Anti-alpp / alppl2 antibodies and uses thereof

Abstract

The application provides an anti-ALPP / ALPPL2 antibody and application thereof, and belongs to the field of biological medicine. Specifically, the application discloses an antibody or antigen binding fragment thereof specifically combined with ALPP / ALPPL2, which comprises heavy chain variable region CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 3, 4 and 5, and light chain variable region CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, 7 and 8. The antibody has high specificity, high sensitivity and can mediate intracellular endocytosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-ALPP-ALPPL2 antibody, its application, and its use in the preparation of drugs for the treatment and / or prevention of tumors. Background Technology

[0002] ALPP (placental alkaline phosphatase) and ALPPL2 (placental-like alkaline phosphatase 2) are two closely related paralogous genes in the alkaline phosphatase family. Both are glycosylphosphatidylinositol (GPI)-anchored membrane-bound proteins involved in the recirculation of ATP from the extracellular space. The ALPP gene is located on chromosome 2q37.1, and ALPPL2 is highly homologous to it. In normal tissues, the expression of ALPP and ALPPL2 is highly restricted, mainly limited to placental trophoblast cells. However, both are abnormally overexpressed in various solid tumors. Studies have shown that ALPP is a marker of poor prognosis in pancreatic cancer, and ALPPL2 has been classified as a rare, truly tumor-specific cell surface antigen.

[0003] The tumor-specific expression profiles of ALPP and ALPPL2 make them ideal targets for targeted cancer therapy. ALPP / ALPPL2 can be efficiently internalized, making them particularly suitable for drug delivery via ADCs. Several ADCs targeting ALPP / ALPPL2 are currently available, such as SGN-ALPV (composed of an anti-ALPP / ALPPL2 monoclonal antibody conjugated to MMAE via a cleavable linker) and HP-004 (a novel ADC simultaneously targeting ALPP and ALPPL2), which have demonstrated excellent antitumor activity in relevant models.

[0004] Although some antibodies targeting ALPP / ALPPL2 have been reported, there is still a need to develop novel antibodies with higher affinity, better specificity, and stronger internalization activity to meet the needs of precision clinical treatment. Summary of the Invention

[0005] This invention relates to an anti-ALPP / ALPPL2 antibody or its antigen-binding fragment and its applications. The antibody of this invention possesses high specificity and high sensitivity, capable of distinguishing ALPP / ALPPL2 from other members of the same family (such as ALPI and ALPL), and exhibits strong internalization activity.

[0006] This invention relates, in one aspect, to an ALPP / ALPPL2 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein,

[0007] The heavy chain variable region includes HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, and HCDR3 as shown in SEQ ID NO:5; the light chain variable region includes LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8.

[0008] In some implementations, the antibody is a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0009] In some implementations, the antibody is a monoclonal antibody.

[0010] In some implementations, the antibody is a chimeric antibody.

[0011] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv fragment, scFv, or di-scFv.

[0012] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7.

[0013] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:7.

[0014] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7.

[0015] In some embodiments, the light chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0016] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:8.

[0017] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0018] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7; and the light chain variable region comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0019] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0020] In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises the IgG1 heavy chain constant region shown in SEQ ID NO:9.

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human κ chain and λ chain.

[0023] In some embodiments, the antibody or its antigen-binding fragment comprises the light chain constant region of the human κ chain shown in SEQ ID NO:10.

[0024] In some embodiments, the antibody is a chimeric antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO:11 and the light chain amino acid sequence shown in SEQ ID NO:12.

[0025] In some embodiments, the antibody or its antigen-binding fragment mediates endocytosis.

[0026] In some embodiments, the antibody or its antigen-binding fragment does not bind to hALPI and ALPL.

[0027] In some embodiments, the antibody or its antigen-binding fragment binds strongly to hALPP, cALPP, and hALPPL2.

[0028] Another aspect of the present invention relates to nucleic acid molecules that encode the aforementioned antibodies or antigen-binding fragments thereof.

[0029] Another aspect of the present invention relates to expression vectors comprising the aforementioned nucleic acid molecules.

[0030] Another aspect of the present invention relates to host cells, which include the aforementioned nucleic acid molecules and / or the aforementioned expression vectors.

[0031] Another aspect of the present invention relates to pharmaceutical compositions comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector and / or the aforementioned host cell.

[0032] In some embodiments, the pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.

[0033] Another aspect of the present invention relates to immune conjugates comprising the aforementioned antibody or antigen-binding fragment thereof conjugated to a therapeutic or diagnostic agent.

[0034] Another aspect of the present invention relates to a kit containing the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, expression vector or host cell.

[0035] In some implementations, the kit is used to detect ALPP / ALPPL2 in biological samples.

[0036] Another aspect of the present invention relates to the use of the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned nucleic acid molecules, expression vectors, host cells or compositions in the preparation of a kit for detecting ALPP / ALPPL2 in biological samples.

[0037] Another aspect of the present invention relates to the use of the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned nucleic acid molecules, expression vectors, host cells and / or pharmaceutical compositions in the preparation of pharmaceuticals.

[0038] In some implementations, the drug is used to treat or prevent ALPP / ALPPL2-mediated diseases.

[0039] In some implementations, the ALPP / ALPPL2-mediated disease is cancer.

[0040] In some implementations, the cancer is stomach cancer.

[0041] Another aspect of the present invention relates to a medicament comprising the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, expression vector, host cell, and / or pharmaceutical composition; the medicament being used to treat or prevent ALPP / ALPPL2-mediated diseases.

[0042] In some implementations, the disease is cancer expressing ALPP / ALPPL2.

[0043] In some implementations, the cancer is stomach cancer. Attached Figure Description

[0044] Figure 1 The graph shows the endocytosis efficiency of antibody J-2 on MKN1 cells. Detailed Implementation

[0045] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0046] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. When a trademark name is used herein, unless the context otherwise indicates, the trademark name includes the product formulation, generic medicine, and active ingredient of the product for which the trademark name is used.

[0047] Certain terms in the invention

[0048] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of a feature, number, operation, material, or combination thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0049] The terms “ALPP / ALPPL2” or “ALPPL2 and / or ALPP” refer to ALPPL2 protein, ALPP protein, or both ALPPL2 and ALPP proteins, including their variants, isotypes, and species homologs of human ALPPL2 / ALPP. The variants or homologs maintain ALPPL2 and / or ALPP activity, for example, at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity. The variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence similarity to naturally occurring ALPPL2 and / or ALPP proteins in their entirety or in parts of the sequence (e.g., 50, 100, 150, or 200 consecutive amino acid sequences).

[0050] The term "CDR" refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a well-defined residue numbering system applicable to antibody variable regions but also residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Each complementarity-determining region may contain amino acid residues from what is defined as a "complementarity-determining region" by Kabat.

[0051] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or different types of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding constant regions of the heavy chain designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, and due to differences in chemical structure and biological function, it can be further divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. The subunit structure and three-dimensional conformation of different immunoglobulin classes are well-known to those skilled in the art. The term "chimeric antibody" refers to an antibody containing sequences derived from two different antibodies, typically from different species. For example, a chimeric antibody contains fragments of human and rodent antibodies, typically the human constant region and the mouse variable region. Methods for generating chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those skilled in the art.

[0052] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

[0053] The term "specific binding" refers to an antibody that preferentially binds to a specific target protein compared to other proteins, but this specificity does not require absolute binding specificity. Antibody binding depends on the presence of the target protein in the sample; for example, if an antibody specifically binds to the target protein in the sample without producing undesirable results such as false positives, then the antibody is considered "specific" to its intended target. The antibodies or their binding fragments used in this invention will bind to the target protein with an affinity at least two times, preferably at least ten times, more preferably at least 20 times, and most preferably at least 100 times higher than that with non-target proteins.

[0054] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody. Humanized antibodies overcome the drawback of chimeric antibodies, which, due to carrying a large amount of mouse protein components, induce a strong immune response. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

[0055] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies is selected. The variable region gene is then cloned from mouse hybridoma cells, followed by the desired human antibody constant region gene. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably containing the heavy chain constant region of human IgG1, IgG2, or IgG4. Alternatively, an IgG1 heavy chain constant region that has been mutated to enhance ADCC (antibody-dependent cell-mediated cytotoxicity) can be used.

[0056] The term "human antibody" includes antibodies containing variable and constant regions of human immunoglobulin sequences. Human antibodies as described herein may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human backbone sequence (i.e., "humanized antibody").

[0057] The term "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0058] The term "expression vector" or "vector" refers to a delivery system that allows for the operative insertion of polynucleotides or nucleic acids encoding a protein, thereby enabling the expression of that protein. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cells.

[0059] The term "host cell" refers to a cell into which a foreign polynucleotide or nucleic acid and / or vector has been introduced. Host cells include "transformers" and "transformed cells," which include the initially transformed cell and its progeny, regardless of the number of passages. Progeny cells may not be identical to parental cells in their nucleic acid content and may contain mutations. This application includes screening or selecting mutant progeny with the same function or biological activity from the initially transformed cells.

[0060] The term “treatment” refers to alleviating a disease or symptom, slowing the onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing a complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.

[0061] The term "prevention" includes the suppression of the occurrence or development of a disease or condition, or symptoms of a particular disease or condition. In some implementations, subjects with a family history of the disease are candidates for preventative programs. Generally, the term "prevention" refers to the administration of a drug before the onset of symptoms or signs, particularly in subjects at risk.

[0062] The term "pharmaceutical composition" herein refers to a composition in which an active substance is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to human or animal subjects. In some embodiments, the active substance is present in a unit dose that is suitable for administration in a treatment regimen and for demonstrating a statistically significant probability of achieving the intended therapeutic effect when used in the relevant population.

[0063] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0064] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, proportions, ratios, or parts are by weight.

[0065] Example 1: Preparation and purification of antibodies

[0066] The nucleotide sequences of the following his-tagged fusion proteins (extracellular region AA 23-506 of human ALPP protein (uniprot ID: P05187), extracellular region AA 20-503 of human ALPPL2 protein (uniprot ID: P10696), extracellular region AA 20-503 of human ALPI protein (uniprot ID: P09923), extracellular region AA 18-501 of human ALPL protein (uniprot ID: P05186), and extracellular region AA 21-504 of monkey ALPP protein (NCBI ID: XP_045223825.1)) were inserted into the pcDNA3.4 expression vector at appropriate restriction sites. HEK293 cells were transfected with PEI, and the supernatant was collected after culture. The his-tagged proteins were purified by affinity chromatography using a Ni column to obtain hALPP protein, hALPPL2 protein, hALPI protein, hALPL protein, and cALPP protein, respectively. The vector pLVX-IRES-puro or pLVX-IRES-neo encoding the full-length nucleotide sequence of the above proteins (hALPP, uniprot ID: P0J-187; hALPPL2, uniprot ID: P10696; hALPI, uniprot ID: P09923; hALPL, uniprot ID: P0J-186; cALPP, NCBI ID: XP_045223825.1) was co-transfected with the backbone vectors pMD2G and psPAX2 into HEK 293T cells. After 6 h of culture, the medium was changed, and the viral supernatant was collected 48 h after transfection and filtered (0.45 μm). The viral supernatant was then added to a solution containing 3 × 10⁻⁶ nucleotides. 5CHO-K1 or HEK293T cells were cultured in 6-well plates with polybrene for 12 h, then the medium was replaced with puromycin or G418 complete F12K / DMEM and cultured for 48 h. Subsequently, 0.5 cells / well were seeded into 96-well plates and cultured for 2 weeks in medium containing the corresponding selection drug. Single clones were selected for amplification to obtain stable overexpressing cells: CHO-K1-hALPP, CHO-K1-hALPPL2, CHO-K1-cALPP, 293T-hALPP, 293T-hALPPL2, 293T-cALPP, 293T-hALPI, and 293T-hALPL.

[0067] Six- to eight-week-old Balb / c, ICR, KM, FVB, and SJL mice were used as immunogens. An equal mixture of hALPP, hALPPPL2, and cALPP proteins was used. The initial immunization was emulsified with complete Freund's adjuvant (CFA), followed by booster immunizations on days 14, 28, and 42 using incomplete Freund's adjuvant (IFA). Blood was collected on day 60, and mice with the highest titers and positive FACS results were selected for a sprint immunization. Spleen cells were aseptically harvested and electrofused with myeloma cells at a 1:1 ratio. After culturing, hybridoma monoclonal cells were selected by ELISA and FACS to obtain cells that bind to three immunogenic proteins, 293T-hALPP overexpressing cell lines, 293T-hALPPL2 overexpressing cell lines, 293T-cALPP overexpressing cell lines, and LOVO cells (naturally expressing hALPP / ALPPL2), but do not bind to 293T-hALPP overexpressing cell lines or 293T-hALPL overexpressing cell lines. These cells were then cryopreserved. Sequencing, recombinant expression, and purification yielded the monoclonal chimeric antibody J-2. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:2, and the CDR region sequence is shown in SEQ ID NO:3-8 in Table 1 (defined according to the Kabat numbering system).

[0068]

[0069] The control antibody was prepared according to the contents of patent CN116997356A, and its heavy chain amino acid sequence is shown in SEQ ID NO:13 and its light chain amino acid sequence is shown in SEQ ID NO:14.

[0070] Example 2: ELISA detection of antibody binding activity

[0071] Recombinant hALPP, hALPPL2, and cALPP proteins were coated onto 96-well plates (incubated overnight at 4 °C), washed three times with PBST (pH 7.4, containing 0.05% Tween-20), and blocked at 37 °C. The blocking buffer was discarded, and the plates were washed three times. Serially diluted antibodies (J-2 antibody, control antibody) were added, and the plates were incubated at 37 °C for 1 h. After washing three times, HRP-labeled goat anti-human IgG (H+L) (Beyotime #A020) was added, and the plates were incubated at 37 °C for 1 h. After washing four times, TMB chromogenic buffer was added until development was complete. The optical density (OD) was measured at 450 nm after the incubation period. The results are shown in Table 2.

[0072] The results show that antibody J-2 can bind to antigens hALPP, hALPPPL2 and cALPP, and the binding ability is comparable to that of the control antibody.

[0073]

[0074] Example 3: FACS detection of antibody binding activity to cell surface ALPP / ALPPL2

[0075] HEK293T / hALPP, 293T / hALPPPL2, 293T / ALPI, and 293T / ALPL cells, respectively, stably expressing hALPP, hALPP L2, cALPP, hALPL, and hALPI, were constructed, and human colon cancer LOVO cell lines were cultured simultaneously. Cell suspensions were collected after digestion, counted, and adjusted to a density of 5E6 cells / mL after pre-cooling PBS. 20 μL / well was added to 96-well plates; then, equal volumes of serially diluted 5-fold assay antibodies (final concentration range 0-50 μg / mL) were added, mixed, and incubated on ice for 1 h. After washing three times with FACS buffer, 20 μL / well of 100-fold diluted PE-labeled anti-human IgG Fc secondary antibody (Biolegend #366904) was added, and the plates were incubated on ice, washed three times, and resuspended in 50 μL FACS buffer. PE fluorescence signals were detected by flow cytometry. The results are shown in Tables 3 and 4.

[0076] The results showed that antibody J-2 did not bind to 293T-hALPI and 293T-hALPL cells, but strongly bound to 293T-hALPP, 293T-cALPP, and 293T-hALPPPL2 cells. J-2 exhibited good binding specificity, and its binding activity on Lovo cells was comparable to that of the control antibody.

[0077]

[0078]

[0079] Example 4: SPR detection of the affinity of antibody for hALPP and hALPPPL2

[0080] The Protein A (Cytiva / 29-1275-56) chip was equilibrated to room temperature and the Prime program was run. Control antibody (4 μg / mL) and antibody J-2 (6 μg / mL) were captured onto the chip surface, respectively. Then, different concentrations (0–60 nM) of hALPP and hALPPL2 proteins were sequentially injected, and binding and dissociation were measured. Data were fitted using a 1:1 binding model with Biacore Evaluation Software to calculate affinity. The results are shown in Table 5.

[0081] The results show that the affinity of J-2 and hALPP proteins is stronger than that of the control antibody, while the affinity of J-2 and hALPPL2 is comparable to that of the control antibody.

[0082]

[0083] Example 5: FACS detection of antibody endocytosis

[0084] MKN1 cells were collected by digestion, washed with pre-chilled PBS, and the density was adjusted to 4E6 cells / mL. 50 μL of the solution was added to each well of a 96-well plate (labeled plate A). An equal volume of the test antibody (final concentration 10 μg / mL) was added, mixed, and incubated on ice for 1 h. The cells were washed four times with pre-chilled PBS buffer, and each well was resuspended with 100 μL of complete culture medium. 50 μL of the suspension was transferred to another 96-well plate (plate B, placed on ice). Plate A was transferred and incubated at 37 °C for 3 h. After incubation, plates A and B were washed once each, with 20 μL of PE-labeled anti-human IgG Fc secondary antibody (2 μg / mL) added each time, and incubated on ice for 30 min. The cells were washed three times, resuspended in 50 μL of FACS buffer, and the PE fluorescence signal was detected by flow cytometry. Endocytosis rate % = (MFI 4 °C - MFI 37 °C) / MFI 4 °C × 100%. Results are shown in Table 6. Figure 1 As shown.

[0085] The results show that the endocytosis efficiency of antibody J-2 on MKN1 cells was 50.83%, while that of the control antibody was 43.98%, indicating that the endocytosis efficiency of antibody J-2 was superior to that of the control antibody.

[0086]

[0087] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An anti-ALPP / ALPPL2 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3-5; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6-8.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody, a single-chain antibody, a chimeric antibody, or a humanized antibody.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv fragment, scFv or di-scFv.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

5. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody is a chimeric antibody, which contains the heavy chain amino acid sequence shown in SEQ ID NO:11 and the light chain amino acid sequence shown in SEQ ID NO:

12.

6. A nucleic acid molecule, characterized in that, The code defines the antibody or antigen-binding fragment thereof according to any one of claims 1-5.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6 and / or the expression vector of claim 7.

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