Chimeric anti-cd27 antibodies

Abstract

The present application provides chimeric anti-CD27 antibody molecules and encoding nucleic acids and expression vectors thereof. The anti-CD27 antibody molecules of the present application can specifically bind to CD27 and have effects of stimulating lymphocyte proliferation, activating lymphocytes, etc.

Description

Technical Field

[0001] This application relates to the field of antibodies, and more specifically, to antibodies against CD27 and their applications. Background Technology

[0002] CD27 co-stimulatory molecule is a type I transmembrane molecule with a molecular weight of 120 kDa, belonging to the TNFR superfamily. It is mainly expressed on most T cells (naive T cells, activated T cells), memory B cells, and some NK cells. When immune cells are activated, CD27 expression levels are upregulated. During T cell activation, CD27 and its ligands coordinate with the TCR. Currently, CD27 is known to have only one ligand molecule—CD70. CD70 is transiently expressed on activated lymphocytes (B cells, T cells) and dendritic cells (DCs). The CD27-CD70 interaction recruits intracytoplasmic TRAFs proteins, thereby activating the NF-κB and JNK signaling pathways, triggering a series of cellular responses including T cell activation, proliferation, survival, and cytotoxicity. Therefore, in the processes of killing tumors and fighting viral infections, CD27-CD70 promotes the activation, differentiation, and memory-based immune processes of T cells. T cell activation not only enhances CD27 expression on the T cell surface but also causes activated T cells to secrete sCD27 (soluble CD27 with a molecular weight of 28–32 kDa). sCD27 binds to CD70, inhibiting the binding of CD70 to CD27 expressed on the cell surface, thereby suppressing the continuous proliferation of T cells and playing a feedback regulatory role. Besides regulating T cell responses, CD27 also promotes the expansion of B cells in germinal centers and the secretion of IFN-γ by some NK cells. Regulating the CD27 target can enhance the host's anti-tumor activity.

[0003] Therefore, the development of CD27-targeting antibodies holds promise for the treatment of CD27-related diseases, including cancer. Currently, only Celldex Therapeutics' Varlilumab (CDX-1127, Phase I / II clinical trial) and CDX-527 (CD27 & PD-L1 bispecific antibody, Phase I clinical trial) are in Phase I / II clinical trials. Since Varlilumab is a high-affinity IgG1 subtype antibody, ADCC and CDC are its main mechanisms of action. Considering the constitutive expression of CD27 on immune cells, high-affinity IgG1 subtype antibodies targeting CD27 may kill normal immune cells due to ADCC and CDC, potentially affecting the body's normal immune function.

[0004] The inventors of this application have developed a new anti-CD27 antibody and studied its properties and related applications. Summary of the Invention

[0005] In a first aspect, this application provides an antibody that specifically binds to a CD27 molecule or its antigen-binding moiety, comprising a heavy chain variable region comprising HCDR1, HCDR2, and / or HCDR3 sequences. In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 or 4. In optional embodiments, the antigen-binding moiety is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, Fd fragments, and single-domain antibodies.

[0006] In some embodiments, the antibody that specifically binds to CD27 or its antigen-binding moiety further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and / or LCDR3 sequences. In some embodiments, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 14.

[0007] In some embodiments, the heavy chain of the antibody that specifically binds to CD27 or its antigen-binding portion comprises an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO: 6, preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 6.

[0008] In some embodiments, the light chain of the antibody or its antigen-binding portion described in the first aspect comprises an amino acid sequence of any one of SEQ ID NOs: 15-22 or an amino acid sequence having at least 80% homology with the above sequences, preferably, the light chain comprises the amino acid sequence shown in SEQ ID NO: 15.

[0009] In some implementations, the antibody that specifically binds to CD27 as described in the first aspect is a monoclonal antibody, such as a murine monoclonal antibody.

[0010] In some implementations, the antibody that specifically binds to CD27 as described in the first aspect is a humanized antibody.

[0011] In some implementations, the antibody that specifically binds to CD27 as described in the first aspect is a human-mouse chimeric antibody.

[0012] In some embodiments, the anti-CD27 antibody disclosed herein, or its antigen-binding portion, binds to the same epitope on CD27 as antibody 6D8-G98A, or competes with 6D8-G98A for binding to CD27. In some embodiments, the heavy chain sequence of the antibody is as shown in SEQ ID NO: 6, and the light chain sequence is as shown in SEQ ID NO: 15.

[0013] In some embodiments, the antibodies disclosed herein, or their antigen-binding portions, are capable of inducing T cell activation. In some embodiments, the antibodies disclosed herein, or their antigen-binding portions, are capable of inhibiting tumor cell growth.

[0014] Secondly, this application provides a nucleotide molecule that encodes the antibody or antigen-binding portion thereof that specifically binds to CD27 as described in the first aspect.

[0015] Thirdly, this application provides an expression vector containing nucleotide molecules as described in the second aspect.

[0016] Fourthly, this application provides a host cell containing the expression vector as described in the third aspect.

[0017] Fifthly, this application provides a pharmaceutical composition comprising the anti-CD27 antibody or its antigen-binding portion as described in the first aspect, and a pharmaceutically acceptable carrier.

[0018] In some embodiments, the composition further comprises one or more other active ingredients. In some embodiments, the active ingredient is an antitumor drug or an antiviral drug.

[0019] In some embodiments, the composition is used to treat CD27-related diseases.

[0020] Sixthly, this application provides the use of the anti-CD27 antibody or its antigen-binding portion as described in the first aspect, or the composition as described in the fifth aspect, in the preparation of a medicament for the prevention or treatment of CD27-related diseases, such as tumors or viral infections.

[0021] In some implementations, the tumor includes colon cancer, liver cancer, lymphoma, stomach cancer, lung cancer, head and neck squamous cell carcinoma, or metastatic cancer thereof.

[0022] In other respects, this application provides methods for preventing or treating CD27-related diseases, comprising administering to an individual in need the antibody or its antigen-binding portion as described in the first aspect, or the pharmaceutical composition as described in the fifth aspect.

[0023] The anti-CD27 antibody or its antigen-binding portion thereof of this application can specifically bind to CD27 and has one or more of the following effects: generating a second signal required for T cell proliferation and activation, promoting the proliferation and activation of antigen-specific T cells; stimulating the body to produce an immune response; promoting the killing effect of T cells on cancer cells; having immune cell stimulation activity independent of ADCC and CDC; and / or inhibiting tumor growth, etc.

[0024] Brief description of the attached figures

[0025] Figure 1 The image shows the results of signal reporter assay of culture supernatant of mouse anti-human CD27 hybridoma cells by signal reporter detection method. It shows the signal reporter assay results of culture supernatant of some mouse anti-human CD27 hybridoma cell lines (Clone116-Clone138).

[0026] Figure 2 This is a functional validation diagram of the candidate mouse anti-human CD27 antibody signal reporter assay, showing the results of antibody assays in the culture supernatants of 6D8, 20C9, and 31G9.

[0027] Figure 3 The graph shows the results of flow cytometry determination of the binding of candidate mouse anti-human CD27 antibodies to human CD27 cell lines, illustrating the binding rate of antibodies in the culture supernatants of 6D8, 20C9, and 31G9 cells to cells expressing hCD27.

[0028] Figure 4 The results of the human CD27 ligand blocking experiment are shown.

[0029] Figure 5 The image shows the ELISA results of the candidate mouse anti-human CD27 antibody binding to monkey CD27, illustrating the binding of candidate monoclonal hybridoma cell line antibodies 6D8, 20C9, and 31G9 to monkey CD27.

[0030] Figure 6 shows the binding and dissociation curves of the candidate mouse anti-human CD27 antibody 6D8 with human CD27 protein. Figure 6A The 6D8 binding dissociation curve is shown. Figure 6B The image shows the binding and dissociation curves of Varlilumab antibody.

[0031] Figure 7A -B shows the results of a cellular function assay using humanized 6D8 antibodies on HEK::CD27 cells. Figure 7A Secondary antibody added. Figure 7B No secondary antibody was added.

[0032] Figure 8The results of the in vivo co-stimulation assay with 6D8-G98A antibody were shown, confirming that 6D8-G98A antibody stimulates T cell proliferation in B-hCD27 mice.

[0033] Figure 9 The curves showing the changes in tumor volume in mice during the in vivo efficacy experiment of the 6D8-G98A antibody are displayed. Detailed Implementation

[0034] This application provides novel anti-CD27 antibodies or antigen-binding moieties thereof that specifically bind to CD27. In a preferred embodiment, the antibody or antigen-binding moieties of this application bind to CD27 on the surface of T cells and activate T cells. This application also provides a polynucleotide encoding the antibody or its antigen-binding fragment, a vector containing the polynucleotide, a host cell containing the polynucleotide or the vector, methods for preparing and purifying the antibody, and medical and biological applications of the antibody or its antigen-binding fragment, such as the prevention or treatment of CD27-related diseases or conditions. This application also covers methods and related kits for detecting CD27 and modulating CD27 activity using the antibody or its antigen-binding fragment.

[0035] To facilitate understanding of this application, some terms used herein are first defined.

[0036] As used in this article, "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds, as well as its multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs).

[0037] As used herein, the term "antigen-binding moiety" of an antibody refers to a portion or segment of the complete antibody molecule responsible for binding an antigen. The antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from complete antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering. Non-limiting examples of antigen-binding moieties include: Fab fragments; F(ab′)2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; single-domain antibodies; dAb fragments; and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues mimicking the hypervariable region of an antibody. The term "antigen-binding moiety" also includes other engineered molecules such as biantibodies, triantibodies, tetraantibodies, and microantibodies.

[0038] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR sequence for VH or VL: the Kabat definition and the Chothia definition, for example, see Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). For a given antibody's variable region sequence, the CDR sequence in the VH and VL sequences can be determined according to either the Kabat or Chothia definition. In the embodiments of this application, the Kabat definition of the CDR sequence is used. In this paper, the CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; the CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

[0039] For a given antibody's variable region sequence, the CDR region sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0040] As used herein, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, or the ability of an antibody to bind to a specific antigen with an affinity at least twice that of a nonspecific antigen. However, it should be understood that antibodies can specifically bind to two or more sequence-related antigens. For example, the antibodies of the present invention can specifically bind to CD27 in both humans and non-humans (e.g., mice or non-human primates).

[0041] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, provided they exhibit the desired biological activity (see, U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0042] As used herein, the term "homology" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of homology if desired. Methods and computer programs used for alignment are well known in the art. "At least 80% homology" as used herein refers to any value between 80% and 100%, such as 85%, 90%, 95%, 99%, etc.

[0043] As used herein, the term "CD27-related disease" includes diseases and / or symptoms associated with the CD27 signaling pathway. Exemplary CD27-related diseases or conditions include viral infections and tumors, such as colon cancer.

[0044] On one hand, this application provides an antibody that specifically binds to CD27 or its antigen-binding portion, which includes a heavy chain variable region and / or a light chain variable region. Tables 1-5 below exemplarily list the CDR, heavy chain, and light chain amino acid sequences applicable to the antibodies disclosed in this application. In some embodiments, the anti-CD27 antibody or its antigen-binding portion includes HCDR1, HCDR2, and / or HCDR3 sequences, independently selected from any one of the HCDR1, HCDR2, or HCDR3 sequences shown in Table 1. In some embodiments, the anti-CD27 antibody of this application may further include a light chain CDR, independently selected from any one of the light chain CDR1, CDR2, or CDR3 sequences shown in Table 2. For example, the anti-CD27 antibody of this application may include any one of the heavy chains shown in Table 3, optionally paired with any one of the light chains shown in Table 4.

[0045] Table 1: Heavy chain CDR amino acid sequence of exemplary anti-CD27 antibodies

[0046]

[0047] Table 2: Light chain CDR amino acid sequence of exemplary anti-CD27 antibodies

[0048]

[0049] Table 3: Heavy chain amino acid sequences of exemplary anti-CD27 antibodies

[0050]

[0051]

[0052] Table 4: Light chain amino acid sequence of exemplary anti-CD27 antibodies

[0053] Antibody number Light chain amino acid sequence 6D8 SEQ ID NO.15 6D8-G98A SEQ ID NO.15 Hu6D8-1, Hu6D8-7, Hu6D8-13, Hu6D8-19 SEQ ID NO.17 Hu6D8-2, Hu6D8-8, Hu6D8-14, Hu6D8-20 SEQ ID NO.18 Hu6D8-3, Hu6D8-9, Hu6D8-15, Hu6D8-21 SEQ ID NO.19 Hu6D8-4, Hu6D8-10, Hu6D8-16, Hu6D8-22 SEQ ID NO.20 Hu6D8-5, Hu6D8-11, Hu6D8-17, Hu6D8-23 SEQ ID NO.21 Hu6D8-6, Hu6D8-12, Hu6D8-118, Hu6D8-24 SEQ ID NO.22

[0054] In some embodiments, the HCDR1 of the antibody or its antigen-binding portion disclosed herein is the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, HCDR2 is the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 3 and 4. For example, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 4.

[0055] The antibodies or antigen-binding regions disclosed in this article may further include light chain variable regions in addition to heavy chain variable regions.

[0056] In some embodiments, the CDR1 (LCDR1) of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, LCDR2 is the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, LCDR3 is the amino acid sequence shown in SEQ ID NO: 14.

[0057] In specific embodiments, the heavy chain of the antibody or its antigen-binding portion disclosed herein has at least 80% homology with the amino acid sequences selected from SEQ ID NOs: 5-11, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology. In more specific embodiments, the antibody heavy chain consists of an amino acid sequence selected from any one of SEQ ID NOs: 5-11. In a specific embodiment, the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO: 6.

[0058] In specific embodiments, the light chain of the antibody disclosed herein has at least 80% homology with sequences selected from SEQ ID NOs: 15-22, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology. In more specific embodiments, the antibody light chain consists of an amino acid sequence selected from any one of SEQ ID NOs: 15-22. In a specific embodiment, the amino acid sequence of the above-described antibody light chain is shown in SEQ ID NO: 15.

[0059] In some implementations, the heavy chain or heavy chain variable region, light chain or light chain variable region of the antibody disclosed herein may be substituted, deleted or added at least one amino acid based on the specific amino acid sequences listed above, and the resulting variants still retain CD27 binding activity.

[0060] In some embodiments, the number of amino acid substitutions, deletions, or additions is 1-30, preferably 1-20, and more preferably 1-10. In a preferred embodiment, the sequence variant differs from the original amino acid sequence by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in substitution, deletion, and / or addition. In a more preferred embodiment, the sequence variant differs from the original amino acid sequence by approximately 1, 2, 3, 4, or 5 amino acids in substitution, deletion, or addition. In a specific embodiment, the amino acid substitution is a conservative substitution.

[0061] In a preferred embodiment, the antibody disclosed herein is antibody 6D8-G98A, wherein the heavy chain sequence of antibody 6D8-G98A is shown as SEQ ID NO: 6, and the light chain sequence is shown as SEQ ID NO: 15.

[0062] In some embodiments, the antibody or its antigen-binding moiety disclosed herein binds to the same epitope on CD27 as antibody 6D8-G98A, or competes with 6D8-G98A for binding to CD27.

[0063] In some embodiments, the antibodies disclosed herein are monoclonal antibodies. In specific embodiments, the antibodies disclosed herein are humanized antibodies. In more specific embodiments, the antibody is an anti-human CD27-IgG4 subtype monoclonal antibody, which, compared to the IgG1 subtype antibody, has immune cell stimulatory activity independent of ADCC and CDC, effectively inhibiting tumor growth in vivo while reducing potential side effects caused by immunotoxicity.

[0064] The antibodies or antigen-binding portions thereof disclosed herein are capable of specifically binding to CD27. In specific embodiments, the antibody or antigen-binding portion thereof specifically binds to primate CD27 or mouse CD27, or CD27 of any species that is highly homologous to primate CD27 or mouse CD27. In a preferred embodiment, the antibody or antigen-binding portion thereof specifically binds to human CD27. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to monkey CD27.

[0065] In some embodiments, the antibodies disclosed herein or their antigen-binding moieties specifically bind to CD27 molecules and have low toxicity (e.g., weak ADCC and CDC), thus making them suitable for the diagnosis or treatment of CD27 activity-related diseases, such as cancer.

[0066] In some embodiments, the antibodies disclosed herein are IgG4 subtype antibodies, such as anti-human CD27-IgG4 subtype monoclonal antibodies. IgG1 subtype antibodies typically exhibit strong antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), with ADCC and CDC being their primary mechanisms of action. Considering the constitutive expression of CD27 on immune cells, high-affinity IgG1 subtype antibodies targeting CD27 may kill normal immune cells due to ADCC and CDC, potentially affecting the body's normal immune function. Therefore, in some specific embodiments, the inventors obtained CD27-IgG4 subtype antibodies by screening antibody hybridoma cell lines with moderate affinity for human CD27 and agonistic activity. Compared to IgG1 subtype antibodies, the CD27-IgG4 subtype antibodies possess immune cell stimulatory activity independent of ADCC and CDC, effectively inhibiting tumor growth in vivo while reducing potential side effects caused by immunotoxicity.

[0067] In some implementations, the antibodies or their antigen-binding portions disclosed herein bind to CD27 on the surface of lymphocytes, stimulating the body to produce an immune response and promoting the proliferation, activation, and killing of T lymphocytes.

[0068] For example, the inventors conducted in vitro and in vivo biological experiments on the anti-CD27 antibody disclosed herein, and the results showed that this antibody can bind well to CD27 molecules, induce lymphocyte activation, and / or inhibit tumor growth.

[0069] This application also provides a nucleotide molecule encoding an antibody or its antigen-binding portion disclosed herein, a vector containing the polynucleotide, a host cell containing the polynucleotide or the vector, and methods for preparing and purifying the antibody.

[0070] In some embodiments, a nucleotide molecule encoding the antibody or its antigen-binding moiety is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the vector.

[0071] In some embodiments, any suitable expression vector may be used in this application. For example, the expression vector may be one of pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF, and pCHO 1.0. The expression vector may include a fusion DNA sequence linked with suitable transcriptional and translational regulatory sequences.

[0072] In some embodiments, the available host cells are cells containing the above-described expression vector, and can be eukaryotic cells. Mammalian or insect host cell culture systems can be used for the expression of the antibody or its antigen-binding portion in this application. For example, HEK293 cells, COS, CHO, NSO, sf9, and sf21 are all suitable for this invention. The host cells can also be prokaryotic cells containing the above-described expression vector, such as DH5α, BL21(DE3), or TG1.

[0073] In some embodiments, the method for preparing the anti-CD27 monoclonal antibody disclosed herein includes: culturing host cells under expression conditions to express the anti-CD27 monoclonal antibody; and isolating and purifying the expressed anti-CD27 monoclonal antibody. Using the above method, the recombinant protein can be purified into a substantially homogeneous substance, for example, appearing as a single band on SDS-PAGE electrophoresis.

[0074] In some implementations, the anti-CD27 antibody disclosed herein can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the anti-CD27 antibody bound to the affinity column.

[0075] In the specific implementation plan, the humanized anti-CD27 monoclonal antibody disclosed herein is obtained through the following methods: mice are immunized with human CD27 protein, and hybridoma technology is used to obtain hybridoma cell lines (strains) capable of expressing anti-human CD27 antibodies. Candidate hybridoma cell lines are then screened using in vitro ELISA, and the antibodies expressed by these candidate hybridoma cell lines are validated through human CD27 protein binding, blocking, and cross-reactivity experiments. Furthermore, in vivo stimulation experiments are conducted on the candidate mouse anti-human CD27 antibodies. Based on the results of the CD27 humanized mouse stimulation experiments, one candidate antibody is selected for humanization design, synthesis, and expression to obtain a humanized anti-CD27 antibody. Affinity, binding, and in vitro functional experiments are then performed to validate the antibody, as well as subsequent in vivo anti-mouse MC38 colon cancer tumor growth experiments. Based on the above experimental results, a new humanized anti-human CD27 antibody, such as a chimeric antibody, is finally obtained.

[0076] This application provides pharmaceutical compositions comprising the antibodies or antigen-binding moieties disclosed herein, and a pharmaceutically acceptable carrier. The aforementioned anti-CD27 antibodies, such as anti-human CD27 monoclonal antibodies, can be formulated into pharmaceutical preparations with pharmaceutically acceptable carriers to achieve more stable therapeutic effects. In some embodiments, these preparations maintain the conformational integrity of the amino acid core sequence of the disclosed anti-CD27 antibodies, such as anti-human CD27 monoclonal antibodies, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamidation, or oxidation). In some embodiments, for liquid preparations, they are typically stable for at least one year when stored at 2°C–8°C. In some embodiments, for lyophilized preparations, they remain stable for at least six months at 30°C.

[0077] This application also provides methods for preventing or treating CD27-related diseases, comprising administering an individual an anti-CD27 antibody, or a composition containing an anti-CD27 antibody, such as an anti-human CD27 monoclonal antibody. In some embodiments, significant anti-tumor effects are observed after administration to animals, including humans. Specifically, the anti-CD27 antibodies disclosed herein are capable of effectively preventing and / or treating cancer and can be used as anti-cancer drugs.

[0078] This application also provides the use of anti-CD27 antibodies, or compositions containing anti-CD27 antibodies, in the preparation of medicaments for the prevention or treatment of CD27-related diseases or symptoms. In some embodiments, the CD27-related diseases or symptoms are viral infections or tumors.

[0079] In some implementation schemes, the aforementioned tumors include colon cancer, liver cancer, lymphoma, stomach cancer, lung cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, chronic lymphocytic leukemia, malignant melanoma, kidney cancer, prostate cancer, ovarian cancer, non-small cell lung cancer or its metastatic cancer, etc.

[0080] When administering the anti-human CD27 antibody and its composition disclosed in this article to animals, including humans, the dosage varies depending on the individual's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and the overall situation can be referenced, and the total dosage should not exceed a certain range.

[0081] The dosage and frequency of administration of antibodies or compositions thereof may vary depending on whether the disease is being prevented or treated. In prophylactic use, a composition containing the antibody or a mixture thereof of this application is administered to a patient who is not yet in a disease state to enhance the patient's resistance; this amount is defined as the "prophylactic effective dose." In this use, the specific dose depends on the patient's health condition and systemic immunity. Generally, relatively low doses are administered for longer periods at relatively infrequent intervals. In therapeutic use, it is sometimes necessary to administer relatively high doses at relatively short intervals until disease progression slows or ceases, and preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient. Those skilled in the art can readily determine the specific dosage and frequency according to actual needs.

[0082] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human.

[0083] In this specification and claims, the words “comprising,” “including,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0084] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0085] The foregoing disclosure generally describes this application. The following embodiments are further illustrations of this application and should not be construed as limiting this application. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, for example, see Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0086] Example

[0087] Example 1. In vitro antibody screening

[0088] Human CD27 protein was purchased from Sino Biological Inc. (Catalog No. 10039-H08B1) and its purity was confirmed to be above 85% by SDS-PAGE. Balb / c mice were immunized with CD27 protein, and the serum antibody titer was measured by ELISA. Mice with suitable serum antibody titers were selected, and hybridoma cell lines were obtained using hybridoma technology. Approximately 1000 monoclonal hybridoma cell lines were obtained by plate-coating. The cell supernatant was analyzed by ELISA (coated with human CD27 protein, with anti-mouse IgG-HRP conjugate as the secondary antibody), and approximately 200 monoclonal cell lines with an OD value greater than 2.0 were selected as candidate hybridoma cell lines.

[0089] Example 2. Screening of candidate hybridoma cell lines

[0090] A cell line stably expressing human CD27 (HEK::CD27 cells) was constructed; candidate hybridoma cell lines were screened using a signaling reporter assay. From approximately 200 hybridoma cell lines, 16 were selected, such as... Figure 1 As shown.

[0091] The cell culture supernatant of the above 16 candidate hybridoma cell lines was purified, and the above detection was repeated. Three hybridoma cell lines (6D8, 20C9 and 31G9) were selected for subsequent screening, as shown in Figure 2.

[0092] Example 3. Human CD27 binding experiment confirms candidate hybridoma cell lines

[0093] The binding affinity of antibodies produced by the three hybridoma cell lines (6D8, 20C9, and 31G9) to cells expressing human CD27 was determined by flow cytometry. The results are as follows: Figure 3 As shown, 6D8, 20C9, and 31G9 all exhibited high binding rates (over 85%) with cells expressing human CD27.

[0094] Example 4. Human CD27 ligand blocking experiment

[0095] Antibodies prepared from the three candidate monoclonal cell lines (6D8, 20C9, and 31G9) were incubated with hCD27-expressing cells sequentially at doses of 1 ng, 10 ng, 100 ng, and 1000 ng. 100 ng of human CD27 ligand (CD70-hFc fusion protein) was then incubated with the cells again. Flow cytometry analysis showed the results as follows: Figure 4As shown, the blocking effect on the binding of hCD27 ligand to hCD27 is enhanced with the increase of antibody amount of the three candidate antibodies; when the amount is above 10ng, the binding of hCD27 ligand to hCD27 is partially or even completely blocked.

[0096] Example 5. Monkey CD27 binding experiment

[0097] The ELISA plate was coated with monkey CD27 protein. 10 μL and 50 μL of culture supernatant from cell lines 6D8, 20C9, and 31G9 were incubated with monkey CD27, respectively. Then, anti-mouse IgG-HRP secondary antibody was added for further incubation. After incubation and color development, the OD value was measured at 450 nm. The results are as follows: Figure 5 As shown, antibodies from three candidate monoclonal cell lines (6D8, 20C9, and 31G9) showed high binding to monkey CD27 protein under 10 μL and 50 μL conditions.

[0098] Example 6. In vitro affinity assay of antibody

[0099] In this embodiment, the affinity of the 6D8 antibody was determined using a Reichert4 SPR instrument.

[0100] First, human CD27 protein (Cat: 10039-H03H, purchased from Sino Biological Inc.) was coated onto an SPR chip (SR7000 GOLD SENSOR SLIDE, MIXED SELF-ASSEMBLED, PART NO: 13206066). Then, different concentrations of 6D8 antibody and anti-CD27 reference antibody (Varlilumab, CDX-1127) were loaded and bound to the chip coated with human CD27 protein. After binding, elution was initiated. The binding and elution processes were monitored and analyzed by the equipment, ultimately obtaining the binding and dissociation curves of the 6D8 antibody. Based on the binding and dissociation curves, the affinity of the 6D8 antibody was calculated. The results are shown in Figure 6 and Table 5. Compared with Varlilumab antibody, the affinity of the 6D8 antibody was moderate at 5.14E-10M, slightly lower than that of Varlilumab (1.9E-12M).

[0101] Table 5. Results of antibody affinity calculation

[0102] 6D8 antibody

[0103] Curve Name Bmax([Signal(uRIU)]) ka(1(M*s)) kd(l / s) KD(M) 6D8(2.50e-8) - Reference Curve Fitting 502.27 1.88E+05 9.69E-05 5.14E-10 6D8(1.25e-8) - Reference Curve Fitting 400.04 1.88E+05 9.69E-05 5.14E-10 6D8(6.25e-9) - Reference Curve Fitting 320.45 1.88E+05 9.69E-05 5.14E-10 6D8(5.00e-8) - Reference Curve Fitting 503.17 1.88E+05 9.69E-05 5.14E-10

[0104] Varlilumab

[0105] Curve Name Bmax([Signal(uRIU)]) ka(1(M*s)) kd(1 / s) KD(M) Varlilumab (1.25e-8) - Reference Curve Fitting 429.85 1.16E+05 2.14E-07 1.85E-12 Varlilumab (6.25e-9) - Reference Curve Fitting 432.38 1.16E+05 2.14E-07 1.85E-12 Varlilumab (2.50e-8) - Reference Curve Fitting 498.18 1.16E+05 2.14E-07 1.85E-12 Varlilumab (5.00e-8) - Reference Curve Fitting 505.48 1.16E+05 2.14E-07 1.85E-12

[0106] Example 7. Humanization of mouse anti-human CD27 monoclonal antibody 6D8

[0107] The light and heavy chain variable region genes of the candidate antibody 6D8 were amplified and sequenced, and the sequences were provided to Nanjing Genscript Biotech Co., Ltd. for humanized sequence design. In short, the mouse CDR region was transplanted into the framework of the light and heavy chain variable regions of the human IgG4 antibody to obtain a chimeric 6D8. Then, computer-aided techniques were used to perform corresponding reversion mutations, resulting in various combinations of light and heavy chains to form multiple humanized antibodies, which were expressed and purified in mammalian cells.

[0108] Finally, the heavy and light chains of the obtained humanized antibodies are shown in Table 6. The 24 humanized antibodies obtained are designated as Hu6D8-1, Hu6D8-2, Hu6D8-3, Hu6D8-4, Hu6D8-5, Hu6D8-6, Hu6D8-7, Hu6D8-8, Hu6D8-9, and Hu6D8-10, respectively. Hu6D8-11, Hu6D8-12, Hu6D8-13, Hu6D8-14, Hu6D8-15, Hu6D8-16, Hu6D8-17, Hu6D8-18, Hu6D8-19, Hu6D8-20, Hu6D8-21, Hu6D8-22, Hu6D8-23, and Hu6D8-24 represent the chimeric 6D8-G98A, obtained by mutating glycine at position 98 to alanine. The specific sequences of the antibody heavy chains are shown in Table 7, and the specific sequences of the light chains are shown in Table 8.

[0109] Table 6

[0110]

[0111]

[0112]

[0113] Example 8. Antibody in vitro binding assay

[0114] In this embodiment, flow cytometry was used to determine the binding rate of several representative 6D8 humanized antibodies (see Table 6) to cells expressing hCD27 (HEK::CD27). The amounts of humanized CD27 antibody were 10 ng and 100 ng, respectively. After incubation with HEK::CD27, the positivity rate of HEK::CD27 cells was detected by flow cytometry. The results are shown in Table 9. Compared with the anti-CD27 reference antibody (Varlilumab, CDX-1127), 6D8-G98A (human-mouse chimeric antibody), 6D8 antibody (mouse anti-human antibody), and the 6D8 humanized antibodies shown in Table 9 could all significantly bind to HEK:CD27 cells expressing human CD27.

[0115] Table 9. Binding rates and median values ​​of humanized 6D8 antibody to HEK::CD27 cells.

[0116]

[0117] Example 9. Antibody Cell Function Experiment

[0118] First, a certain amount of HEK::CD27 cells were added to a 96-well plate. Then, humanized antibodies Hu6D8-1 to Hu6D8-24 (see Table 2) were added sequentially to achieve a final concentration of 1 μg / mL for each humanized antibody. Anti-human IgG Fc (secondary antibody) at 3 times the concentration of the humanized antibody was added. The plate was incubated at 37°C and 5% CO2 for 20 h. 40 μL of cell culture supernatant was added to a 96-well plate containing 160 μL of Quanti Blue, mixed thoroughly, and incubated at 37°C for 1 h. The OD value was measured at 620 nm.

[0119] As shown in Figure 7, compared with the anti-CD27 reference antibody (Varlilumab, CDX-1127), 6D8-G98A (human-mouse chimeric antibody), 6D8 antibody (mouse anti-human antibody), and humanized antibodies (Hu6D8-1 to Hu6D8-24) all showed comparable or superior in vitro activity, especially without the addition of secondary antibody.

[0120] Example 10. Antibody in vivo stimulation experiment

[0121] CD27 humanized mice (B-hCD27 mice), homozygous, female, 6-8 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Mice were randomly divided into a control group and a 6D8-G98A group (n=3 per group) according to their body weight. At D0 (date of first administration) and D2, mice were administered 300 μg / mouse via intraperitoneal injection. At D0 and D10, 100-200 μL of peripheral blood was collected from the mice and anticoagulated with EDTA·K2. Then, peripheral blood lymphocytes (PBMCs) were separated using mouse Ficoll lymphocyte separation medium and incubated with CD3, CD8, CD44, and CD62L antibodies. The proportion of CD3+CD8+CD44+CD62L- in peripheral blood was detected by flow cytometry.

[0122] The results are as follows Figure 8 As shown in Table 10, at D0, the proportion of CD3+CD8+CD44+CD62L- in the peripheral blood of both groups of mice was below 7%; at D10, the proportion of CD3+CD8+CD44+CD62L- in the peripheral blood of the control group mice was slightly increased, while that in the 6D8-G98A group mice was significantly increased (average above 30%). Statistical analysis showed a significant difference between the two groups (P < 0.01), indicating that the 6D8-G98A antibody can significantly stimulate the body's immune response and activate a large number of T cells.

[0123] Table 10 Individual data on T cell proliferation in B-hCD27 mice stimulated by 6D8-G98A antibody.

[0124]

[0125] Example 11. In vivo antitumor pharmacodynamic experiment of anti-hCD27 antibody

[0126] CD27 humanized mice (B-hCD27 mice), homozygous, female, 6-8 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0127] Establishment and grouping of MC38 mouse colon cancer model: Each mouse was subcutaneously inoculated with 5×10 5 1 cell / 0.1 mL; mouse tumor volume 100–150 mm 3 At that time, the tumors were randomly divided into two groups according to their tumor volume: a control group and a 6D8-G98A group. Five animals in each group were given intraperitoneal medication. The dose of 6D8-G98A was 3 mg / kg, and the medication was given once every three days (Q3D) for a total of 6 doses.

[0128] Indicator measurements: 1) Weight change: The weight of mice in each group was measured every three days, and the mice's condition was observed; 2) Tumor volume change: The length and width of the tumor in each group were measured every three days, and the tumor volume was calculated (volume = length × width 2 / 2).

[0129] The experimental results showed that the body weight of mice in both groups increased slowly, with no difference between the groups. Over time, the tumor volume in the control group mice gradually increased, while the tumor volume in the 6D8-G98A group mice increased slowly (see [link to experimental data]). Figure 9 (See Table 11). Calculation of tumor growth inhibition rate (TGI%) revealed that the 6D8-G98A group significantly inhibited tumor growth, with a TGI% greater than 50%. These results indicate that the 6D8-G98A antibody has an inhibitory effect on colon cancer in MC38 mice.

[0130] Table 11 Changes in tumor volume between the control group and the 6D8-G98A antibody treatment group.

[0131]

[0132] It is understood that although the inventions described in this application are in the specific forms described above, these inventions are not limited to the specific content described in these specific forms. It will be apparent to those skilled in the art that various equivalent changes can be made to the technical features contained in the inventions described herein without departing from the spirit of the inventions described herein, and all such changes should fall within the scope of the inventions.

[0133] References

[0134] [1] Watts, TH2005. TNF / TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol. 23: 23-68.

[0135] [2] Hintzen, RQ, SMLens, MP Beckmann, RG Goodwin, D. Lynch, and RAvan Lier. 1994. Characterization of the human CD27 ligand, a novel member of the TNF gene family. J. Immunol. 152: 1762-1773.

[0136] [3]Tesselaar,K.,L.A.Gravestein,G.M.van Schijndel,J.Borst,and R.A.vanLier.1997.Characterization of murine CD70,the ligand of the TNF receptorfamily member CD27.J.Immunol.159:4959-4965.

[0137] [4]Hintzen,R.Q.,S.M.Lens,K.Lammers,H.Kuiper,M.P.Beckmann,and R.A.vanLier.1995.Engagement of CD27 with its ligand CD70 provides a second signalfor T cell activation.J.Immunol.154:2612-2623.

[0138] [5]Taraban,V.Y.,T.F.Rowley,D.F.Tough,and A.Al-Shamkhani.2006.Requirement for CD70 in CD4+Th cell-dependent and innatereceptor-mediated CD8+T cell priming.J.Immunol.177:2969-2975.

[0139] [6]Keller,A.M.,A.Schildknecht,Y.Xiao,M.van den Broek,andJ.Borst.2008.Expression of costimulatory ligand CD70 on steady-statedendritic cells breaks CD8+T cell tolerance and permits effectiveimmunity.Immunity 29: 934-946.

[0140] [7]Keller,A.M.,Y.Xiao,V.Peperzak,S.H.Naik,andJ.Borst.2009.Costimulatory ligand CD70 allows induction of CD8+T-cellimmunity by immature dendritic cells in a vaccination setting.Blood 113:5167-5175.

[0141] [8]Bullock,T.N.,and H.Yagita.2005.Induction of CD70 on dendriticcells through CD40 or TLR stimulation contributes to the development of CD8+Tcell responses in the absence of CD4+T cells.J.Immunol.174:710-717.

[0142] [9]Ahrends,T.,N.Ba, Y.Xiao,H.Yagita,H.van Eenennaam,and J.Borst.2016.CD27 agonism plus PD-1 blockade recapitulates CD4+T-cell help intherapeutic anticancer vaccination.Cancer Res.76:2921-2931.

[0143]

[10] Arens,R.,K.Schepers,M.A.Nolte,M.F.van Oosterwijk,R.A.van Lier,T.N.Schumacher,and M.H.van Oers.2004.Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+T cellformation.J.Exp.Med.199∶1595-1605.

[0144]

[11] Dong,H.,N.A.Franklin,D.J.Roberts,H.Yagita,M.J.Glennie,andT.N.Bullock.2012.CD27 stimulation promotes the frequency of IL-7receptorexpressing memory precursors and prevents IL-12-mediated loss of CD8(+)T cell memory in the absence of CD4(+)T cell help.J.Immunol.188: 3829-3838.

[0145]

[12] Hendriks,J.,L.A.Gravestein,K.Tesselaar,R.A.van Lier,T.N.Schumacher,and J.Borst.2000.CD27 is required for generation and long-term maintenance of T cell immunity.Nat.Immunol.1:433-440.

[0146]

[13] Hendriks,J.,Y.Xiao,and J.Borst.2003.CD27 promotes survival ofactivated T cells and complements CD28 in generation and establishment ofthe effector T cell pool.J.Exp.Med.198∶1369-1380.

[0147]

[14] Schildknecht,A.,I.Miescher,H.Yagita,and M.van denBroek.2007.Priming of CD8+T cell responses by pathogens tyPically depends onCD70-mediated interactions with dendritic cells.Eur.J.Immunol.37: 716-728.

[0148]

[15] Welten, S.P., A. Redeker, K.L. Franken, C.A. Benedict, H. Yagita, F.M. Wensveen, J. Borst, C.J. Melief, R.A. van Lier, K.P. van Gisbergen, and R. Arens. 2013. CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection. J. Virol. 87: 6851-6865.

[0149]

[16] Hintzen RQ, de Jong R, Hack CE, Chamuleau M, de Vries EF, ten Berge IJ, Borst J, van Lier RA. A soluble form of the human T cell differentiation antigen CD27 is released after triggering of the TCR / CD3 complex. J Immunol. 1991, 147(1): 29-35.

[0150]

[17] He LZ, Prostak N, Thomas LJ, et al. Agonist anti-human CD27 monoclonal antibody induces T cell activation and tumor immunity in human CD27-transgenic mice. J Immunol. 2013, 191(8): 4174-4183.

[0151]

[18] Vitale LA, He LZ, Thomas LJ, et al: Development of a human monoclonal antibody for potential therapy of CD27-expressing lymphoma and leukemia. Clin Cancer Res. 2012;18(14): 3812-3821. Sequence Listing <110> Taizhe Huikang Biomedical Co., Ltd. Beijing ImmunoArk Pharmaceutical Technology Co., Ltd. <120> Chimeric anti-CD27 antibody <130> 21C12521CN <160> twenty two <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Asn Tyr Gly Met Asn 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <400> 2 Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <400> 3 Glu Gly Asp Gly Phe Asp Ser 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <400> 4 Glu Gly Asp Ala Phe Asp Ser 1 5 <210> 5 <211> 116 <212> PRT <213> Artificial Sequence <400> 5 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 6 <211> 116 <212> PRT <213> Artificial Sequence <400> 6 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Thr Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asp Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 7 <211> 116 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Asp Ser Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Asp Ser Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 9 <211> 116 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Asp Ser Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 116 <212> PRT <213> Artificial Sequence <400> 10 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Asp Ser Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 11 <211> 116 <212> PRT <213> Artificial Sequence <400> 11 Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Asp Ser Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <400> 12 Ser Ala Thr Ser Ser Val Asn Tyr Met His 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <400> 13 Asp Thr Ser Arg Leu Ala Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Gln Gln Trp Asn Thr Asn Pro Trp Thr 1 5 <210> 15 <211> 106 <212> PRT <213> Artificial Sequence <400> 15 Gln Phe Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 106 <212> PRT <213> Artificial Sequence <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 106 <212> PRT <213> Artificial Sequence <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 106 <212> PRT <213> Artificial Sequence <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 106 <212> PRT <213> Artificial Sequence <400> 19 Gln Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 106 <212> PRT <213> Artificial Sequence <400> 20 Gln Phe Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 106 <212> PRT <213> Artificial Sequence <400> 21 Gln Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 106 <212> PRT <213> Artificial Sequence <400> 22 Gln Phe Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Thr Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105

Claims

1. A chimeric antibody or its antigen-binding moiety that specifically binds to CD27, comprising heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3 sequences; wherein the HCDR1 sequence is the amino acid sequence shown in SEQ ID NO: 1: NYGMN; the HCDR2 sequence is the amino acid sequence shown in SEQ ID NO: 2: WINTNTGEPTYADDFKG; and the HCDR3 sequence is the amino acid sequence shown in SEQ ID NO: 4: EGDAFDS; and the LCDR1 sequence is the amino acid sequence shown in SEQ ID NO: 12: SATSSVNYMH; the LCDR2 sequence is the amino acid sequence shown in SEQ ID NO: 13: DTSRLAS; and the LCDR3 sequence is the amino acid sequence shown in SEQ ID NO: 14: QQWNTNPWT; and The chimeric antibody mentioned above is a human-mouse chimeric antibody.

2. The chimeric antibody or its antigen-binding portion as described in claim 1, wherein the antigen-binding portion is selected from the Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, and Fd fragment.

3. The chimeric antibody or its antigen-binding portion as described in claim 1 or 2, wherein the antibody or its antigen-binding portion specifically binds to human CD27 or monkey CD27.

4. The chimeric antibody or its antigen-binding portion as described in claim 1 or 2, wherein the antibody or its antigen-binding portion is capable of inducing T cell activation.

5. The chimeric antibody or its antigen-binding portion as claimed in claim 1, wherein the heavy chain sequence of the antibody is as shown in SEQ ID NO: 6, and the light chain sequence is as shown in SEQ ID NO:

15.

6. A nucleotide molecule encoding an antibody or its antigen-binding portion as described in any one of claims 1-5.

7. An expression vector comprising the nucleotide molecule of claim 6.

8. A host cell comprising the nucleotide molecule of claim 6 or the expression vector of claim 7.

Images