Anti-CD26 antibody and application thereof

Abstract

The invention relates to an antibody or an antigen binding fragment combined with human CD26, and an application of the antibody or the antigen binding fragment in preparation of a medicine for treating tumors with high CD26 expression. The CD26 murine antibody and the antigen binding fragment have relatively high specificity, affinity and binding force, and have certain killing activity on different tumor cells. A humanized bispecific antibody, a full-length humanized antibody and an antibody coupling drug are successfully constructed by using the CD26 murine antibody, have higher affinity to tumor cells compared with other murine-derived bodies, have higher affinity to tumor cells compared with other murine-derived bodies, and have higher binding and tumor cell internalization levels and lower non-tumor cell internalization levels; and the compound shows better drug effect and safety in ADC (Analog to Digital Converter) molecules.

Description

Anti-CD26 antibodies and their applications Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to antibodies or antigen-binding fragments that bind to human CD26, and their use in preparing drugs for treating tumors that highly express CD26. Background Art

[0002] CD26 is a multifunctional type II transmembrane glycoprotein that can also be found in a soluble form in plasma. CD26 typically exists as a homodimer, with a monomer consisting of 766 amino acids and a relative molecular mass of approximately 110 kDa. CD26 amino acid residues are divided into five regions from the inside out: an intracellular region (1-6), a transmembrane region (7-28), a heavily glycosylated region (29-323), a cysteine-rich region (324-551), and a C-terminal catalytic domain (552-766). Its three-dimensional molecular structure is closely related to its function. CD26 (DPP4) inhibitors have been used clinically for decades to treat type 2 diabetes. The expression of CD26 is significantly increased on the surface of various tumor cells, such as malignant mesothelioma, renal cancer, prostate cancer, lung cancer, etc. For these types of tumors with high CD26 expression, CD26 is an important target (CD26 / DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).

[0003] Among the most rapidly developing targeted anticancer drugs targeting human CD26 is YS110, a monoclonal antibody from Y'S Therapeutics. The completed Phase II clinical study of YS110 demonstrated good tolerability, but the disease control rate fell short of expectations, consistent with the low activity demonstrated in preclinical studies.

[0004] Although the literature reports that CD26 is widely distributed, the use of YS110 still has good safety, which shows that its safety in clinical use is well guaranteed. Among the 30 patients in the YS110 clinical trial, no patient died. The side effects were mainly 16.1% of patients had adverse reactions to infusion, 9.7% of patients had hiccups, and 6.5% of patients had diarrhea. The more serious adverse reactions included 2 cases of interstitial lung disease, 1 case of bacterial pneumonia, 1 case of tumor lysis syndrome, 1 case of hypercapnia, and 1 case of hypoxia. (Phase 2 Study of YS110, a Recombinant Humanized Anti-CD26 Monoclonal Antibody, in Japanese Patients With Advanced Malignant Pleural Mesothelioma, JTO Clinincal and Research Reports (2021), 2(6))

[0005] In summary, although the safety of the CD26 target has been verified in clinical trials, existing antibodies targeting CD26 are difficult to achieve good therapeutic effects on tumors.

[0006] Antibody-drug conjugates (ADCs) consist of three parts: an antibody, a linker, and a cytotoxic small molecule. They utilize the antibody's specific recognition of antigens to transport toxin molecules to the tumor site and effectively release drug molecules, thereby achieving the therapeutic purpose of killing tumor cells. Antibody-drug conjugates combine the specific recognition of antibodies with the tumor-killing effect of toxin molecules. The main function of the antibody part is to bind to the target antigen, giving the drug targeted properties. Currently, 15 ADC drugs have been approved for marketing worldwide, with the most ADCs targeting HER2.

[0007] Generally speaking, the antibody portion of an ADC should possess properties such as high specificity, high affinity, low immunogenicity, high endocytosis, and a long half-life. Currently, there are no ADC drugs targeting CD26 that have been marketed or entered clinical stages, and the potential of CD26 as an ADC drug target needs to be further explored.

[0008] Summary of the Invention

[0009] To solve the above problems, the first object of the present invention is to provide an anti-CD26 antibody or antigen-binding fragment with higher specificity, affinity, and better internalization.

[0010] The present invention provides an antibody or antigen-binding fragment that specifically binds to human CD26, comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3; and LCDR1 shown in SEQ ID NO: 4, LCDR2 with the sequence YRS (Y, R, and S represent tyrosine, arginine, and serine, respectively), and LCDR3 shown in SEQ ID NO: 5.

[0011] The antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that shown in SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that shown in SEQ ID NO: 9.

[0012] The antibody or antigen-binding fragment, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids in the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 are inserted, deleted or substituted, and the amino acid substitutions are conservative amino acid substitutions.

[0013] The antibody or antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO: 9.

[0014] The present application relates to an antibody-drug conjugate targeting CD26, comprising the above-mentioned anti-CD26 antibody.

[0015] The present invention also relates to nucleotide sequences encoding the amino acid sequences of the antibodies or antigen-binding fragments.

[0016] The present invention also relates to a vector comprising the nucleotide sequence and a host cell comprising the nucleic acid.

[0017] The present invention also relates to a method for producing an antibody or an antigen-binding fragment thereof, which comprises culturing the host cell and recovering the antibody or antigen-binding fragment from the culture.

[0018] The present invention also relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment, which further comprises a pharmaceutically acceptable excipient.

[0019] The present invention also relates to a method for treating tumors, comprising administering an effective amount of the antibody or antigen-binding fragment to a patient; the tumors highly express CD26, including but not limited to renal cancer, mesothelioma, lung cancer, liver cancer, prostate cancer, etc. The antibody or antigen-binding fragment is administered alone or in combination with other anticancer agents.

[0020] Compared with the existing technology, this application has the following advantages:

[0021] The anti-CD26 antibodies screened by the present invention have high specificity, affinity, and binding capacity for CD26-positive tumor cells, and the full-length humanized antibody derived from mouse antibody No. 79 has higher affinity, binding capacity and internalization level for tumor cells than full-length humanized antibodies derived from other mouse antibodies, and is more suitable as the antibody portion of the ADC molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: Flow cytometry analysis of antibody binding differences in mixed cell systems

[0023] Figure 2: Flow cytometry detection of fluorescence intensity changes after antibody internalization in 786-0 cells

[0024] Figure 3: Flow cytometry detection of fluorescence intensity changes after antibody internalization in PBMC cells

[0025] Figure 4: Flow cytometry analysis of fluorescence intensity changes after antibody internalization in different PBMC cells

[0026] Figure 5: Cell survival induced by DT3C-coupled 21G428-429, 21G430-431 and YS110 in UT16 cells (72 h)

[0027] Figure 6: Cell survival induced by DT3C-coupled 21G428-429, 21G430-431 and YS110 in 786-0 cells (72 h) DETAILED DESCRIPTION

[0028] Unless otherwise specifically defined herein, the technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

[0029] Singular forms such as "a," "an," and "the" include their corresponding plural referents.

[0030] The term "or" means and is used interchangeably with "and / or".

[0031] The term "CD26" is also known as dipeptidyl peptidase 4 (DPP4). The amino acid sequence of human CD26 can be found in Genbank under the accession number NP_001926.2, and its cDNA sequence can be found in Genbank under the accession number NM_001935.3.

[0032] The term "conservative amino acid substitution" refers to the replacement of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, such as its binding affinity for CD26. Common conservative amino acid substitutions are well known in the art. For example, Ala can be conservatively substituted with Gly or Ser; Arg can be conservatively substituted with Lys or His; Asn can be conservatively substituted with Gln or His, etc. The term "affinity" refers to the strength of the interaction between an antibody and an antigen. Antibody variable regions interact with antigens through non-covalent forces; the greater the interaction, the stronger the affinity.

[0033] The term "antibody" refers to a family of immunoglobulins that can non-covalently, reversibly, and specifically bind to their respective antigens. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain: CL. VH and VL can be further subdivided into highly variable complementarity determining regions (CDRs, also known as hypervariable regions) and more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. HCDR1, HCDR2, and HCDR3 are the three heavy chain complementary determining regions, and LCDR1, LCDR2, and LCDR3 are the three light chain complementary determining regions. The positions of the CDRs and framework regions can be determined using various numbering systems well known in the art, such as Kabat, Chothia, IMGT, etc., and this application uses IMGT to determine. The heavy chain variable region (VH) and light chain variable region (VL) are responsible for antigen recognition, especially the complementary determining regions (CDRs) therein, which are generally specific for different epitopes of the antigen. The constant region is primarily responsible for effector functions.

[0034] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to an antigen, such as a fragment that retains one or more CDR regions, including but not limited to Fab fragments, FV fragments, diabodies, linear antibodies, single-chain antibodies, nanobodies, multispecific antibodies, etc.

[0035] The term "monoclonal antibody" means a substantially homogeneous antibody population, i.e., the amino acid sequences of the antibody molecules contained in the population are identical except for a small amount of possible naturally occurring mutations. In contrast, polyclonal antibodies typically include a variety of different antibodies having different amino acid sequences in their variable domains (particularly their complementary determining regions). Monoclonal antibodies can be obtained by methods known to those skilled in the art. The present disclosure uses hybridoma technology to obtain monoclonal antibodies, which is one of the many methods known to obtain monoclonal antibodies.

[0036] The term "murine antibody" refers to an antibody that contains only rat or mouse immunoglobulin sequences.

[0037] The term "chimeric antibody" refers to an antibody in which the variable regions are derived from non-human (eg, murine) antibody sequences, with the remainder derived from human antibody sequences.

[0038] The term "humanized antibody" refers to a antibody in which the CDR regions are derived from non-human (eg, murine) antibody sequences, while the remainder are derived from human antibody sequences.

[0039] The term "bispecific antibody" refers to an antibody that has two identical or different specific antigen-binding sites.

[0040] The term "BiTE" refers to a bispecific antibody, or "bispecific T cell engager," composed of two antigen-specific single-chain variable fragments (scFv) connected by a linker. One scFv targets a tumor-associated surface antigen, while the other targets CD3 on the surface of T cells. Single-chain variable fragments (scFv), also known as single-chain antibodies, are composed of an antibody heavy chain variable region and a light chain variable region connected by a linker. A "linker" is an amino acid sequence used to connect different protein fragments, and much research has been conducted on the design and selection of linkers. In a BiTE, the linker connects a heavy chain variable region and a light chain variable region to form an scFv. It also connects two scFvs in series and allows them to rotate freely. It is generally composed of a repeating structure of GGGGS (S represents serine and G represents glycine). The small molecular weight and short side chain of glycine can increase the flexibility of the side chain, while the strong hydrophilicity of serine can increase the hydrophilicity of the peptide chain. Optimizing the number of repeat structures can not only help the light chain and heavy chain in the scFv to associate in the correct conformation, but also promote the optimal interaction between T cells and target cells in the immune synapse by affecting the distance between the two scFvs (Zhou Chong et al. Research Progress of Bispecific Single-Chain Antibody BiTE [J]. Journal of Biology, 2018). Common linkers connecting the heavy chain variable region and the light chain variable region in scFv are KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSTSGGGSGGGSGGGGSS (US 20180326032), GSTSGSGKPGSGEGSTKG (Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22), etc. In a specific embodiment of the present application, the flexible linker connecting a heavy chain and a light chain to form an scFv is composed of three GGGGS repeating structures, and the flexible linker connecting two scFvs in series is GGGGS. Those skilled in the art can select and determine from known linkers based on the teachings of existing technology through routine experimental methods and limited trials, and can optimize the number of GGGGS repeats in the two flexible linkers, or use flexible linkers other than GGGGS.

[0041] CD3 is an integral part of T cell signaling. When BiTE molecules bind to T cells and tumor cells at the same time, T cells are activated, promoting CD8+ T cells to directly secrete perforin and granzymes, and CD4+ T cells to secrete cytokines to further recruit activated killer T cells, thereby killing tumor cells. The CD3 antibody sequence can be selected from OKT-3, L2K, TR66, UCHT1, SP34, IORT3, Catumaxomab, Blinatumomab, Solitomab and other CD3 antibody sequences known in the art. In the specific embodiments of the present application, the CD3 antibody sequence in Solitomab is used. Those skilled in the art can select and determine from other known CD3 antibodies by conventional experimental methods and limited experiments, and are not limited to the specific CD3 antibody sequences in the specific embodiments of the present application.

[0042] The term "antibody-drug conjugate" (ADC) refers to the process of connecting a small molecule toxin to an antibody through a linker, and the antibody acts as a carrier to transport the small molecule toxin into target cells.

[0043] The terms "administration," "administering," "treating," and "treatment" refer to the contacting of an exogenous pharmaceutical, therapeutic, or diagnostic agent with a tissue, organ, cell, or biological fluid of a subject. "Treatment" refers to slowing or arresting the development of clinical symptoms of a disease, or to alleviating or improving at least one physical parameter, or to preventing the progression of a disease.

[0044] Example 1 Screening of CD26-positive and CD26-negative cells

[0045] The target cells were cultured in T75 cell culture flasks. When the cells were more than 80% confluent, the cells were digested with trypsin and collected. The cells were washed once with PBS and counted using a hemocytometer. The cells were divided into 5 × 10 5 cells per aliquot; anti-CD26 monoclonal antibody was used as the primary antibody and incubated with the target cells at room temperature for approximately 40 minutes. After the incubation period, the supernatant was discarded by centrifugation, the cell pellet was resuspended with PBS, centrifuged again and the supernatant was discarded, and the cell pellet was collected; the corresponding antibody Alexa Fluor 488 mouse anti-human IgG1 was then used as the secondary antibody, the cell pellet was resuspended, and the cells were incubated in the dark at room temperature for approximately 30 minutes. After the incubation period, the cells were washed twice with PBS, centrifuged and the supernatant was discarded, and the cell pellet was collected; the cell pellet was resuspended in approximately 200 ul of PBS solution, and the CD26 positivity rate was detected and analyzed by flow cytometry within 1 hour.

[0046] Flow cytometry analysis revealed that the CD26-positive cell lines included 786-0, OS-RC-2, UT16, and NCI-H226, while G401 and A-375 were CD26-negative cell lines. The positive rates are shown in Table 1. The CD26-positive cell lines were consistent with those in the reference (Chinese Patent Application No. CN200680034937.4, CD26 / DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).

[0047] Table 1 CD26 positive rate of target cells

[0048] Example 2: Preparation and screening of anti-human CD26 hybridoma cell lines

[0049] Step 1: Animal immunization

[0050] An rCD26 expression vector was designed based on the CD26 cDNA sequence published in GenBank (GenBank Accession No. NM_001935.3). A histidine tag was introduced at the 5' end of the codon, and the gene was synthesized. The gene was then ligated into the pCHO1.0 plasmid for expression and purification in CHO-S host cells. The recombinant CD26 target protein was immunized in female BALB / c mice according to standard immunization procedures. For detailed immunization procedures, please refer to the "Antibody Preparation and Use Laboratory Guide." Serum titers from immunized mice were monitored using an indirect ELISA assay. The mice with the highest serum titers were selected for fusion experiments using mouse spleen cells and mouse myeloma cells.

[0051] Step 2: Cell fusion

[0052] (1) Preparation of spleen cells

[0053] The immunized mice were sacrificed by cervical dislocation and soaked in 75% (v / v) alcohol for 10 minutes. The spleens were removed on a sterile operating table and placed on a cell sieve. The cells were thoroughly ground, passed through the sieve, and washed several times by centrifugation with sterile 1640 culture medium (purchased from Gibco). The cells were resuspended to make a single-cell suspension, counted, and set aside.

[0054] (2) Preparation of feeder cells

[0055] An 8- to 10-week-old female BALB / c mouse was eyeballed to obtain negative serum. The mouse was sacrificed by cervical dislocation and then immersed in 75% (v / v) alcohol for 10 minutes. The abdominal skin was aseptically opened to expose the peritoneum. Approximately 10 mL of 1640HT medium (purchased from SIGMA) was injected into the mouse's peritoneal cavity using a syringe. The abdomen was gently massaged and pipetted several times. The macrophage-containing medium was aspirated and injected into 20% 1640HAT medium for later use.

[0056] A 2- to 3-week-old female BALB / c mouse was sacrificed by cervical dislocation and then immersed in 75% (v / v) alcohol for 10 minutes. The thymus was aseptically removed and placed on a cell sieve, ground, and passed through the sieve to obtain thymocytes, which were placed in the above-mentioned 20% 1640HAT medium containing macrophages and set aside.

[0057] (3) Cell fusion

[0058] Select mouse myeloma cell line SP2 / 0 in the logarithmic growth phase, collect and count. 8 The above spleen cells were mixed with 2×10 7 Add the above SP2 / 0 cell lines to a fusion tube, mix, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and gently rub the tube back and forth on the palm of your hand to loosen the pellet. Add 1 mL of preheated PEG1450 (available from SIGMA) slowly, then rapidly, over 60 seconds. Stop the reaction by adding 30 mL of 1640HT medium. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, gently rub the pellet to loosen it, and add it to the 20% 1640HT medium obtained in step 2.

[0059] After thoroughly mixing the above HAT medium, aliquot 200 μL / well into a 96-well cell culture plate and culture in a cell culture incubator at 37°C and 5% CO2. After one week, replace the 20% 1640HAT medium with 10% 1640HT medium. After 3 days, collect the supernatant for analysis.

[0060] Step 3: Screening of anti-CD26 specific hybridoma cell lines

[0061] (1) Preparation of the test plate: Dilute the recombinant CD26 target protein to 1 μg / ml with CB coating solution, coat the 96-well ELISA plate at 100 μl / well, coat overnight at 2-8°C, wash once, and pat dry; block with PBST buffer containing 2% BSA (200 μl / well) at 37°C for 2 hours; pat dry and set aside.

[0062] (2) Screening of positive clones: 100 μl / well of the culture supernatant of the cells to be tested was added to the above-mentioned detection plate, incubated at 37°C for 30 minutes, washed and patted dry, 100 μl / well of diluted HRP-labeled goat anti-mouse IgG was added, incubated at 37°C for 30 minutes, washed and patted dry, 100 μl / well of TMB color development solution was added, incubated at 37°C in the dark for 15 minutes, 50 μl of 2M H2SO4 was added to each well to terminate the reaction, and the OD 450 Read the value at the positive well. 450 The positive value / negative control value is ≥ 2.1. Positive clones are selected for cell cloning screening. After three to four rounds of cloning screening, a single clone with a 100% positive rate is identified as a stable cell line and the cell line is then confirmed.

[0063] Step 4: Activity evaluation of CD26 mouse monoclonal antibody (mouse anti-79)

[0064] Tumor cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. Tumor cells were counted at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, and 3 replicates were set up for each group. 50 μl of anti-CD26 antibody with a final concentration of 100 ng / ml was added to the reaction wells of the corresponding sample wells, 50 μl of culture medium was added to the blank control wells, and 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells. The cells were incubated in a 37°C incubator for 30 min, and then 6×10 6 cells / ml of PBMC cells (peripheral blood mononuclear cells), the reaction system was incubated at 37 ° C under carbon dioxide culture for 5 hours. After the reaction was completed, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0065] Cell lysis rate formula = (V sample -V vehicle control ) / (V TritonX-100 -V vehicle control )×100%. Where V sample V is the average value of the fluorescence signal readings measured under the conditions of excitation light wavelength and emission light wavelength in the drug treatment group. vehicle control V is the average value of the fluorescence signal readings of the blank control group measured under the conditions of excitation light wavelength and emission light wavelength. TritonX-100 It is the average value of the fluorescence signal readings measured in the positive control group under the conditions of excitation light wavelength and emission light wavelength.

[0066] Table 2 Detection of cytotoxic effect of PBMC mediated by mouse antibody No. 79 on target cells

[0067] From the above results, it can be seen that under the condition of antibody concentration of 100 ng / ml and action for 5 h, the cell lysis rate of PBMC mediated by mouse antibody No. 79 on target cell 786-0 was 48.8% (the cell lysis rates of PBMC mediated by other mouse antibodies such as mouse antibodies No. 62, 72, 160, 51, and 212 on target cell 786-0 under the same conditions were 53.1%, 8.6%, 13.0%, 12.3%, and 11.3%, respectively). The cell lysis rate of PBMC mediated by mouse antibody No. 79 on target cell OS-RC-2 was 48.8%. The cell lysis rate of PBMC against target cells OS-RC-2 mediated by mouse antibody No. 79 was 34.4% (the cell lysis rates of PBMC against target cells OS-RC-2 mediated by other mouse antibodies such as mouse antibodies No. 47, 76, 62, and 82 under the same conditions were 15.4%, 19.9%, 49.9%, and 30.8%, respectively). The cell lysis rate of PBMC against target cells NCI-H226 mediated by mouse antibody No. 79 was 25.5% (the cell lysis rates of PBMC against target cells NCI-H226 mediated by other mouse antibodies such as mouse antibodies No. 47, 51, 62, and 82 under the same conditions were 6.7%, 21%, 14.3%, and 17.5%, respectively). Mouse antibody No. 79 has killing activity against different CD26-positive cell lines.

[0068] Example 3: Sequencing and screening of variable region sequences of hybridoma cell lines

[0069] Step 1. Extraction of total RNA from CD26 hybridoma cells

[0070] The hybridoma cell lines were passaged into T75 culture flasks and cultured until the cells reached approximately 90% confluence. The cells were then digested and collected by centrifugation. Total RNA was extracted from the monoclonal hybridoma cell lines using an RNA extraction kit (purchased from Roche). The first-strand cDNA was then reverse transcribed and amplified using a cDNA reverse transcription kit (purchased from Thermo) using the total RNA as a template. The reaction products were stored at -20°C. For long-term storage, they should be stored at -70°C.

[0071] Step 2. PCR amplification of heavy and light chain variable region genes

[0072] Using the first-strand cDNA from hybridoma cells as a template, a 50 μl reaction system was added with 1 μl of cDNA, 5 μl of 10× PCR buffer, 1 μl of each upstream and downstream primer (25 pmol), 1 μl of dNTPs, 1 μl of 25 mmol / L MgCl₂, and 39 μl of H₂O. The reaction was pre-denatured at 95°C for 10 min, followed by the addition of 1 μl of Taq enzyme and PCR amplification. The reaction conditions were 94°C, denaturation for 1 min, 58°C, annealing for 1 min, and extension at 72°C for 1.5 min for 30 cycles, followed by a 10-min incubation at 72°C. Five μl of the PCR product was subjected to electrophoresis on a 1.2% agarose gel.

[0073] Step 3. Cloning and sequencing of heavy and light chain variable region genes

[0074] According to the instructions of the pGM-T Fast ligation kit (Beijing Tiangen Biochemical Technology Co., Ltd., VT207-02), the heavy and light chain variable region genes were ligated with the pGM-T vector, transformed into Escherichia coli Top10 competent cells, screened by blue-white spots, and cultured at 37°C for 12-16 h.

[0075] The resulting white colonies were inoculated into 1-5 ml of LB medium containing ampicillin at a final concentration of 100 μM. After shaking at 37°C for 3-4 hours, clones containing the correct insertion sequence were screened by PCR. Positive clones were sequenced and the results were compared and analyzed using the IMGT online gene library to obtain the amino acid sequences of the antibody's heavy and light chain variable regions and CDR regions.

[0076] Mouse antibody No. 79 comprises the HCDR1 set forth in SEQ ID NO: 1, the HCDR2 set forth in SEQ ID NO: 2, and the HCDR3 set forth in SEQ ID NO: 3; the LCDR1 set forth in SEQ ID NO: 4, the LCDR2 with the sequence YRS, and the LCDR3 set forth in SEQ ID NO: 5. Its heavy chain variable region is set forth in SEQ ID NO: 6, and its light chain variable region is set forth in SEQ ID NO: 7.

[0077] Example 4: Construction of humanized bispecific antibodies

[0078] The amino acid sequences of the heavy and light chain variable regions of humanized mouse antibody No. 79 are: 19G300-VH (SEQ ID NO: 8), 19G300-VL (SEQ ID NO: 9).

[0079] The humanized heavy and light chain variable regions were connected via a linker peptide (SEQ ID NO: 10). A linker peptide (SEQ ID NO: 11) was added downstream and optimized according to the codon preference of mammalian CHO cells to obtain the optimized CD26 single-chain antibody gene sequence (SEQ ID NO: 12). An AvrII restriction site and a Kozak sequence were introduced upstream, and a CD3 single-chain antibody gene (SEQ ID NO: 13), a stop codon, and a BstZ17I restriction site were added downstream. The resulting humanized CD26-CD3 BiTE gene sequence was directly synthesized and constructed into the pUC57 plasmid, named pUC57-19G300.

[0080] The target gene was amplified, and the PCR product was recovered by 1% agarose gel electrophoresis. The PCR product and the pZHK2.0 vector were double-digested with AvrII and BstZ17I. The double-digested product was ligated into the pZHK2.0 vector with T4 ligase and transformed into Top10 competent cells. The cells were plated on LB plates containing kanamycin resistance and cultured overnight at 37°C. The next day, positive clones were screened and sequenced for complete consistency with the expected sequence, thus obtaining the humanized CD26-CD3 BiTE bispecific antibody expression plasmid.

[0081] A mammalian cell line stably expressing a highly expressed CD26-CD3 humanized BiTE bispecific antibody was inoculated in Dynamis medium and cultured in a fed-batch format at 37°C, 8% CO2, and 130 rpm. The supernatant was harvested and centrifuged at 12,000 rpm for 15 minutes. The supernatant was filtered through a 0.45 μm filter to obtain the treated supernatant, which was then purified by chromatography. The target product was approximately 55 kDa.

[0082] Example 5: Affinity Analysis of Humanized Bispecific Antibodies to CD26 and CD3 Proteins

[0083] 1. Affinity study with CD26 protein

[0084] First, the CD26 protein was biotinylated with EZ-link NHS-PEG12-Biotin; the biotinylated CD26 protein was immobilized using an SA biosensor and equilibrated in a 1× Kinetics solution; the protein was bound to a solution of the antibody to be detected at a concentration of 500 nM; and the protein was dissociated in a 1× Kinetics solution. Data were analyzed using Fortebio Data Analysis 8.0 software to calculate the affinity constant.

[0085] Table 3 Affinity of humanized bispecific antibody 19G300 for CD26 protein

[0086] 2. Affinity study of antibodies and CD3 protein

[0087] First, the CD3 protein was biotinylated with EZ-link NHS-PEG12-Biotin. The biotinylated CD3 protein was immobilized using an SA biosensor and equilibrated in a 1× Kinetics solution. The protein was then bound to a humanized bispecific antibody solution at a detection concentration of 500 nM and dissociated in a 1× Kinetics solution. Data were analyzed using Fortebio Data Analysis 8.0 software to calculate affinity constants.

[0088] Table 4 Affinity of humanized bispecific antibody 19G300 for CD3 protein

[0089] In summary, the affinity of 19G300 to CD26 protein is 1.14E-09M, and the affinity of 19G300 to CD3 protein is 1.61E-09M.

[0090] Example 6: Construction of a full-length humanized antibody

[0091] 21G430-431 is a full-length humanized anti-CD26 antibody derived from mouse antibody No. 79, with the heavy and light chain variable region amino acid sequences of 19G300-VH (SEQ ID NO: 8) and 19G300-VL (SEQ ID NO: 9); 21G428-429 is a full-length humanized anti-CD26 antibody derived from mouse antibody No. 62, with the heavy and light chain variable region amino acid sequences of 19G294-VH (SEQ ID NO: 17) and 19G294-VL (SEQ ID NO: 19); the heavy and light chain variable region amino acid sequences of YS110 are YS110-VH (SEQ ID NO: 18) and YS110-VL (SEQ ID NO: 20).

[0092] A signal peptide sequence (SEQ ID NO: 14) was added upstream of 19G300-VH (SEQ ID NO: 8), 19G294-VH (SEQ ID NO: 17), and YS110-VH (SEQ ID NO: 18), and a human IgG1 heavy chain constant region (SEQ ID NO: 15) was added downstream, respectively, to obtain the IgG1 heavy chain sequences of the full-length humanized anti-CD26 antibodies 21G430-431, 21G428-429, and YS110. The sequences were optimized according to the codon preference of mammalian cells (CHO) to obtain the optimized gene sequences. An AvrII restriction site and a Kozak sequence were introduced upstream, and a stop codon and a BstZ17I restriction site were added downstream. After direct synthesis, the sequences were constructed into the AvrII and BstZ17I restriction sites of the pCHO1.0 vector.

[0093] A signal peptide sequence (SEQ ID NO: 14) was added upstream of 19G300-VL (SEQ ID NO: 9), 19G294-VL (SEQ ID NO: 19), and YS110-VL (SEQ ID NO: 20), and a human kappa light chain constant region (SEQ ID NO: 16) was added downstream to obtain the IgG1 light chain sequences of the full-length humanized anti-CD26 antibodies 21G430-431, 21G428-429, and YS110. The sequences were optimized according to the codon preference of mammalian cells (CHO) to obtain the optimized gene sequences. An EcoRV restriction site and a Kozak sequence were introduced upstream, and a stop codon and a PacI restriction site were added downstream. The sequences were directly synthesized and constructed into the EcoRV and PacI restriction sites of the pZHK5.18 vector.

[0094] The pCHO1.0 vector comes from Thermo Corporation and has a puromycin resistance gene. The pZHK5.18 vector is a modified pCHO1.0 vector that replaces the puromycin resistance gene with the hygromycin resistance gene.

[0095] The heavy chain vector and the light chain vector were co-transfected into CHO-S cells. After double pressure screening with hygromycin and puromycin, a CHO cell line stably expressing the full-length antibody was obtained. The stably expressing CHO cell line was inoculated into Dynamis medium and cultured in a fed-batch manner at 37°C, 8% CO2, and 130 rpm.

[0096] The culture supernatant was harvested and centrifuged at 12,000 rpm for 15 minutes. The supernatant was then filtered through a 0.45 μm filter to obtain the treated culture supernatant. Purification was performed using Cytiva MabSelect Sure affinity chromatography medium. The purified product was adjusted to pH 5.5 and then refined using Cytiva SP HP cationic chromatography medium. The target product was approximately 150 kDa in size.

[0097] Example 6: Differences in binding ability of full-length humanized antibodies in mixed cell systems

[0098] 786-0 cells were collected and counted, and the cell density was adjusted to 6 × 10 5 PBMC cells were collected and counted, and the cell density was adjusted to 6×10 5 786-0 cells / mL. Add 50 μL each of 786-0 and PBMC cells to an EP tube to form a mixed cell system. Add antibody diluted to a final concentration of 0.3 μg / mL, mix thoroughly with the mixed cell system, and incubate at 4°C for 1 hour.

[0099] After incubation, cells were collected by centrifugation, washed twice with PBS, and resuspended in PBS. Goat anti-human IgG fluorescent secondary antibody was added, and cells were incubated at 4°C for 1 hour. Cells were then washed twice with PBS, resuspended in PBS, and analyzed using a BD Accuri C6 flow cytometer.

[0100] The test results show (Figure 1) that the full-length humanized anti-CD26 antibody 21G430-431 exhibited significantly higher binding levels in cancer cells 786-0 than the full-length humanized anti-CD26 antibody 21G428-429. Relatively speaking, 21G430-431 exhibited comparable binding levels to 21G428-429 in PBMC cells. On the one hand, this indicates that the full-length humanized antibodies 21G428-429 and 21G430-431 have better selectivity for recognizing the CD26 target on tumor cells than on PBMC cells, preferentially binding to the CD26 target on tumor cells and having extremely low binding to the CD26 target on PBMC cells, thereby avoiding killing normal cells. On the other hand, it indicates that the full-length humanized antibody 21G430-431 has higher tumor cell binding than 21G428-429.

[0101] Example 7: Comparison of internalization levels of full-length humanized antibodies

[0102] Antibody-drug conjugates (ADCs) are conjugated to cytotoxic molecules, combining the specificity of antibodies to recognize target cells with the powerful tumor-killing effect of toxins. The antibody portion should have: (1) high tumor tissue specificity and strong affinity / binding capacity to ensure that the ADC efficiently recognizes tumor cells and reduces off-target toxicity; (2) after binding to the target antigen, it can be effectively internalized into tumor cells and quickly release toxin molecules after entering endosomes or lysosomes, thereby exerting the small molecule cytotoxic effect.

[0103] To monitor antibody internalization into lysosomes, the antibodies were fluorescently labeled according to the pHrodo iFL Green STP ester instructions. This fluorescent label is pH-sensitive, significantly enhancing fluorescence at low pH. The pH of the lysosomal environment is approximately 5.0, significantly lower than the cytoplasmic pH of 7.2. Therefore, when the fluorescently labeled antibody enters tumor cells and is engulfed by lysosomes, its fluorescence increases significantly due to the significant change in pH from 7.4 to 5.0. This serves as an indicator of antibody internalization into lysosomes.

[0104] 786-0 / PBMC cells were collected and counted, and the cell density of both cells was adjusted to 2×10 5 Cells were collected at 400 μg / mL. 50 μL of cells were added to an EP tube as a mixed cell system. Fluorescently labeled antibodies were added, diluted to a final concentration of 5 μg / mL, mixed thoroughly, and incubated at 37°C for 24 hours. Another group of cells was collected without fluorescently labeled antibodies and mixed thoroughly before testing, designated as time 0. Before testing, the samples were centrifuged to remove the serum-containing culture medium and replaced with PBS. The mean fluorescence intensity was determined using a flow cytometer.

[0105] The test results showed (Figure 2) that the anti-CD26 full-length humanized antibody 21G430-431 exhibited significantly higher internalization levels in cancer cells 786-0 than the anti-CD26 full-length humanized antibody 21G428-429, indicating that 21G430-431 is more suitable as an antibody in ADC molecules than 21G428-429.

[0106] In addition, to avoid off-target effects of ADC molecules, low internalization levels in non-effector cells can help reduce the risk of side effects. As shown in Figure 3, anti-CD26 antibodies exhibit lower internalization levels in PBMC cells than in cancer cells. Comparing the difference in mean fluorescence intensity after 24 hours of internalization with that at 0 hours, 21G430-431 exhibits slightly lower internalization levels in PBMC cells than 21G428-429.

[0107] As shown in Figure 4, PBMCs from three different individuals were selected for internalization experiments. In the PBMCs from different individuals, 21G430-431 showed a lower internalization level than 21G428-429.

[0108] Example 8: Killing of cancer cells by anti-CD26 antibody coupled with biotoxin

[0109] As shown in Examples 7 and 8, 21G430-431 exhibited superior affinity, binding, and internalization levels to 21G428-429, suggesting its potential as the antibody component of an ADC. Therefore, in this example, DT3C (diphtheria toxin), a protein that binds to the Fc terminus of an antibody and, after internalization, is hydrolyzed by intracellular proteases to release diphtheria toxin, was used as a tool. This protein was incubated with the anti-CD26 antibodies 21G430-431, 21G428-429, and YS110 to construct simple antibody-drug conjugates, preliminarily verifying the toxicity of the antibody as a drug conjugate.

[0110] DT3C and each antibody molecule were added to the culture medium to prepare a solution of 40ug / mL DT3C and 20ug / mL antibody. After mixing, the mixture was placed in an incubator and incubated for one hour. During the incubation period, 786-0 cells and UT16 cells were collected and counted, and the cell density was adjusted to 5×10 4 / mL. Add cells to a 96-well plate at a volume of 50 microliters per well. After the incubation of 40ug / mL DT3C and 20ug / mL antibody solution is completed, add 50uL per well to the well plate and continue incubating at 37°C for 72h. After the incubation is completed, add 10uL of CCK8 solution to each well, place it in an incubator and incubate for one hour, and use an enzyme reader to detect the absorbance at 450nm. The absorbance of each sample needs to be deducted from the background signal in the culture medium without cells. The cell survival rate is calculated using the group without antibody as the control group. The calculation formula is as follows: (A Sample -A Medium ) / A Blank (where A Sample The absorbance at 450 nm of the wells incubated with samples is A Medium A is the absorbance of the pure culture solution at 450 nm. Blank Absorbance at 450 nm in the wells containing the antibody solution

[0111] The test results show (Figures 5 and 6) that the three anti-CD26 antibody-drug conjugates all exhibited a certain ability to kill target cells. DT3C-conjugated 21G430-431 showed superior killing ability to DT3C-conjugated 21G428-429, with significant differences. This indicates that antibodies with higher cell affinity, binding affinity, and stronger internalization levels showed higher levels of killing target cells after being conjugated to toxin molecules. In this case, YS110, which has progressed to clinical phase II, was also used as a control antibody. It can be seen that 21G430-431 exhibited a stronger ADC effect than YS110, with significant differences.

[0112] Based on the results of the above examples, the anti-CD26 antibody derived from mouse antibody No. 79 has high specificity, affinity, and binding ability for CD26-positive tumor cells, and the full-length humanized antibody derived from mouse antibody No. 79 has higher affinity and binding ability for tumor cells, higher tumor cell internalization level, and lower non-tumor cell internalization level than other full-length humanized antibodies derived from mouse antibodies, thereby showing better efficacy and safety in ADC molecules and can be used as the antibody portion of ADC molecules.

Claims

1. An antibody or antigen-binding fragment that specifically binds to human CD26, comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and LCDR1 as shown in SEQ ID NO: 4, LCDR2 with a sequence of YRS, and LCDR3 as shown in SEQ ID NO:

5.

2. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

9.

3. The antibody or antigen-binding fragment of claim 2, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids in the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 are inserted, deleted or substituted, and the amino acid substitutions are conservative amino acid substitutions.

4. The antibody or antigen-binding fragment of claim 2, comprising a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO:

9.

5. An antibody-drug conjugate targeting CD26, comprising the anti-CD26 antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. A nucleotide sequence encoding the amino acid sequence of the antibody or antigen-binding fragment of any one of claims 1 to 4.

7. A vector comprising the nucleotide sequence of claim 6.

8. A host cell comprising the vector of claim 7.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 4 10. A method for treating a tumor that highly expresses CD26, comprising administering to a patient an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 4, or the antibody-drug conjugate of claim 6, wherein the tumor highly expresses CD26, including but not limited to renal cancer, mesothelioma, lung cancer, liver cancer, prostate cancer, etc.

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