An antibody against gp350 and its preparation method and application

Abstract

The application provides an antibody against gp350 and a preparation method and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1-3, SEQ ID No. 13-15 or SEQ ID No. 25-27, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4-6, SEQ ID No. 16-18 or SEQ ID No. 28-30. The anti-gp350 antibody screened in the application can specifically combine with the EBV virus envelope glycoprotein gp350 antigen, and the affinity is between 10 ‑9 ~ 10 ‑ 7 M orders of magnitude. The antibody of the application can be used to prepare a medicine for preventing and / or treating tumors or autoimmune diseases, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-gp350 antibody, its preparation method, and its application. Background Technology

[0002] Epstein-Barr virus (EBV) is a major member of the human herpesvirus family, infecting nearly 90% of adults worldwide. Although most infected individuals present with asymptomatic or mild infectious mononucleosis, EBV infection is closely associated with the development of various malignant diseases, such as nasopharyngeal carcinoma (NPC), Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and several immunodeficiency-related lymphoproliferative disorders. Due to the severity of these diseases and the widespread global prevalence of EBV, the treatment and prevention of EBV have become a crucial focus of current medical research.

[0003] gp350 is the most abundant glycoprotein on the outer membrane of EBV, accounting for more than 35% of EBV surface proteins. gp350 mediates viral entry into host B cells by binding to the host cell's complement receptor CR2 / CD21, making it an indispensable target in the EBV infection process. Studies have shown that gp350 possesses good antigenicity and can induce the production of highly efficient neutralizing antibodies in vivo, thereby inhibiting the viral invasion process. Therefore, gp350 is not only an important candidate molecule for vaccine development but also an ideal target for passive immunotherapy. Although research targeting gp350 has made some progress, existing therapeutic strategies targeting gp350 still have significant limitations. For example, while existing vaccines can induce antibody production, their neutralization efficiency is not ideal; some anti-gp350 antibodies can inhibit viral entry into host cells, but their in vivo stability and affinity still need optimization. Furthermore, the application of gp350 as a target is mainly focused on passive immunotherapy, with relatively little exploration of genetically engineered immunocellular therapies. Therefore, further research and development of therapeutic molecules targeting gp350, especially in combination with novel cell therapy strategies, is of significant medical importance and clinical value.

[0004] Chimeric antigen receptor T-cell (CAR-T) therapy is an immunotherapy that uses genetic engineering to modify T cells to specifically recognize and attack tumors or infectious pathogens. CAR-T therapy has achieved significant efficacy in treating hematologic malignancies such as B-cell lymphoma. However, traditional autologous CAR-T therapy suffers from long production cycles, high costs, and requires customized production for each patient, limiting its widespread application. Universal CAR-T technology uses CRISPR gene editing to remove TRAC and B2M molecules from CAR-T cells, avoiding the host's immune rejection of allogeneic cells, thus enabling "off-the-shelf" production. This technology not only reduces production costs but also significantly shortens treatment time, providing more patients with the possibility of timely treatment. The core idea of ​​universal CAR-T cell therapy targeting gp350 is to use a high-affinity anti-gp350 single-chain antibody (scFv) as the recognition domain of the CAR molecule, enabling universal CAR-T cells to specifically recognize and attack infected or tumor cells expressing gp350. This universal CAR-T cell does not require matching with the patient's own cells and can be rapidly deployed as an "out-of-the-box" cell therapy.

[0005] Therefore, there is an urgent need to develop an anti-gp350 antibody with high affinity, high specificity, and good in vivo stability, and to explore its application in various therapeutic approaches, including universal CAR-T therapy, to meet clinical needs. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to provide an anti-gp350 antibody, its preparation method and application.

[0007] The present invention provides an antibody or antigen-binding fragment against gp350, the antibody or antigen-binding fragment against gp350 comprising a heavy chain variable region and a light chain variable region;

[0008] The heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment includes amino acid sequences such as HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 1 to 3, and the light chain variable region includes amino acid sequences such as LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No. 4 to 6.

[0009] Alternatively, the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment may include amino acid sequences such as HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 13-15, and the light chain variable region may include amino acid sequences such as LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No. 16-18.

[0010] Alternatively, the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment may include amino acid sequences such as HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No. 25-27, and the light chain variable region may include amino acid sequences such as LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No. 28-30.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 9;

[0012] Alternatively, the amino acid sequence of the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment is as shown in SEQ ID No. 19, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 21;

[0013] Alternatively, the amino acid sequence of the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment is shown in SEQ ID No. 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 33.

[0014] Furthermore, preferably, the antibody or antigen-binding fragment further includes a backbone region and a constant region;

[0015] The constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; preferably, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese or humans.

[0016] Furthermore, the structure of the heavy chain variable region is: skeleton region-HCDR1-skeleton region-HCDR2-skeleton region-HCDR3-skeleton region.

[0017] Further, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, heavy chain antibodies, humanized antibodies, single-chain antibodies, and full-length antibodies; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; more preferably, the antibody is a single-chain antibody, and the amino acid sequence of the single-chain antibody is shown in SEQ ID No. 11, SEQ ID No. 23, or SEQ ID No. 35;

[0018] The antigen-binding fragment is any one of F(ab')2, Fab', Fab, and Fv.

[0019] Furthermore, the aforementioned antigen-binding fragments, i.e., the functional fragments of the antibody, typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the aforementioned functional fragments of the antibody can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0020] The aforementioned antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0021] F(ab')2, Fab', Fab, and Fv are different antigen-binding fragments of the antibody. The F(ab')2 fragment is obtained by digesting IgG antibody with pepsin, contains two antigen-binding regions (Fab), linked by disulfide bonds in the hinge region, and has a molecular weight of approximately 110 kDa.

[0022] The Fab' fragment is obtained by reducing the F(ab')2 fragment and contains an antigen-binding region (Fab) and a partial hinge region, with a molecular weight of approximately 55 kDa.

[0023] The Fab fragment was obtained by digesting IgG antibodies with papain. It contains a complete light chain (variable and constant regions) and a partial heavy chain (variable and a constant region), with a molecular weight of approximately 47–48 kDa.

[0024] The Fv fragment is the smallest antigen-binding fragment of an antibody, containing only the variable regions (VH and VL) of the antibody, which bind through non-covalent interactions, and has a molecular weight of approximately 25 kDa.

[0025] The present invention also provides a method for preparing the above-mentioned anti-gp350 antibody or antigen-binding fragment, the method comprising the following steps: transducing a recombinant vector containing the above-mentioned anti-gp350 antibody or antigen-binding fragment into host cells by transfection or infection, culturing, and purifying to obtain the antibody or antigen-binding fragment.

[0026] The present invention also provides a recombinant chimeric antibody or recombinant protein, wherein the antigen-binding domain of the recombinant chimeric antibody or recombinant protein includes the above-mentioned anti-gp350 antibody or antigen-binding fragment; preferably, it further includes a signal peptide, a hinge region, a transmembrane region and a signal transduction domain.

[0027] More preferably, the recombinant protein targets CAR-T cells, the CAR-T cells including at least one of universal CAR-T cells and autologous CAR-T cells.

[0028] The "chimeric antibody" described in this invention is an antibody formed by fusing the variable region of a non-human antibody with the constant region or backbone region of a human antibody, which can reduce the immune response induced by non-human antibodies.

[0029] Furthermore, the signal transduction domain includes CD3ζ; preferably, the signal transduction domain further includes a 4-1BB intracellular region.

[0030] The present invention also provides a gene or gene fragment encoding the above-mentioned anti-gp350 antibody or antigen-binding fragment, the above-mentioned recombinant chimeric antibody or recombinant protein; preferably, the nucleotide sequence of the gene or gene fragment is as shown in SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 32, SEQ ID NO. 34 or SEQ ID NO. 36.

[0031] The present invention also provides a pharmaceutical composition comprising at least one of the above-mentioned anti-gp350 antibody or antigen-binding fragment, the above-mentioned recombinant chimeric antibody or recombinant protein, and further comprising other active agents; the other active agents include at least one of immune checkpoint-related agents, antibody-drug conjugates, bispecific antibodies, multispecific antibodies, radionuclides or kinase inhibitors.

[0032] Furthermore, the other therapeutic agents include at least one of chemotherapeutic drugs, radionuclides, photosensitizers, photothermal agents, immune checkpoint inhibitors, toxins, factors, kinase inhibitors, antibodies that inhibit second signaling molecules, PD-L1 inhibitors, and PD-1 / PD-L1 monoclonal antibody drugs.

[0033] The present invention also provides the use of the above-mentioned anti-gp350 antibody or antigen-binding fragment, the above-mentioned recombinant chimeric antibody or recombinant protein, the above-mentioned gene or gene fragment, and the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention or / or treatment of tumors or autoimmune diseases.

[0034] Furthermore, the tumor is EBV-positive nasopharyngeal carcinoma, gastric carcinoma, Burkitt lymphoma, or Hodgkin lymphoma;

[0035] The autoimmune disease is an inflammatory autoimmune disease caused by EBV infection, preferably multiple sclerosis.

[0036] Furthermore, the drug may be in the following forms: immune cells, reagents, kits, or compositions.

[0037] The present invention has achieved the following beneficial effects:

[0038] This invention screened three anti-gp350 antibodies from mouse hybridomas, which can specifically bind to the EBV viral envelope glycoprotein gp350 antigen, with antibody affinity of 10. -9 ~10 -7 These are all high-affinity antibodies, ranging from the M order of magnitude. Furthermore, this invention utilizes anti-gp350 antibodies to construct universal CAR-T (UCAR-T) cells targeting gp350. These cells all exhibit cytotoxic activity against target cells, among which gp350UCAR-T (19 # The antibody exhibits the strongest cytotoxic activity. This invention's antibody can be formulated into a drug for the prevention and / or treatment of tumors or autoimmune diseases, and has broad application prospects in the fields of tumor or autoimmune disease prevention and treatment.

[0039] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0040] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0041] Figure 1 To identify hybridoma-positive clones using ELISA.

[0042] Figure 2 The purified recombinant chimeric antibody was identified by SDS-PAGE.

[0043] Figure 3 To identify the reactivity of recombinant chimeric antibodies with gp350 antigen using ELISA.

[0044] Figure 4 For recombinant single-chain antibodies (gp350-8#-scFv, gp350-19#-scFv, gp350-35) # -scFv) and gp350 + - HeLa cell-specific binding identification.

[0045] Figure 5 To detect the efficiency of CRISPR Cas9 / RNPs electroporation knockout of TRAC and B2M on the surface of T cells by flow cytometry.

[0046] Figure 6 To detect gp350-targeting UCAR-T cells (8) using RTCA. # UCAR-T, 19 #UCAR-T, 35 # UCAR-T) for gp350 + -HeLa's lethal effect.

[0047] Figure 7 To evaluate gp350 UCAR-T(19) using an immune-reconstituted mouse xenograft model # In vivo antitumor activity of cells: (A) represents the change in tumor volume in NCG mice; (B) represents the in vivo antitumor activity of gp350UCAR-T (19 # The tumor inhibition rate. Detailed Implementation

[0048] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0049] In this invention, "overnight" refers to a reaction time of 12 ± 5 hours.

[0050] The method for preparing gp350 antigen according to the present invention is as follows:

[0051] Using the gp350 gene as a template, the dominant epitope region containing amino acid Met1-Pro490 of the gp350 gene was selected as the immune targeting fragment and cloned into the pFastBacHT B expression vector carrying a His tag at the C-terminus. Then, it was expressed using an insect baculovirus expression system, and the recombinant gp350 protein was subsequently purified using the His tag. The purified recombinant protein is the gp350 antigen.

[0052] Example 1: Mouse B cell fusion and hybridoma screening

[0053] 1. Animal immunization

[0054] Five- to six-week-old female Balb / c mice were used as immunization animals, with an immunization dose of 100 μg per mouse. For the initial immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant gp350 protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Two weeks later, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, thoroughly emulsified, and then injected subcutaneously at multiple sites for booster immunization. A total of four booster immunizations were administered. On day 10 after the last booster immunization, blood samples were collected to measure antibody titers. Three days before cell fusion, mice were administered a 100 μg recombinant protein intraperitoneal pulse.

[0055] 2. Cell fusion and hybridoma screening

[0056] Under aseptic conditions, the spleens of mice after immunization were harvested, and a suspension rich in B cells was prepared. Cells were then fused with SP2 / 0 cells using the classic PEG (Sigma) method (Greenfield, EA (2018). Polyethylene Glycol Fusion for Hybridoma Production. Cold Spring Harbor Protocols, 2018(3), pdb-prot103176.). The fused cells were resuspended in HAT medium for culture. On days 5 and 10 post-fusion, the medium was partially replaced with fresh HAT medium. ELISA, immunofluorescence, and flow cytometry were performed from days 11 to 15 post-fusion to screen for positive clones.

[0057] The specific steps are as follows: ELISA screening was performed using a 96-well plate. The recombinant gp350 protein was coated onto the bottom of the well at a rate of 100 ng / well and incubated overnight at 4°C. The plate was then developed using HRP-conjugated anti-mouse IgG antibody and chemiluminescent reagent (Beyotime Biotechnology Co., Ltd.), and the values ​​were read at a wavelength of 450 nm using an ELISA reader.

[0058] Based on the above ELISA analysis results, three positive hybridoma clones were finally identified (named 8 respectively). # 19 # 35 # ()( Figure 1 (This information is used for subsequent experiments such as sequence cloning and affinity analysis.)

[0059] 3. Sequencing analysis of specific antibodies

[0060] Hybridoma antibody variable region sequence cloning: The three hybridoma clones in logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara). The resulting cDNA was amplified by PCR using mouse Ig-Primer Set (Novagen) and then sequenced to obtain the variable region sequences of the heavy and light chains. The CDR sequences of the variable regions of the heavy and light chains are shown in Table 1.

[0061] Table 1. CDR sequences and single-chain antibody sequences (scFv) contained in the heavy and light chain variable regions of mouse monoclonal antibodies.

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Example 2: Preparation of gp350 recombinant chimeric antibody

[0068] A fusion protein expression platform was constructed based on the pcDNA3.1 eukaryotic expression vector to express fusion proteins from hybridoma clones 8. # The light and heavy chain sequences (nucleotide sequences as shown in SEQ ID NO. 12) were cloned into the pcDNA3.1 vector. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system to obtain the gp350 recombinant chimeric antibody (denoted as gp350-8). # ).

[0069] Example 3: Preparation of gp350 recombinant chimeric antibody

[0070] Referring to the method of Example 2, the only difference is that 8 # The light chain and heavy chain sequences were replaced with 19 # The light and heavy chain sequences (nucleotide sequences as shown in SEQ ID NO. 24) were used to obtain the gp350 recombinant chimeric antibody (denoted as gp350-19). # ).

[0071] Example 4: Preparation of gp350 recombinant chimeric antibody

[0072] Referring to the method of Example 2, the only difference is that 8 # The light chain and heavy chain sequences were replaced with 35. # The light chain and heavy chain sequences (nucleotide sequences as shown in SEQ ID NO. 36) were used to obtain the gp350 recombinant chimeric antibody (denoted as gp350-35). # ).

[0073] Example 5: Preparation of recombinant single-chain antibodies

[0074] A single-chain antibody (scFv) expression platform was constructed based on a eukaryotic expression vector, and hybridoma clone 8 was used to express the antibody. # The VH and VL gene fragments (nucleotide sequences shown in SEQ ID NO. 8 and SEQ ID NO. 10) were tandemly constructed into the pcDNA3.1 vector using a (G4S)3-linker. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system to obtain a recombinant single-chain antibody (denoted as gp350-8). # -scFv). gp350-8 # The amino acid sequence of -scFv is shown in SEQ ID NO.11.

[0075] Example 6: Preparation of recombinant single-chain antibodies

[0076] Referring to the method of Example 5, the only difference is that 8 # The VH and VL gene segments were replaced with 19 # The VH and VL gene fragments (nucleotide sequences shown in SEQ ID NO.20 and SEQ ID NO.22) were used to obtain a recombinant single-chain antibody (denoted as gp350-19). # -scFv). gp350-19 # The amino acid sequence of -scFv is shown in SEQ ID NO.23.

[0077] Example 7: Preparation of recombinant single-chain antibodies

[0078] Referring to the method of Example 5, the only difference is that 8 # The VH and VL gene segments were replaced with 35 # The VH and VL gene fragments (nucleotide sequences shown in SEQ ID NO.32 and SEQ ID NO.34) were used to obtain a recombinant single-chain antibody (denoted as gp350-35). # -scFv). gp350-35 # The amino acid sequence of -scFv is shown in SEQ ID NO.35.

[0079] Example 8: Construction of universal CAR-T (UCAR-T) cells targeting gp350

[0080] The single-chain antibody gp350-8 from Example 5 was selected. # -scFv constructs a CAR with the following structure: CD8αsignal-peptide-scFv(gp350)-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EG FP (where scFv(gp350) in the structure is gp350-8) # HEK293T cells were used as the cells for lentiviral packaging. Lentiviral packaging was performed using a three-plasmid packaging system (psPAX2, pMD2.G, CAR-T vector). After 48 hours, the supernatant viral solution was collected, concentrated by ultracentrifugation, pre-coated with RetroNectin protein, and then added to infect T cells. After 48 hours of infection, the transfection efficiency of CAR-T cells was assessed by flow cytometry and found to be above 80.0%.

[0081] Using CRISPR-Cas9 / RNP electroporation, sgRNA targeting TRAC and B2M, along with Cas9 protein, were electroporated into CAR-T cells to prepare universal gp350-targeting UCAR-T cells. After 72 hours of expansion, flow cytometry was used to detect the expression of TCR and β-2-Microglobulin in CAR-T cells. Flow cytometry results showed that 60.9% of T cells had TRAC gene knockout, while 57.6% had B2M knockout. Figure 5 Subsequently, TRAC was obtained through magnetic bead sorting. - / B2M - Negative CAR-T cells, denoted as 8 # UCAR-T (also known as gp350 UCAR-T (8) # )).

[0082] Example 9: Construction of universal CAR-T (UCAR-T) cells targeting gp350

[0083] The method described in Example 8 is the same, except that the single-chain antibody gp350-8 is used instead. # Replace -scFv with single-chain antibody gp350-19 # -scFv, to get TRAC - / B2M - Negative CAR-T cells, designated as 19 # UCAR-T (also known as gp350 UCAR-T (19) # )).

[0084] Example 10: Construction of universal CAR-T (UCAR-T) cells targeting gp350

[0085] The method described in Example 8 is the same, except that the single-chain antibody gp350-8 is used instead. # Replace -scFv with single-chain antibody gp350-35 # -scFv, to get TRAC - / B2M - Negative CAR-T cells, denoted as 35 # UCAR-T (also known as gp350 UCAR-T (35) # )).

[0086] The following experimental examples demonstrate the beneficial effects of the present invention.

[0087] Experimental Example 1: Determination of the reactivity and affinity of gp350 recombinant chimeric antibody with antigen

[0088] 1. Experimental Methods

[0089] To assess the reactivity of the recombinant chimeric antibody with the antigen, 200 ng / well of gp350 recombinant protein was pre-coated onto an ELISA plate, incubated overnight at 4°C, and then the plate was blocked. Different amounts of recombinant chimeric antibody (dilution: 10) were then added. -5 ~10 2 The sample was washed with secondary antibody (μg / mL), the color was developed, the reaction was terminated, and the optical density (OD450) at 450 nm was measured using an ELISA reader. The binding capacity was determined by fitting a four-parameter nonlinear regression curve.

[0090] The three recombinant chimeric antibodies (gp350-8) of gp350 were validated by surface plasmon resonance. # GP350-19 # GP350-35 # Affinity was determined, and its binding kinetic constant (Kd) was determined.

[0091] 2. Experimental Results

[0092] SDS-PAGE results showed that the gp350 recombinant chimeric antibody with high purity was obtained after affinity chromatography purification, and the band sizes all met the expected requirements. Figure 2 Furthermore, all three recombinant chimeric antibodies against gp350 showed high specificity in binding to the recombinant gp350 protein. Figure 3 ).

[0093] Affinity assay results showed that all three recombinant chimeric antibodies against gp350 could specifically bind to the gp350 antigen, with an affinity of 10. -9 ~10 -7 All three recombinant chimeric antibodies are in the order of M and belong to high-affinity antibodies. Kinetic characteristics show that all three have relatively slow dissociation rates. Specific detection data are shown in Table 2.

[0094] Table 2 Summary of affinity data for gp350 recombinant chimeric antibodies

[0095] Recombinant chimeric antibody Ka(1 / Ms) kd(1 / s) KD(M) <![CDATA[gp350-8 # ]]> <![CDATA[2.57×10 5 ]]> <![CDATA[5.22×10 -2 ]]> <![CDATA[2.03×10 -7 ]]> <![CDATA[gp350-19 # ]]> <![CDATA[6.35×10 6 ]]> <![CDATA[9.26×10 -3 ]]> <![CDATA[1.46×10 -9 ]]> <![CDATA[gp350-35 # ]]> <![CDATA[3.69×10 6 ]]> <![CDATA[5.12×10 -2 ]]> <![CDATA[1.39×10 -8 ]]>

[0096] Experiment Example 2: Verification of the reactivity of recombinant single-chain antibody with antigen

[0097] 1. Experimental Methods

[0098] To identify recombinant single-chain antibodies (gp350-8#-scFv, gp350-19#-scFv, gp350-35) # The reactivity of -scFv with the antigen, pre-emptively adding gp350 +HeLa cells were seeded in 96-well plates and cultured overnight. The next day, purified recombinant single-chain antibodies (gp350-8#-scFv, gp350-19#-scFv, gp350-35#-scFv) were added to each well. # After incubating with 594 fluorescently labeled goat anti-human secondary antibody for 1 hour, the mixture was washed with PBS and incubated for another hour. After washing, the results were observed using a fluorescence microscope.

[0099] 2. Experimental Results

[0100] The results showed that three recombinant single-chain antibodies against gp350 (gp350-8#-scFv, gp350-19#-scFv, gp350-35#-scFv, gp350-35#-scFv) were effective against gp350. # -scFv) and gp350 + -HeLa both showed good specific binding ( Figure 4 ).

[0101] Experiment Example 3: Evaluation of the in vitro killing effect of gp350-targeting UCAR-T cells on target cells.

[0102] 1. Experimental Methods

[0103] gp350 + C666-1 cells (nasopharyngeal carcinoma cells) were added at a density of 5000 cells per well and 100 μL per well (replicas) to both a label-free killer detection instrument (RTCA) 96-well plate and a regular 96-well cell culture plate. The RTCA 96-well plate was placed in the instrument and cultured until the Cell Index value was between 1.0 and 2.0. Then, the prepared gp350-targeted UCAR-T cells were co-incubated at an effector-to-target ratio of 2:1. The RTCA monitoring time was 65 h to evaluate their killing activity.

[0104] 2. Experimental Results

[0105] The results are as follows Figure 6 As shown, three types of UCAR-T cells targeting gp350 (8 # UCAR-T, 19 # UCAR-T, 35 # UCAR-T cells showed cytotoxic activity against C666-1 cells, and 19 # UCAR-T has the strongest lethality.

[0106] Experiment Example 4: Evaluation of the in vivo antitumor activity of gp350-targeting UCAR-T cells using an immunodeficient mouse xenograft model.

[0107] 1. Experimental Methods

[0108] The in vivo antitumor activity of gp350-targeting UCAR-T cells was evaluated using a xenograft mouse model. An immunodeficient (NCG) mouse model of EBV-positive nasopharyngeal carcinoma cells was established using the C666-1 cell line for evaluation.

[0109] 1×10⁻⁶ mice were subcutaneously injected into the right hind limb of NCG mice. 6 A mouse EBV-positive nasopharyngeal carcinoma xenograft tumor model was established using C666-1-mCherry.ffLuc cells to verify the optimal in vitro killing activity of gp350 UCAR-T(19) cells. # The in vivo efficacy of NCG was assessed. On day 5 post-vaccination, NCG mice were randomly divided into three groups of five, and each group was injected with 1×10⁻⁶ NCG via the tail vein. 7 Mock-T cells, 1×10 7 One GP350 UCAR-T (19 # Mice were divided into groups and a control group was set up that received PBS injections. The growth and survival of mice were observed daily, and the mice were weighed and tumor size measured every 3 days. The average tumor volume was calculated using the formula V = 1 / 2(L × W). 2 The calculation is performed, where L represents the length of the tumor and W represents the width of the tumor. When the mouse tumor volume reaches 1500 mm²... 3 If obvious ulceration occurs on the tumor surface, the mouse is euthanized, and the animal experiment ends. The gp350 UCAR-T(19) tumor volume is calculated based on the tumor volume of the experimental and control groups. # The tumor inhibition rate is calculated using the following formula: Tumor inhibition rate (%) = [1 - (tumor volume in the experimental group / tumor volume in the control group)] × 100%.

[0110] 2. Experimental Results

[0111] The results are as follows Figure 7 As shown in A and 7B, compared with the control group, gp350 UCAR-T (19 # The tumor volume in the ) group was significantly smaller than that in the control group (PBS and Mock-T), confirming that gp350 UCAR-T (19 # The drug significantly inhibited the growth of C666-1 cells. Data analysis was performed using one-way ANOVA. ns indicates no statistically significant difference, and **** indicates P < 0.0001. Figure 7 ).

[0112] In summary, this invention provides an anti-gp350 antibody, its preparation method, and its application. The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1–3, SEQ ID No. 13–15, or SEQ ID No. 25–27, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4–6, SEQ ID No. 16–18, or SEQ ID No. 28–30. The anti-gp350 antibody screened by this invention can specifically bind to the EBV viral envelope glycoprotein gp350 antigen, with an affinity of 10. -9 ~10 -7 The antibodies of this invention are on the order of M. They can be formulated into drugs for the prevention and / or treatment of tumors or autoimmune diseases, and have broad application prospects.

Claims

1. An antibody or antigen-binding fragment against gp350, characterized in that, Anti-gp350 antibodies or antigen-binding fragments include heavy chain variable regions and light chain variable regions; The heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment includes amino acid sequences such as HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 1 to 3, and the light chain variable region includes amino acid sequences such as LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No. 4 to 6. Alternatively, the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment may include amino acid sequences such as HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 13-15, and the light chain variable region may include amino acid sequences such as LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No. 16-18. Alternatively, the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment may include amino acid sequences such as HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No. 25–27, and the light chain variable region may include amino acid sequences such as LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No. 28–30.

2. The antibody or antigen-binding fragment against gp350 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

9. Alternatively, the amino acid sequence of the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment is as shown in SEQ ID No. 19, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 21; Alternatively, the amino acid sequence of the heavy chain variable region of the anti-gp350 antibody or antigen-binding fragment is shown in SEQ ID No. 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

33.

3. The antibody or antigen-binding fragment against gp350 according to claim 1, characterized in that, Preferably, the antibody or antigen-binding fragment further includes a backbone region and a constant region; The constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; preferably, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese or humans.

4. The antibody or antigen-binding fragment against gp350 according to claim 1, characterized in that, The antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, heavy chain antibodies, humanized antibodies, single-chain antibodies, and full-length antibodies; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; more preferably, the antibody is a single-chain antibody, and the amino acid sequence of the single-chain antibody is shown in SEQ ID No. 11, SEQ ID No. 23, or SEQ ID No.

35. The antigen-binding fragment is any one of F(ab')2, Fab', Fab, and Fv.

5. A method for preparing the anti-gp350 antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The method includes the following steps: transducing a recombinant vector containing an antibody or antigen-binding fragment against gp350 as described in any one of claims 1 to 4 into host cells via transfection or infection, culturing, and purifying to obtain the desired result.

6. A recombinant chimeric antibody or recombinant protein, wherein the antigen-binding domain of the recombinant chimeric antibody or recombinant protein comprises the anti-gp350 antibody or antigen-binding fragment as described in any one of claims 1 to 4; Preferably, it also includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain; More preferably, the recombinant protein targets CAR-T cells, the CAR-T cells including at least one of universal CAR-T cells and autologous CAR-T cells.

7. A gene or gene fragment, characterized in that, It encodes the antibody or antigen-binding fragment against gp350 as described in any one of claims 1 to 4, or the recombinant chimeric antibody or recombinant protein as described in claim 6; preferably, the nucleotide sequence of the gene or gene fragment is as shown in SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 32, SEQ ID NO. 34 or SEQ ID NO.

36.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the anti-gp350 antibody or antigen-binding fragment of any one of claims 1 to 4, the recombinant chimeric antibody or recombinant protein of claim 6, and further comprises other active agents; the other active agents include at least one of immune checkpoint-related agents, antibody-drug conjugates, bispecific antibodies, multispecific antibodies, radionuclides or kinase inhibitors.

9. Use of the antibody or antigen-binding fragment against gp350 according to any one of claims 1 to 4, the recombinant chimeric antibody or recombinant protein according to claim 6, the gene or gene fragment according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the prevention or / or treatment of tumors or autoimmune diseases.

10. The use according to claim 9, characterized in that, The tumor is an EBV-positive nasopharyngeal carcinoma, gastric carcinoma, Burkitt lymphoma, or Hodgkin lymphoma. The autoimmune disease is an inflammatory autoimmune disease caused by EBV infection, preferably multiple sclerosis.

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