Antibody or antigen binding fragment specifically binding to trop2

Abstract

The invention discloses an antibody or an antigen binding fragment specifically binding to trop2. The antibody or the antigen binding fragment comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.2, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO.4, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO.5, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO.6. The expression condition of trop2 in human breast cancer and pancreatic cancer cell membrane tissue samples can be detected through an immunohistochemical method, and the adjoint diagnostic kit for the humanized trop2 antibody coupling medicine has the advantages of assisting in precise medication and avoiding invalid treatment.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an antibody or antigen-binding fragment that specifically binds to trop2. Background Technology

[0002] Trop2, also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein and an intracellular calcium signal transmembrane transducer.

[0003] Trop2 was first discovered in human placental trophoblasts and is expressed in various normal tissues. Subsequently, it was found that many tumor cells exhibit high expression of Trop2. For example, under normal conditions, Trop2 is mainly expressed in epithelial cells and plays a crucial role in embryonic development. Trop2 expression can be detected in normal tissues such as the skin, cornea, salivary glands, respiratory tract, and lungs, but in limited quantities. However, Trop2 is an important tumor-promoting factor, highly expressed in various malignant tumors, commonly including breast cancer, urothelial carcinoma, cervical cancer, and pancreatic cancer, thus raising the question of Trop2 as a target for tumor therapy.

[0004] Antibody-drug conjugates (ADCs) are a novel therapeutic approach that utilizes the specificity of monoclonal antibodies to deliver cytotoxic drugs by recognizing target antigens in cancer cells. They not only achieve precise drug delivery and minimize toxicity to normal tissues, but also broaden the therapeutic window and enhance pharmacokinetic and pharmacodynamic properties. In ADCs, antibodies need to possess characteristics such as high specificity, low immunogenicity, long half-life, and good stability, which are key factors determining drug efficacy.

[0005] Based on the above considerations, this invention believes that the prerequisite for humanized Trop2 antibody-conjugated therapeutic drugs for breast cancer, urothelial carcinoma, cervical cancer, and pancreatic cancer cells lies in ensuring the specific recognition of tumor cells expressing Trop2. To overcome this problem, this invention utilizes antibody expression and screening technology, undergoing immune response, antibody sorting, gene recombination, high-throughput expression, and performance evaluation, to screen out the Trop2 antibody with the highest sensitivity and strongest specificity. This antibody is then used in the development of Trop2 companion diagnostic kit antibodies for humanized Trop2 antibody-conjugated cell therapy drugs for breast cancer and pancreatic cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an antibody or antigen-binding fragment that specifically binds to trop2. By immunizing rabbits with trop2 protein constructed in vitro as an immunogen, hybridoma cell lines expressing antibodies are obtained through cell fusion and screening.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An antibody or antigen-binding fragment that specifically binds to trop2, comprising three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO.7 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO.8; or, a variant having one or more CDRs with a conserved variation of no more than three amino acids in each CDR region with respect to the aforementioned light and heavy chain CDR regions.

[0009] Furthermore, when antibody HCDRs are encoded according to the Kabat coding rules, the antibody or antigen-binding fragment includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; the amino acid sequence of HCDR1 is shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is shown in SEQ ID NO.2, the amino acid sequence of HCDR3 is shown in SEQ ID NO.3, the amino acid sequence of LCDR1 is shown in SEQ ID NO.4, the amino acid sequence of LCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.6; or, variants having a single or multiple CDRs with a conserved variation of no more than 3 amino acids in each CDR region compared to the above 6 CDR regions.

[0010] Preferably, the aforementioned antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences of which are selected from the following:

[0011] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, or has at least 75%, 85%, 95% or 99% sequence identity with SEQ ID NO.7;

[0012] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, or has at least 75%, 85%, 95%, or 99% sequence identity with SEQ ID NO.8.

[0013] Preferably, the aforementioned antibody or antigen-binding fragment further includes a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.9 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO.10.

[0014] Preferably, the aforementioned antibody or antigen-binding fragment is selected from the group consisting of: monoclonal antibody, chimeric antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, scFv and dsFv.

[0015] The amino acid sequences of the above antibody or antigen-binding fragments are shown in Table 1:

[0016] Table 1. Amino acid sequence listing of primers and probes

[0017]

[0018] A nucleic acid molecule encoding an antibody or antigen-binding fragment as described above. The preparation method of the nucleic acid is a conventional method in the art, comprising the following steps: obtaining the nucleic acid molecule encoding the aforementioned antibody through gene cloning technology, or obtaining the nucleic acid molecule encoding the aforementioned antibody through artificial full-sequence synthesis.

[0019] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the above-mentioned antibody can be appropriately substituted, deleted, altered, inserted, or added to provide a homologue of a polynucleotide. The homologue of the polynucleotide in this invention can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the antibody sequence, while maintaining antibody activity.

[0020] A recombinant vector comprising the aforementioned nucleic acid molecule. The recombinant vector can be obtained by conventional methods in the art, i.e., constructed by linking the nucleic acid molecule of this application to various vectors. The vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule.

[0021] A recombinant host cell comprising the aforementioned nucleic acid molecule or the aforementioned vector. The host cell can be any conventional host cell in the art, as long as it enables the recombinant vector to replicate stably and autonomously, and the nucleic acid it carries can be effectively expressed.

[0022] A companion diagnostic kit for humanized trop2 antibody-drug conjugates (ADCs) comprises the aforementioned antibody or its antigen-binding fragment. The kit can assist clinicians in screening cancer patients suitable for humanized trop2 ADCs, enabling precise medication and avoiding ineffective treatment.

[0023] The aforementioned antibody or antigen-binding fragments are used in the preparation of trop2 companion diagnostic immunohistochemical assays for targeted therapy of breast and pancreatic cancer. These assays include quantitative methods, immunogenicity assays, flow cytometry, ELISA, or IHC.

[0024] The aforementioned antibody or antigen-binding fragments may be used in the diagnosis or companion diagnostics of tumors, or in the preparation of diagnostic or companion diagnostic kits for tumors.

[0025] The aforementioned antibody or antigen-binding fragments are used in the preparation of preclinical and clinical diagnostic reagents for trop2.

[0026] A method for detecting trop2 expression for non-diagnostic purposes, using the aforementioned antibody or antigen-binding fragment, or the aforementioned immunohistochemical antibody reagent or kit, to detect in vitro biological samples.

[0027] The advantages of this invention are: the antibody or antigen-binding fragment in this invention can specifically recognize the trop2 antigen recognition site, providing an immunohistochemical antibody for detecting trop2 protein that is highly specific, sensitive, and has lower immunohistochemical background staining; the antibody can be widely used for in vitro detection of trop2 protein, and for preparing trop2 protein companion diagnostic immunohistochemical detection products for breast cancer and pancreatic cancer patients before antibody-drug conjugate therapy; and it has an auxiliary role in precise drug use for companion diagnostic kits of humanized trop2 antibody-drug conjugates, avoiding ineffective treatment. Attached Figure Description

[0028] Figure 1 This refers to the IHC detection results of antibody 7R2 in breast cancer in Example 1;

[0029] Figure 2 This refers to the IHC detection results of antibody 7R2 in pancreatic cancer in Example 1;

[0030] Figure 3 This is the IHC result of antibody 13T5 in pancreatic cancer in Example 1. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: Preparation and screening of specific antibodies

[0033] In this embodiment, the trop2-specific antibody was prepared according to the following method:

[0034] (1) Preparation of trop2 protein immunogen: A trop2 protein expression plasmid with His and Twin-Strep Tag was constructed by gene synthesis method. The expression plasmid was transfected into HEK293 cells using Invitrogen Lipofectamine 2000 transfection reagent. After culturing for 72 hours, the supernatant was collected and purified trop2 protein was obtained by affinity chromatography.

[0035] (2) Immunization of New Zealand White Rabbits: Using the trop2 protein prepared in step (1) as an immunogen, two New Zealand White Rabbits were immunized with trop2 protein via in vivo injection. After immunization, the serum of the immunized animals was detected by ELISA to determine the level of immune response. After routine immunization, if the animal serum could reach the level of immune response against the immunogen (OD value > 1.0, titer 1:128.00), cell fusion could be performed.

[0036] (3) Cell fusion and plating: Cell fusion was performed once using the electrofusion method. Half of the fused cells were plated into solid culture medium, and the other half of the fused cells were frozen for preservation.

[0037] (4) Picking single-clonal cell lines: Pick single cell clusters cultured in solid culture medium and culture them in 96-well culture plates.

[0038] (5) Screening: The supernatant of fused cells was screened by ELISA to select cells that were positive for binding to trop2 protein.

[0039] (6) Clonal expansion culture and retesting: Positive clone cells were transferred to 48-well plates for expansion culture. 0.5 ml of cell supernatant was collected from each expanded clone for detection by indirect ELISA.

[0040] (7) Clonal expansion culture and retesting: Positive clones were transferred to 12-well plates for expansion culture. 1.5 ml of supernatant was collected from each expanded clone for indirect ELISA detection. Based on antigen recognition confirmation, two optimal stable cell lines (clones 7R2 and 13T5) were screened and cryopreserved. 4 ml of supernatant was collected from each clone before cryopreservation, and all clones were subtype identified and preserved.

[0041] (8) Gene sequencing of hybridoma cell antibody 7R2: Total RNA was extracted from hybridoma cells and reverse transcribed into cDNA by RT-PCR. The antibody light and heavy chain sequences were cloned and constructed into a T vector. The antibody gene sequence was then obtained by DNA sequencing analysis. According to the Kabat coding rules, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of antibody 7R2 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0042] (9) Antibody production and purification: The antibody gene obtained in step 8 was transfected into HEK293 cells for expansion culture, the antibody was purified by protein A / G affinity chromatography, and the purified antibody was stored in phosphate buffer (PBS).

[0043] Antibody 7R2 was applied to breast cancer cell samples and pancreatic cancer cell samples, respectively, while antibody 13T5 was applied to pancreatic cancer cell samples. IHC assays were then performed, and the results are shown below. Figures 1-3 The specific testing steps are shown in Table 2.

[0044] Table 2 IHC Detection Procedure

[0045]

[0046] pass Figures 1-3 As can be seen, clear cell membrane staining is observed on breast cancer and pancreatic cancer cell samples using antibody 7R2. Combined with the distribution of trop2 on the cells, it can be determined to be trop2 staining. Clear cell membrane staining is also observed on pancreatic cancer samples using antibody 13T5, similar to the staining sites of antibody 7R2, but the staining intensity is slightly lower. This indicates that antibody 7R2 in this patent has a better staining effect and can achieve better detection results when developing companion diagnostic test kits for breast cancer and pancreatic cancer.

[0047] Example 2: Antibody 7R2 Specificity Analysis

[0048] In this embodiment, monoclonal antibody 7R2 was obtained according to the method described in Example 1 above. The antibody was then subjected to specific analysis using an enzyme-linked immunosorbent assay (ELISA). The analysis steps are as follows:

[0049] (1) Dilute trop2 and trop1 proteins to 6 μg / mL with PBS and add 80 μL to each well of an ELISA plate. Seal the plate with sealing film and incubate at 4°C overnight (about 20 h).

[0050] (2) Remove the remaining liquid in the wells, air dry the microplate, wash the plate with PBST washing solution, soak 300 μL / well for 1 min, air dry the microplate, and perform the next washing. Wash the plate a total of 3 times.

[0051] (3) Add 100 μL of blocking agent (PBST washing solution containing 10% BSA) to each well, seal the plate with sealing film, and incubate at 37°C for 1.5 h.

[0052] (4) Repeat step 2 to wash the plate 3 times.

[0053] (5) Dilute the above antibody to 1 μg / mL with sample dilution buffer (PBST washing buffer containing 1% BSA). Add 100 μL to each well of the ELISA plate. Seal the plate with sealing film and incubate at 37°C for 1.0 h.

[0054] (6) Repeat step 2 to wash the plate 3 times.

[0055] (7) Dilute HRP-Goat-Anti-rabbit IgG to 1:20000 with sample dilution buffer, add 100 μL to each well, seal with sealing film, and incubate at 37°C for 1.0 h.

[0056] (8) Repeat step 2 to wash the plate 3 times.

[0057] (9) Add 100 μL of colorimetric solution to each well. Seal the plate with a sealing film and incubate at 37°C in the dark for 20 min.

[0058] (10) Add 50 μL of stop solution to each well and gently shake the microplate until the color development is uniform.

[0059] (11) Use an ELISA reader to read the absorbance values ​​at 470 nm and 650 nm. Subtract OD650 from OD470 to obtain the absorbance value (OD value). The specific detection results are shown in Table 3.

[0060] Table 3. OD values ​​of antibodies detected by ELISA

[0061]

[0062] Experimental results show that the above-mentioned antibodies have good specificity and high activity against trop2 protein, and do not cross-react with trop1 protein.

[0063] Example 3: Assisting in the screening of suitable patients for humanized trop2 antibody-drug conjugates

[0064] In this embodiment, a trop2 companion diagnostic kit prepared using the monoclonal antibody 7R2 obtained through the method described in Example 1 was used to screen clinically enrolled patients who expressed trop2 positively for antibody-drug conjugates containing humanized trop2. The specific procedures are as follows.

[0065] Tumor tissue samples were obtained from breast cancer patients via biopsy. Then, trop2 expression was analyzed by immunohistochemistry using a trop2 companion diagnostic kit prepared with the monoclonal antibody 7R2 obtained according to the method described in Example 1. The results showed positive trop2 expression, indicating the availability of humanized trop2 antibody-drug conjugates. After a period of treatment, the tumor in the patient significantly shrank.

[0066] Similarly, another tumor tissue sample was obtained from a cancer patient via biopsy and tested using the same method. The results showed no trop2 expression, indicating that humanized trop2 antibody-drug conjugates could not be used. However, after treatment with this drug for a period of time, the tumor in the patient's body did not shrink significantly and even became larger.

[0067] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically binds to trop2, characterized in that, The antibody or antigen-binding fragment includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; the amino acid sequence of HCDR1 is shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is shown in SEQ ID NO.2, the amino acid sequence of HCDR3 is shown in SEQ ID NO.3, the amino acid sequence of LCDR1 is shown in SEQ ID NO.4, the amino acid sequence of LCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

6.

2. The antibody or antigen-binding fragment that specifically binds to trop2 according to claim 1, characterized in that, The antibody or antigen-binding fragment further includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO.

8.

3. An antibody or antigen-binding fragment that specifically binds to trop2 according to claim 1 or 2, characterized in that, The antibody or antigen-binding fragment further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.9 and the amino acid sequence of which is shown in SEQ ID NO.

10.

4. The antibody or antigen-binding fragment that specifically binds to trop2 according to claim 1, characterized in that, The antibody or antigen-binding fragment is selected from the group consisting of: monoclonal antibody, chimeric antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, scFv and dsFv.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment as described in any one of claims 1 to 4.

6. A recombinant vector, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 5.

7. A recombinant host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A companion diagnostic kit for humanized trop2 antibody-drug conjugates, characterized in that, It includes the antibody or antigen-binding fragment as described in any one of claims 1 to 4.

9. The use of the antibody or antigen-binding fragment according to any one of claims 1 to 4 in the preparation of a trop2 companion diagnostic immunohistochemical detection product for targeted drug therapy in breast cancer and pancreatic cancer, wherein the targeted drug is a humanized trop2 antibody-drug conjugate.

10. A method for detecting trop2 expression for non-diagnostic purposes, characterized in that, The antibody or antigen-binding fragment according to any one of claims 1 to 4, or the companion diagnostic kit according to claim 8, is used to detect in vitro biological samples.

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