EGFR antibody mutant with high recognition specificity and application thereof
By designing multiple mutations in the amino acid sequence of cetuximab, an EGFR antibody mutant capable of specifically recognizing EGFR resistance mutations was developed, solving the problem of on-target/off-tumor toxicity in solid tumor CAR-T therapy and improving the application scope and safety of solid tumor CAR-T products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN LAIMANG BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-12-27
- Publication Date
- 2026-06-30
AI Technical Summary
Existing EGFR inhibitors, such as cetuximab, cannot effectively identify EGFR extracellular domain mutations when faced with EGFR resistance mutations, leading to on-target/off-tumor toxicity in CAR-T therapy for solid tumors. There is a lack of antibodies that specifically recognize EGFR resistance mutations.
By designing mutations at multiple sites in the amino acid sequence of cetuximab, a highly specific EGFR antibody mutant was developed. This mutant can specifically recognize drug-resistant mutations in the extracellular region of the EGFR antigen and reduce the ability to recognize unmutated EGFR antigens.
It has broadened the application scope and safety of CAR-T products for solid tumors, reduced the killing of normal tissues, significantly improved the killing rate of tumor cells with EGFR resistance mutations, and reduced the toxic side effects on normal cells.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to an AI-optimized EGFR antibody mutant with high recognition specificity and its applications. Background Technology
[0002] EGFR (epidermal growth factor receptor) is a cell membrane receptor tyrosine kinase, primarily found on the surface of epithelial cells, fibroblasts, and keratinocytes. Under normal conditions, EGFR activates intracellular signaling pathways by binding to exogenous epidermal growth factor, thereby participating in many signaling pathways related to cell growth, differentiation, and apoptosis. EGFR overexpression and abnormal activation are closely related to the occurrence and development of various tumors, including lung cancer, breast cancer, and colorectal cancer. Therefore, inhibitors targeting EGFR, including small molecule tyrosine kinase inhibitors and monoclonal antibodies, can effectively inhibit tumor cell growth and spread, achieving significant clinical efficacy. However, due to the presence of tumor drug resistance mutations, the efficacy of EGFR inhibitors is often limited by these mutations. Taking cetuximab as an example, cetuximab is an EGFR antibody widely used in indications such as metastatic colorectal cancer, head and neck tumors, non-small cell lung cancer, and recurrent or metastatic head and neck squamous cell carcinoma. It exerts its anti-tumor effect by binding to the extracellular domain of EGFR, preventing EGFR from binding to its ligands. However, mutations in the extracellular domain of EGFR (G465R, S492R) prevent it from binding to cetuximab, leading to drug resistance in tumor cells. Patent CN111499749B discloses a cetuximab mutant and its application. The mutant's mutation sites include heavy chain W52D and Y104D mutation sites. These mutants can recognize the G465R and S492R EGFR extracellular domain mutations while maintaining a relatively high affinity for natural EGFR, representing a promising approach for targeting EGFR-resistant mutations in tumor treatment. However, due to its relatively high affinity for natural EGFR, the corresponding antibody will face the problem of insufficient selection specificity in the development of CAR-T therapy for solid tumors.
[0003] CAR-T therapy is an emerging cell-based immunotherapy approach that engineers a patient's T lymphocytes to express chimeric antigen receptors (CARs) that specifically recognize and kill tumor cells carrying corresponding antigens. CAR-T therapy has demonstrated significant efficacy in treating relapsed / refractory leukemia and lymphoma, among other hematological malignancies. However, it faces numerous challenges in solid tumors, including tumor heterogeneity, tumor invasion, and a lack of tumor-specific targets. One major challenge is that many targets in current CAR-T research for solid tumors are tumor-associated antigens (TAAs). For example, EGFR is highly expressed not only on tumor surfaces but also in normal tissues. This can lead to EGFR-targeted CAR-T products for solid tumors also recognizing and killing normal cells, resulting in severe on-target / off-tumor toxicity. Clinically, EGFR resistance mutations (G465R, S492R) induced by cetuximab treatment provide an excellent tumor-specific target for CAR-T therapy in solid tumors. These resistance mutations are not expressed in normal tissues, effectively mitigating on-target / off-tumor toxicity. Although the cetuximab mutant disclosed in patent CN111499749B can effectively identify EGFR resistance mutations, its high affinity for natural EGFR cannot avoid the on-target / off-tumor toxicity issues encountered in solid tumor CAR-T development. Currently, there is still a lack of antibodies that can specifically identify resistance mutations in the extracellular domain of EGFR without recognizing EGFR antibodies expressed in normal tissues, thus addressing the safety concerns of EGFR-targeted CAR-T therapy. Summary of the Invention
[0004] The purpose of this invention is to provide an AI-optimized EGFR antibody mutant with high recognition specificity and its applications, thereby addressing the problems existing in the prior art. This invention designs mutations at multiple different sites in the amino acid sequence of cetuximab, enabling the antibody mutant to specifically recognize drug-resistant mutations in the extracellular region of the EGFR antigen, while not recognizing unmutated EGFR antigens. This improves the application scope and safety of subsequent immunotherapy products, particularly CAR-T products for solid tumors.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides an AI-optimized EGFR antibody mutant with high recognition specificity, which is obtained by performing the following mutations on cetuximab: any combination of three or four of the following mutations: H52D, H104D, H101M, H56E, H99D, L50D and L34Y.
[0007] The amino acid sequences of the heavy and light chains of the cetuximab are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
[0008] H52D refers to the mutation of the 52nd amino acid in the heavy chain of the cetuximab to amino acid D;
[0009] H104D refers to the mutation of the 104th amino acid in the heavy chain of the cetuximab to amino acid D;
[0010] H101M refers to the mutation of the 101st amino acid in the heavy chain of the cetuximab to amino acid M.
[0011] H56E refers to the mutation of the 56th amino acid in the heavy chain of the cetuximab to amino acid E.
[0012] H99D refers to the mutation of the 99th amino acid in the heavy chain of the cetuximab to amino acid D;
[0013] The L50D refers to the mutation of the 50th amino acid in the light chain of the cetuximab to amino acid D.
[0014] The L34Y refers to the mutation of the 34th amino acid in the light chain of the cetuximab to amino acid Y.
[0015] Preferably, the EGFR antibody mutant is an H52D / H104D / H101M mutant, an H52D / H104D / H56E mutant, an H52D / H104D / H101M / L50D mutant, an H52D / H104D / H56E / L34Y mutant, or an H52D / H104D / H53I / L26N mutant;
[0016] The amino acid sequences of the heavy and light chains of the H52D / H104D / H101M mutant are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively;
[0017] The amino acid sequences of the heavy and light chains of the H52D / H104D / H56E mutant are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively;
[0018] The amino acid sequences of the heavy and light chains of the H52D / H104D / H101M / L50D mutant are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.
[0019] The amino acid sequences of the heavy and light chains of the H52D / H104D / H56E / L34Y mutant are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively;
[0020] The amino acid sequences of the heavy and light chains of the H52D / H104D / H53I / L26N mutant are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.
[0021] The present invention also provides a coding gene for the above-mentioned EGFR antibody mutant.
[0022] The present invention also provides a recombinant vector comprising the above-described encoding gene.
[0023] The present invention also provides a recombinant host cell comprising the above-described recombinant vector.
[0024] The present invention also provides the use of the above-mentioned encoding gene, recombinant vector or recombinant host cell in the preparation of the above-mentioned EGFR antibody mutant.
[0025] The present invention also provides the use of the above-mentioned EGFR antibody mutant in the preparation of CAR-T, CAR-NK or engineered antibody drugs.
[0026] The present invention also provides a CAR-T cell therapy drug for solid tumors, characterized in that the active ingredient includes the above-mentioned EGFR antibody mutant.
[0027] The solid tumor CAR-T cell therapy drug also includes pharmaceutically acceptable excipients.
[0028] The excipients include any one or a combination of at least two of the following: carrier, wetting agent, solubilizer, osmotic pressure regulator, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.
[0029] The solid tumor is any one of melanoma, colon cancer, lung cancer, esophageal cancer, ovarian cancer, cervical cancer, or breast cancer.
[0030] The present invention also provides the application of the above-mentioned EGFR antibody mutant in the preparation of EGFR inhibitors.
[0031] The present invention also provides an EGFR inhibitor, characterized in that the active ingredient comprises the above-mentioned EGFR antibody mutant.
[0032] The present invention discloses the following technical effects:
[0033] This invention designs mutations at multiple different sites in the amino acid sequence of cetuximab, enabling the antibody mutant to specifically recognize drug-resistant mutations in the extracellular region of EGFR antigen and reduce the ability to recognize unmutated EGFR antigen, thereby improving the application scope and safety of subsequent immunotherapy products, especially CAR-T products for solid tumors. Attached Figure Description
[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0035] Figure 1 The graph shows the full concentration gradient BLI test curves for the EGFR antibody H52D / H104D / H101M mutant. Among them, A and C are the full concentration gradient BLI test curves for the antibody mutant and three antigens: EGFR natural antigen, EGFR-G465R mutant antigen, and EGFR-S492R mutant antigen, respectively. The different colored curves in each subgraph represent the detection results of the antigens used in the test.
[0036] Figure 2 The graph shows the full concentration gradient BLI test curves for the EGFR antibody H52D / H104D / H56E mutant. Among them, A and C are the full concentration gradient BLI test curves for the antibody mutant and three antigens: EGFR natural antigen, EGFR-G465R mutant antigen, and EGFR-S492R mutant antigen, respectively. The different colored curves in each subgraph represent the detection results of different concentrations of antigen used in the test.
[0037] Figure 3 This is a BLI test curve for the full concentration gradient of the EGFR antibody H52D / H104D / H101M / L50D mutant; where A and C are the full concentration gradient BLI test curves for the antibody mutant and three antigens: EGFR natural antigen, EGFR-G465R mutant antigen, and EGFR-S492R mutant antigen, respectively. The different colored curves in each sub-graph represent the detection results of different concentrations of antigen used in the test.
[0038] Figure 4The graphs show the full concentration gradient BLI test curves for the EGFR antibody H52D / H104D / H56E / L34Y mutant. In the graphs, A and C represent the full concentration gradient BLI test curves for the antibody mutant and three antigens: EGFR natural antigen, EGFR-G465R mutant antigen, and EGFR-S492R mutant antigen, respectively. The different colored curves in each subgraph represent the test results using different concentrations of antigen.
[0039] Figure 5 The graph shows the full concentration gradient BLI test curves for the EGFR antibody 52D / H104D / H53I / L26N mutant. In the graph, A and C are the full concentration gradient BLI test curves for the antibody mutant and three antigens: EGFR natural antigen, EGFR-G465R mutant antigen, and EGFR-S492R mutant antigen, respectively. The different colored curves in each subgraph represent the detection results of the antigens used in the test.
[0040] Figure 6 The figures show the results of in vitro killing specificity experiments of CAR-T cells against tumor cells overexpressing EGFR natural antigen and two cetuximab resistance mutant antigens, G465R and S492R, at an effector cell / target cell ratio (E:T) of 1:1. A shows the killing results of different T cell control groups (T cell medium), blank T cells (BlankT) not expressing CAR structure, CAR-T cells expressing unmutated cetuximab scFv (WT CAR-T), and CAR-T cells expressing H52D / H104D / H101M / L50D mutant cetuximab scFv (Mutant CAR-T) against COLO 320DM cells not expressing EGFR antigen; B shows the killing results of several T cells (same as A) against COLO 320DM cells overexpressing EGFR natural antigen; C shows the killing results of several T cells (same as A) against COLO cells overexpressing EGFR G465R mutant antigen. Results of killing experiments on 320DM cells; D represents the results of killing experiments on COLO 320DM cells overexpressing EGFR S492R mutant antigen by several types of T cells (same as A);
[0041] Figure 7 The results of the CAR-T cell anti-tumor experiment in mice are shown. Among them, A shows the tumor growth in mice under the following conditions: no T cell infusion control group, CAR-T cells infused with unmutated cetuximab scFv, and CAR-T cells infused with cetuximab scFv with H52D / H104D / H101M / L50D mutations; B shows the monitoring data of body weight changes in different groups of mice. Detailed Implementation
[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0047] Based on the cetuximab sequence, this invention, through AI computational model optimization combined with experimental verification, designed a batch of EGFR antibody mutant sequences that specifically recognize two EGFR cetuximab resistance mutations, G465R and S492R, thus solving the safety issues in the current development of CAR-T drugs targeting EGFR in solid tumors. The method of AI computational optimization combined with experimental verification is as follows:
[0048] First, using AF2-multimer, mutations were introduced into the PDB structure (PDB: 1YY9) of the natural EGFR antigen (EGFR-WT) and cetuximab antibody (Ctx) complex to obtain the complex structures of Ctx with two mutant antigens, EGFR-S492R and EGFR-G465R. The rationality of the structures was evaluated based on existing experimental results in the literature.
[0049] Secondly, single-point saturation mutation scanning was performed on all antibody amino acids at the antigen-antibody binding interface. Using Rosetta flexddG and an AI model, the affinity of the mutated antibodies for the three antigens EGFR-WT, EGFR-S492R, and EGFR-G465R was predicted. Based on the calculated predictions, approximately 100 single-point mutated antibody sequences were selected for expression and purification, and the affinity of the mutated antibodies for the three antigens was determined using a BLI single-point assay.
[0050] The third step involves selecting sites with enhanced specificity from the single-point mutation experiment results for combined mutation. Rosetta flexddG and AI models are then used to predict the affinity of the combined antibody mutations for the three antigens EGFR-WT, EGFR-S492R, and EGFR-G465R. Approximately 150 combined mutant antibody sequences with enhanced specificity from the calculated prediction results are selected for expression and purification. The affinity of the mutant antibodies for the three antigens is then determined using the BLI single-point assay.
[0051] Finally, the highly specific antibody sequence obtained from the BLI single-point experiment was selected, and accurate affinity values were obtained by BLI multi-concentration detection.
[0052] Example 1
[0053] Mutants of EGFR antibodies are derived from cetuximab (amino acid sequences SEQ ID NO.1 and SEQ ID NO.2) with the following mutations: any combination of three or four of the following: H52D, H104D, H101M, H56E, H99D, L50D, and L34Y. In the description of the mutation sites, the first letter H or L represents the heavy and light chains of the antibody, respectively; the numbers represent the specific sites on the heavy and light chains; and the last letter represents the mutated amino acid code.
[0054] The cetuximab sequence is as follows:
[0055] Heavy chain amino acid sequence (SEQ ID NO.1):
[0056] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVS A;
[0057] Light chain amino acid sequence (SEQ ID NO.2):
[0058] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.
[0059] EGFR antibody mutant sequence expression:
[0060] (1) The EGFR antibody mutant protein sequence was reverse-translated into nucleic acid sequence, and codon optimization was performed for the expression system used. The obtained nucleic acid sequence was synthesized into a whole genome and inserted into a pcDNA3.4 plasmid vector containing a fragment of mouse IgG1 / kappa subtype antibody to obtain a recombinant plasmid;
[0061] (2) The recombinant plasmid obtained in step (1) was transformed into a mammalian CHO cell expression system for cell culture. Then, the cell culture supernatant was collected for protein purification to obtain the mutant of EGFR antibody.
[0062] The amino acid sequence of the mutant EGFR antibody is as follows:
[0063] H52D / H104D / H101M mutant sequence:
[0064] Heavy chain amino acid sequence (SEQ ID NO.3):
[0065] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIDSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTMYDDEFAYWGQGTLVTVSA;
[0066] Light chain amino acid sequence (SEQ ID NO.4):
[0067] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.
[0068] H52D / H104D / H56E mutant sequence:
[0069] Heavy chain amino acid sequence (SEQ ID NO.5):
[0070] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIDSGGETD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDDEFAYWGQGTLVTVSA;
[0071] Light chain amino acid sequence (SEQ ID NO.6):
[0072] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.
[0073] H52D / H104D / H101M / L50D mutant sequence:
[0074] Heavy chain amino acid sequence (SEQ ID NO.7):
[0075] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIDSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTMYDDEFAYWGQGTLVTVSA;
[0076] Light chain amino acid sequence (SEQ ID NO.8):
[0077] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKDASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.
[0078] H52D / H104D / H56E / L34Y mutant sequence:
[0079] Heavy chain amino acid sequence (SEQ ID NO.9):
[0080] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIDSGGETD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDDEFAYWGQGTLVTVSA;
[0081] Light chain amino acid sequence (SEQ ID NO.10):
[0082] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIYWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.
[0083] H52D / H104D / H53I / L26N mutant sequence:
[0084] Heavy chain amino acid sequence (SEQ ID NO.11):
[0085] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIDIGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDDEFAYWGQGTLVTVSA;
[0086] Light chain amino acid sequence (SEQ ID NO.12):
[0087] DILLTQSPVILSVSPGERVSFSCRANQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.
[0088] Example 2
[0089] Affinity and specificity assays of EGFR mutant antibodies with EGFR natural antigen and cetuximab resistance mutant antigen:
[0090] The EGFR antibody mutants with high expression levels from Example 1 were used, and their affinities with the natural EGFR antigen and two cetuximab resistance mutant antigens, G465R and S492R, were measured. First, EGFR antibody mutants at a concentration of 10 μg / mL were used to capture mouse Fc cells and fix the antibody using an AMQ chip. Then, three EGFR antigens at serially diluted concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.70 nM, and 1.23 nM were used as analytes, and Fortebio's [analyte name missing] ... Biomembrane interferometry (BLI) was used to detect binding and dissociation curves at multiple concentrations, and the dissociation constant KD was calculated. The experimental results are shown in Table 1. The full-concentration gradient BLI test curves for the EGFR antibody mutants H52D / H104D / H101M, H52D / H104D / H56E, H52D / H104D / H101M / L50D, H52D / H104D / H56E / L34Y, and H52D / H104D / H53I / L26N are shown in the figure. Figures 1-5 As shown.
[0091] The results in Table 1 show that the unmutated cetuximab binds strongly to the unmutated EGFR antigen, but binds weakly to the G465R and S492R mutant antigens. The EGFR antibody mutants obtained through mutation design in Example 1 showed a significantly decreased binding ability to the unmutated EGFR antigen, or even no binding at all, while exhibiting increased affinity for the G465R and S492R mutant antigens. Therefore, the relative specificity of both affinity was significantly improved.
[0092] Table 1. Affinity of EGFR antibody mutants to three EGFR antigens
[0093]
[0094] Example 3
[0095] A randomly selected EGFR antibody mutant (H52D / H104D / H101M / L50D) validated in Example 2 was used as the scFv sequence for CAR-T cells, and the killing specificity of CAR-T cells was determined:
[0096] CAR sequences were constructed using the scFv sequence corresponding to the EGFR antibody mutant, and CD3+ isolated from peripheral blood mononuclear cells (PBMCs) of healthy volunteers were processed using lentiviral vectors. + T lymphocytes were activated and transduced to express the constructed CAR sequence. Target cell lines overexpressing three antigens—EGFR-WT, EGFR-G465R, and EGFR-S492R—were constructed using the COLO 320DM tumor cell line. Untransduced T cells, CAR-T cells expressing the WT antibody scFv sequence, and CAR-T cells expressing the EGFR mutant antibody scFv sequence were respectively subjected to killing experiments with target cells overexpressing different EGFR antigens and blank control cells at an effector cell / target cell ratio (E:T) of 1:1 to verify the killing specificity of CAR-T cells against target cells overexpressing different EGFR antigens. The experimental results are as follows: Figure 6 As shown.
[0097] The results showed that the CAR-T cells corresponding to the H52D / H104D / H101M / L50D mutant sequence had a reduced killing rate of approximately 25% against target cells overexpressing EGFR WT, which was only 30% of the killing ability of WT CAR-T. However, the killing rates against target cells overexpressing the two mutant antigens EGFR G465R and S492R increased to approximately 85% and 81%, respectively, which were three times the killing ability of WT CAR-T. The killing rate against target cells of COLO 320DM that did not express EGFR was the same as that of WT CAR-T, demonstrating very high tumor antigen killing specificity. It can effectively eliminate target cells expressing the two cetuximab resistance mutations of EGFR antigens G465R and S492R, but has no killing effect on target cells expressing unmutated EGFR antigens, thus effectively solving the safety issues of solid tumor CAR-T.
[0098] Example 4
[0099] Furthermore, using the EGFR antibody mutant (H52D / H104D / H101M / L50D) validated in Example 3 as the scFv sequence for CAR-T cells, the antitumor activity of CAR-T cells in mice was determined:
[0100] 2.5×10 6 G465R mutant COLO 320DM (EGFR positivity rate approximately 99%) was subcutaneously injected into 8-week-old female NSG mice. When the tumor area was 20-25 mm, 2 The patients were divided into three groups and treated with different CAR-T cells via tail vein injection: (1) PBS (control group without T cell infusion); (2) WT CAR-T (CAR-T cells expressing unmutated cetuximab scFv); (3) Mutant CAR-T (CAR-T cells expressing H52D / H104D / H101M / L50D mutated cetuximab scFv). The CAR-T infusion dose was 0.5 × 10⁻⁶ cells. 6 Subsequently, the tumor size was measured every three days using calipers, and the mouse's weight changes were recorded. The experimental results are as follows: Figure 7 As shown.
[0101] The results showed that the therapeutic effect of WT CAR-T was not significantly different from that of the PBS control group, while Mutant CAR-T corresponding to the H52D / H104D / H101M / L50D mutant sequence could significantly delay tumor growth. There was no significant difference in mouse body weight among the groups, and no obvious toxicity was observed.
[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An AI-optimized EGFR antibody mutant with high recognition specificity, characterized in that, The EGFR antibody mutant is obtained by performing the following mutations on cetuximab: any combination of three or four of the following mutations: H52D, H104D, H101M, H56E, H99D, L50D and L34Y. The amino acid sequences of the heavy and light chains of the cetuximab are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. H52D refers to the mutation of the 52nd amino acid in the heavy chain of the cetuximab to amino acid D; H104D refers to the mutation of the 104th amino acid in the heavy chain of the cetuximab to amino acid D; H101M refers to the mutation of the 101st amino acid in the heavy chain of the cetuximab to amino acid M. H56E refers to the mutation of the 56th amino acid in the heavy chain of the cetuximab to amino acid E. H99D refers to the mutation of the 99th amino acid in the heavy chain of the cetuximab to amino acid D; The L50D refers to the mutation of the 50th amino acid in the light chain of the cetuximab to amino acid D. The L34Y refers to the mutation of the 34th amino acid in the light chain of the cetuximab to amino acid Y.
2. The EGFR antibody mutant according to claim 1, characterized in that, The EGFR antibody mutants are H52D / H104D / H101M mutant, H52D / H104D / H56E mutant, H52D / H104D / H101M / L50D mutant, H52D / H104D / H56E / L34Y mutant, or H52D / H104D / H53I / L26N mutant; The amino acid sequences of the heavy and light chains of the H52D / H104D / H101M mutant are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The amino acid sequences of the heavy and light chains of the H52D / H104D / H56E mutant are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The amino acid sequences of the heavy and light chains of the H52D / H104D / H101M / L50D mutant are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The amino acid sequences of the heavy and light chains of the H52D / H104D / H56E / L34Y mutant are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively; The amino acid sequences of the heavy and light chains of the H52D / H104D / H53I / L26N mutant are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.
3. The encoding gene of the EGFR antibody mutant as described in claim 1.
4. A recombinant vector, characterized in that, Includes the coding gene as described in claim 3.
5. A recombinant host cell, characterized in that, Includes the recombinant vector as described in claim 4.
6. The use of the encoding gene as described in claim 3, the recombinant vector as described in claim 4, or the recombinant host cell as described in claim 5 in the preparation of the EGFR antibody mutant as described in claim 1.
7. The use of an EGFR antibody mutant as described in claim 1 or 2 in the preparation of CAR-T, CAR-NK or engineered antibody drugs.
8. A CAR-T cell therapy drug for solid tumors, characterized in that, The active ingredient includes the EGFR antibody mutant as described in claim 1 or 2.
9. The use of an EGFR antibody mutant as described in claim 1 or 2 in the preparation of an EGFR inhibitor.
10. An EGFR inhibitor, characterized in that, The active ingredient includes the EGFR antibody mutant as described in claim 1 or 2.