin-VH-R50A) to HER2 is 3.88 × 10⁻⁶. -7 M, yield approximately 56.4 mg / L Protein A, with a purity of 99% after affinity purification. While maintaining a low affinity for HER2, it significantly improved expression levels and purity.

[0089] Table 4. List of transient expression levels, purity, and HER2 binding affinity of Herceptin mutants after expression optimization.

[0090]

[0091] Example 4. In vitro killing experiment of Herceptin mutant in combination with NK92

[0092] MC38-HER2 is a humanized MC38 colon cancer cell line with the HER2 gene (purchased from Nanmo Biotechnology, catalog number #NM-S13-TM13), which can express HER2 on the cell membrane surface. In this embodiment, MC38-HER2 was used as the target cell to analyze the killing effect of Herceptin mutant combined with NK92 on HER2-positive tumor cells. The Cell Counting Kit-8 (CCK-8 kit, purchased from Solarbio Biotechnology Co., Ltd., catalog number #CA1210) is a rapid and highly sensitive assay kit based on WST-8, widely used for cytotoxicity analysis. WST-8 works by being reduced by mitochondrial dehydrogenases in the presence of electron coupling reagents to generate a highly water-soluble orange-yellow formazan product. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at 450 nm using a microplate reader, indirectly reflecting the number of viable cells. In this embodiment, CCK-8 was used to detect the killing effect; the specific experimental procedure is as follows:

[0093] 1. Preparation of target cells

[0094] Add 100 µL of complete culture medium (DMEM (Gibco, C11995500BT) + 10% FBS (sigma, F8687)) to a 96-well plate, seed MC38-HER2 cells at a density of 20,000 cells per well, and incubate at 37°C in a 5% CO2 incubator for 6 hours to allow the cells to adhere.

[0095] 2. Add Herceptin mutant and NK92

[0096] The experimental design is as follows, with each group containing 5 duplicate wells:

[0097] Group A: Control group, with only NK92 cells added, effector-to-target ratio of 5:1;

[0098] Group B: Experimental group, with wild-type Herceptin and NK92 cells added, Herceptin final concentration was 10 µg / mL, effector-target ratio was 5:1;

[0099] Group C: Experimental group, with Herceptin mutant (Mutant4-1 used in this example) added to NK92 cells, the final concentration of Herceptin mutant was 10 µg / mL, and the effector-target ratio was 5:1.

[0100] Herceptin mutant and NK92 cells were added to 96-well plates according to the experimental groups, and the volume of each well was adjusted to 200 µL using complete culture medium.

[0101] 3. Co-culture and detection

[0102] After inoculation, the mixture was thoroughly combined and co-cultured at 37°C for 4 h and 16 h in a 5% CO2 incubator, respectively. After co-culture, 20 μL of CCK8 was added to each well and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0103] 4. Result Calculation

[0104] Lethality (%) = [A (measured value) - A (background value)] / [A (no lethality value) - A (background value)] × 100;

[0105] A (measured value): Absorbance measured in the control group and experimental group;

[0106] A (Background value): Absorbance of wells containing only culture medium and CCK-8 solution;

[0107] A (No-kill value): Absorbance of the pore containing only the target cells and CCK-8 solution.

[0108] The killing results are shown in Figure 1. Herceptin, when used in combination with NK cells, effectively enhanced the killing efficiency of NK cells against tumor cells, increasing from 10.5% to 31.1% at 4 hours and from 16.6% to 46.7% at 16 hours. This improvement is attributed to the ADCC effect mediated by Herceptin on NK cells. However, when using a Herceptin mutant with decreased affinity for HER2, the ADCC effect was significantly reduced, decreasing from 31.1% to 18.9% at 4 hours and from 46.7% to 33.9% at 16 hours. The tumor cell killing efficiency when used in combination with NK cells was only 60% of that of wild-type Herceptin.

[0109] Example 5. In vitro killing experiment of Herceptin mutant coupled with NK92

[0110] In this embodiment, Herceptin and its mutant (Mutant4-1) are covalently coupled to NK92 cells. Before coupling, the antibody needs to be modified by attaching a linker containing guanosine diphosphate-fucose (GDP-Fucose) to several Lys bonds. Then, through an enzymatic reaction using the recombinant truncated form of α-1,3-fucosyltransferase (FucTd), the antibody is coupled to the N-GlcNac on the NK92 cell membrane surface, forming a stable glycosidic bond, thus producing a structurally stable cell antibody-cell conjugate that has no effect on antibody function and cell activity and function. The specific coupling process is as follows:

[0111] First, the NHS active ester on TCO-PEG4-NHS reacts with the primary amine of the antibody protein to form a stable amide bond. In a 100 μL reaction system, 1 mg of purified Herceptin or its mutant protein and 15 μg of TCO-PEG4-NHS (Shanghai Pairui Pharmaceutical Technology Co., Ltd., product number A34125) were added. The volume was adjusted to 100 μL using 20 mM HEPES buffer (pH 7.0–7.5, Thermo Fisher Scientific (China) Co., Ltd., product number 15630), and incubated at room temperature for 30 min. The reaction was terminated by adding 5 μL of 1 M Tris buffer (pH 8.0), and incubated at room temperature for 5 min. The reaction product was then added to a PD SpinTrap G-25 desalting column (Cytiva, product number 28918004), centrifuged at 800×g to remove unreacted and small molecules generated during the reaction, yielding pure Herceptin or its mutant linked to TCO-PEG4.

[0112] Secondly, taking advantage of the reverse electron requirement between trans-cyclooctene (TCO) and tetrazine, Herceptin or its mutant linked to TCO-PEG4 was coupled with GDP-Fucose-PEG4-Methyltetrazine (synthesized by Yantang Biotechnology Co., Ltd., product number YT-HJP-3-29). The Herceptin or its mutant linked to TCO-PEG4 obtained in the previous step was reacted with 30 μg of GDP-Fucose-PEG4-Methyltetrazine. After incubation at room temperature for 30 min, the reaction product was added to a PD SpinTrap G-25 desalting column and centrifuged at 800×g to remove unreacted and small molecules generated during the reaction, yielding pure GDP-Fucose-coupled Herceptin or its mutant.

[0113] Finally, Herceptin carrying GDP-Fucose or its mutant was coupled to N-GlcNac on the surface of NK92 cells using fucosyltransferase (the recombinant truncated form of α-1,3-fucosyltransferase of Helicobacter pylori disclosed in CN114369585A used in this invention). At 5 × 10⁻⁶ cells / years, the fucosyltransferase was used to bind the herceptin to GDP-Fucose or its mutant form to N-GlcNac on the cell membrane. 7 50 μg / mL fucosyltransferase and 200 μg / mL Herceptin or its mutant containing GDP-Fucose were added to NK92 cells, and incubated at room temperature for 30 min. After the reaction, the cells were washed with pH 7.4 PBS, centrifuged at 500×g for 5 min, and the supernatant was removed. NK92 cells conjugated with Herceptin or its mutant were resuspended in PBS for subsequent killing experiments.

[0114] In this embodiment, MC38-HER2 cells were used as target cells to analyze the killing effect of NK92 cells coupled with Herceptin or its mutant on HER2-positive tumor cells. The specific experimental procedure and data analysis of the killing experiment were the same as in Example 4. The experimental groups were as follows, with 5 replicates per group:

[0115] Group A: Control group, with the addition of uncoupled NK92 cells, effector-to-target ratio of 5:1;

[0116] Group B: Experimental group, with NK92 cells conjugated with wild-type Herceptin added, with an effector-to-target ratio of 5:1;

[0117] Group C: Experimental group, NK92 cells conjugated with Herceptin mutant (the Herceptin mutant used in this example is Mutant4-1) were added, with an effector-to-target ratio of 5:1.

[0118] The killing results are shown in Figure 2. Compared with NK92 cells alone, NK92 cells conjugated with Herceptin showed a more than three-fold increase in killing efficiency within 4 hours (11.9% vs 37.7%) and within 16 hours (16.2% vs 53%). Furthermore, NK92 cells conjugated with a low-affinity Herceptin mutant exhibited even higher killing efficiency, increasing from 37.7% to 40.5% within 4 hours and from 53% to 60.6% within 16 hours. This is related to the fact that NK92 cells can promptly separate from target cells after exerting their killing effect, thereby reducing their own apoptosis.

[0119] Next, we used 7-ADD (7-Aminoactinomycin D) fluorescent DNA dye (Beijing Bio-Lab Technology Co., Ltd., product number #KFS190) to label apoptotic cells in NK92 cell antibody-conjugates after incubation with target cells for 4 hours, and detected them by flow cytometry. The staining results are shown in Figure 3. The results showed that the number of apoptotic cells in NK cells conjugated with low-affinity Herceptin was less than that in NK cells conjugated with wild-type Herceptin (7% vs 11%), and close to the number of apoptotic cells in fresh NK cells (this group of NK cells was not subjected to killing experiments and was directly taken from the culture medium for detection) (7% vs 5%).

[0120] Example 6. In vivo tumor suppression experiment in mice after Herceptin mutant was coupled with NK92.

[0121] NK92 cells conjugated with Herceptin or its mutant were prepared according to Example 5 (the Herceptin mutant used in this example was Mutant4-1).

[0122] Twelve 6-8 week old NOG mice were randomly divided into four groups of three mice each. The experimental groups are as follows:

[0123] Group A: Control group, intraperitoneal injection of 200 μL PBS;

[0124] Group B: Herceptin combined with NK92, with simultaneous intraperitoneal injection of wild-type Herceptin (20 μg / animal / dose) and NK92 cells (5×10⁻⁶). 6 (Cells / each / dose), adjust the injection volume to 200 μL using PBS;

[0125] Group C: NK92 cells conjugated with wild-type Herceptin, intraperitoneally injected with NK92 cells conjugated with wild-type Herceptin (5 × 10⁻⁶). 6 Cells / dosage / dose; NK92 cells co-conjugated with 20 μg wild-type Herceptin; adjust the injection volume to 200 μL using PBS;

[0126] Group D: NK92 cells conjugated with low-affinity Herceptin mutant, intraperitoneally injected with NK92 cells conjugated with low-affinity Herceptin mutant (5 × 10⁻⁶ cells). 6 Cells / dosage / dose; NK92 cells co-conjugated with 20 μg of low-affinity Herceptin mutant; adjust the injection volume to 200 μL using PBS;

[0127] NOG mice were purchased from Beijing Vitonda Biotechnology Co., Ltd. and housed at Vitonda Biotechnology. The target cells used in this example were MC38-HER2-luc cells, which stably express firefly luciferase. These cells were obtained by transfecting MC38-HER2 cells with a lentivirus (purchased from Chongqing Yingmaoshengye Biotechnology Co., Ltd.) containing the luciferase expression gene. After inoculation into the mice, the firefly luciferase content was detected using luciferin as a substrate, and the distribution and size of tumor cells in the mice were analyzed using in vivo imaging technology. Three days before administration, each mouse was intraperitoneally injected with 3 × 10⁻⁶ mmol / L of luciferase. 5 MC38-HER2-luc cells were used. On day 3 post-tumor inoculation, the drugs were administered intraperitoneally according to experimental groups, twice a week for a total of four administrations. Tumor changes were recorded weekly using a PerkinElmer IVIS in vivo imaging system. Ten minutes before each imaging session, a fluorescein substrate (MedChemExpress, catalog number #HY-12591A) was injected intraperitoneally at a dose of 150 mg / kg.

[0128] The experimental results are shown in Figures 4 and 5 and Table 5. At the same Herceptin dosage, the NK92-conjugated group showed better killing effect on HER2-positive cells in vivo compared to the Herceptin-NK92 combination group. The NK92 conjugated with the low-affinity Herceptin mutant exhibited a more sustained killing ability.

[0129] Table 5. Statistical table of fluorescein in vivo imaging data of NOG mice in each experimental group after drug administration (fluorescein intensity unit: photons / sec)

[0130]

[0131] Example 7. Half-life evaluation experiment of Herceptin mutant coupled with NK92

[0132] NK92 cells conjugated with Herceptin or its mutant were prepared according to Example 5 (the Herceptin mutant used in this example was Mutant4-1).

[0133] Twenty-four 6-8 week old SCID Beige mice were randomly divided into two groups of 12 mice each. The experimental groups are as follows:

[0134] Group A: NK92 cells conjugated with wild-type Herceptin were injected intratumorally, and the injection volume was adjusted to 50 μL using PBS.

[0135] Group B: NK92 cells conjugated with a low-affinity Herceptin mutant were injected intratumorally, and the injection volume was adjusted to 50 μL using PBS.

[0136] In this embodiment, SCID Beige mice were obtained from Beijing Vital River Laboratory Animal Co., Ltd. and housed in the Nestling Animal Laboratory. MC38-HER2 cells were used as target cells, and 1×10⁶ cells were used. 6 MC38-HER2 was subcutaneously injected into the right scapula of mice, and tumor volume was measured along three orthogonal axes (a, b, and c), calculated as tumor volume = abc / 2. When the tumor grew to 100–150 mm... 2At approximately 7-9 days, NK92 cells conjugated with Herceptin or its mutant were injected intratumorally into mouse tumors according to the experimental groups. Tumors were harvested on days 0, 1, 3, and 7, with 3 mice from each group harvested each time. The harvested tumors were minced and digested with type II collagenase at 37°C for 2 hours to prepare a single-cell suspension. The obtained cells were stained and labeled with PE-anti-hCD45 antibody (Biolegend, catalog number #368510, 1:200 dilution), FITC-anti-hCD3 antibody (Elabscience, catalog number #E-AB-F1230C, 1:200 dilution), and APC-anti-hCD56 antibody (Elabscience, catalog number #E-AB-F1239E, 1:200 dilution). The number of NK cells (human CD45 positive, human CD3 negative, and human CD56 positive) was detected and analyzed by flow cytometry.

[0137] The results are shown in Figure 6. On day 7 after intratumoral injection, the number of NK cells conjugated with the low-affinity Herceptin mutant was twice that of wild-type Herceptin in the tumor microenvironment. Using nonlinear regression, the half-life of NK92 cells conjugated with wild-type Herceptin in the tumor microenvironment was calculated to be approximately 2.5 days, while the half-life of NK92 cells conjugated with the low-affinity Herceptin mutant was approximately 4.2 days. This indicates that NK cells conjugated with low-affinity antibodies can more easily separate from target cells due to the low affinity of their conjugate antibodies, thereby improving the survival ability of NK cells in the tumor microenvironment.

Claims

1. A mutant of the Herceptin antibody, characterized in that, The Herceptin antibody mutant is a light chain variable region F53L mutant, a light chain variable region R66G mutant, a light chain variable region H91Q mutant, a heavy chain variable region R50G mutant, a heavy chain variable region R50A mutant, a heavy chain variable region R50V mutant, a heavy chain variable region R50L mutant, a heavy chain variable region R50I mutant, a heavy chain variable region R59T mutant, a heavy chain variable region G103R mutant, a heavy chain variable region F104L mutant, a light chain variable region H91Q and a heavy chain variable region R50G mutant, a heavy chain variable region R50G and a G103R mutant, a heavy chain variable region G103R and a F104L mutant, or a heavy chain variable region R50G, R59T and a G103R mutant, based on the light chain variable region as shown in the amino acid sequence of SEQ ID NO. 1, positions 1-107, and the heavy chain variable region as shown in the amino acid sequence of SEQ ID NO. 2, positions 1-120.

2. The Herceptin antibody mutant according to claim 1, characterized in that, The amino acid sequence of the light chain constant region of the Herceptin antibody mutant is shown in SEQ ID NO. 1, positions 108-214, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 2, positions 121-450.

3. A polynucleotide encoding the Herceptin antibody mutant of claim 1 or 2.

4. The polynucleotide of claim 3, wherein, The polynucleotide sequence encoding the heavy chain variable region of the Herceptin antibody heavy chain variable region R50G mutant is shown in SEQ ID NO. 6, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, positions 49-369, or The polynucleotide sequence encoding the heavy chain variable region of the Herceptin antibody heavy chain variable region R50A mutant is shown in SEQ ID NO. 7, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, positions 49-369, or The polynucleotide sequence encoding the heavy chain variable region of the Herceptin antibody heavy chain variable region R50V mutant is shown in SEQ ID NO. 8, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, positions 49-369, or The polynucleotide sequence encoding the heavy chain variable region of the Herceptin antibody heavy chain variable region R50L mutant is shown in SEQ ID NO. 9, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, positions 49-369, or The polynucleotide sequence encoding the heavy chain variable region of the Herceptin antibody heavy chain variable region R50I mutant is shown in SEQ ID NO. 10, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, positions 49-369, or The polynucleotide sequence encoding the light chain variable region of the Herceptin antibody light chain variable region H91Q and heavy chain variable region R50G mutant is shown in SEQ ID NO. 11, and the polynucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 12, or The polynucleotide sequence encoding the light chain variable region of the Herceptin antibody light chain variable region H91Q and heavy chain variable region R50G mutant is shown in SEQ ID NO. 11, and the polynucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 12, or The polynucleotide sequence encoding the heavy chain variable region of the R50G and G103R mutant of the Herceptin antibody is shown in SEQ ID NO. 13, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 3, 49-369.

5. The polynucleotide of claim 3, wherein The polynucleotide sequence encoding the heavy chain constant region of the mutant of the Herceptin antibody is shown in SEQ ID NO. 3, 1690-2679, and the polynucleotide sequence encoding the light chain constant region is shown in SEQ ID NO. 3, 370-690.

6. A vector expressing the mutant of the Herceptin antibody according to claim 1 or 2, characterized in that, The vector contains the polynucleotide according to any one of claims 3-5.

7. A host cell expressing the mutant of the Herceptin antibody according to claim 1 or 2, characterized in that, The host cell contains the vector according to claim 6.

8. The mutant of the Herceptin antibody according to claim 1 or 2 for use in the preparation of a medicament for treating tumors.

9. An effector cell conjugated with the mutant of the Herceptin antibody according to claim 1 or 2, which has the activity of inhibiting and / or killing tumors.

10. The effector cell of claim 9, wherein The effector cell is an NK cell.

11. The effector cell according to claim 9 or 10 for use in the preparation of a medicament for treating tumors.

12. A method of preparing a conjugate of an Herceptin antibody or an Herceptin antibody mutant of claim 1 or 2 with an effector cell, comprising the steps of, The method comprises the following steps: (1) modifying the Herceptin antibody or its mutant to obtain a Herceptin antibody or antibody mutant conjugated with TCO-PEG4; (2) reacting the Herceptin antibody or antibody mutant conjugated with TCO-PEG4 obtained in step (1) with guanosine 5'-diphosphate-fucose-triazole-tetramethylene glycol-methyl tetrazine to obtain a Herceptin antibody or antibody mutant conjugated with GDP-Fucose; (3) using fucosyltransferase to conjugate the Herceptin antibody or antibody mutant conjugated with GDP-Fucose obtained in step (2) to N-GlcNac on the surface of the membrane of an NK cell.

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