Antibody targeting CD70 or antigen binding fragment binding CD70 as well as preparation method and application thereof

Abstract

The invention relates to a CD70-targeting antibody or a CD70-binding antigen binding fragment, and a preparation method and application of the CD70-targeting antibody or the CD70-binding antigen binding fragment. The antibody targeting CD70 or the antigen binding fragment binding CD70 can block CD70 / CD27 signals in a targeted manner, and kill cancer cells expressing CD70 by enhancing antibody-dependent cell-mediated cytotoxic effect, antibody-dependent cell-mediated phagocytosis and complement-dependent cytotoxic effect, so that immune monitoring on solid tumors is recovered.

Description

Antibodies targeting CD70 or antigen-binding fragments binding to CD70 and preparation methods and applications thereof Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antibody targeting CD70 or an antigen-binding fragment binding to CD70, and a preparation method and application thereof. Background Art

[0002] T cell activation requires the coordinated action of two signals: the first signal, provided by the binding of antigen-specific MHC class II molecules to the CD3 / TCR protein complex; and the second signal, provided by the binding of co-stimulatory molecules on the surface of antigen-presenting cells or target cells to corresponding receptors on the T cell surface. These co-stimulatory molecules responsible for providing this second signal can be divided into two major categories: the tumor necrosis factor-tumor necrosis factor receptor (TNF-TNFR) superfamily and the immunoglobulin superfamily. CD27 and its ligand CD70 are two key members of the TNF-TNFR superfamily. Their interaction can activate the Wnt and NF-κB signaling pathways, regulating the proliferation and differentiation of T, B, and NK cells. CD70 is a type II transmembrane protein with an extracellular C-terminal portion, consisting of 193 amino acids and a molecular weight of 50 kDa.

[0003] The CD70 protein is typically present on the cell membrane as a trimer. Because its expression is highly regulated by antibodies, co-stimulatory molecules, and cytokines under physiological conditions, CD70 expression on the surface of activated T and B cells is short-lived, lasting only a few days, and is restricted to effector lymphocytes to maintain their normal physiological functions. In addition to these cells, CD70 is also expressed on mature dendritic cells (DCs) and natural killer cells (NK cells) to a limited extent, and is generally absent from normal non-lymphocyte tissues. CD70 expression is high in hematopoietic tumors, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, and Waldenstrom's macroglobulinemia. Multiple myeloma (MM) accounts for 1% of all cancers and approximately 10% of all hematologic malignancies. In the United States, over 32,000 new cases are diagnosed annually, and approximately 13,000 patients die from the disease. For decades, the age-adjusted annual incidence rate in the United States has remained stable at approximately 0.4 / 100,000 people. The incidence of multiple myeloma is slightly higher in men than in women, and twice as high in African Americans as in whites. The median age of patients at diagnosis is approximately 65 years. Among solid tumors, CD70 is also highly expressed in undifferentiated nasopharyngeal carcinoma, germ cell carcinoma, thymoma, Wilms tumor, glioblastoma, astrocytoma, and ovarian cancer. It is particularly expressed in renal cancer. Currently, CD70 is considered a new specific tumor marker for renal cancer. Statistics from 2020 show that there are approximately 430,000 new cases of renal cancer worldwide. In terms of histology, renal cell carcinoma (RCC) accounts for the vast majority of renal cancer cases (90%), mainly including clear cell renal cell carcinoma (ccRCC; 70%), papillary renal cell carcinoma (pRCC; 10-15%), and chromophobe renal cell carcinoma (5%). In summary, CD70 is expressed on the surface of activated T cells and B cells for a short time and is continuously highly expressed in tumors. Therefore, CD70 is a good target for some tumor targeting and immunotherapy.

[0004] Currently, over 30 new CD70-targeting drugs are registered for clinical trials, including CAR-T therapy, monoclonal antibodies, small molecule drugs, and ADCs. The most advanced is in Phase 2 clinical trials. Cusatuzumab, a naked CD70 antibody, is the most advanced of these drugs. Developed by Argenx under the development codename ARGX-110, Cusatuzumab acquired the rights to ARGX-110 in December 2018, with Janssen's subsidiary, Cilag GmbH International, securing a $300 million upfront payment. The Phase 2 AML trial achieved a complete remission rate of 67%, with no dose-limiting toxicities. In China, two naked CD70 antibodies are available: SEA-CD70, a collaboration between BeiGene and Seagen, which received clinical approval from the China Food and Drug Administration (CFDA) in 2021, and IMM40H, developed by Yimin Onco, which received clinical approval from both the FDA and the CFDA in August 2022. ARX305, a CD70-ADC jointly developed by Xinma Bio and Ambrx, was approved for clinical trials by CDE in July 2022, and PRO1160 of Pufang Bio is in preclinical development.

[0005] The resistance of leukemia stem cells (LSCs) to traditional therapies, including chemotherapy, is a key factor in leukemia relapse. Acute myeloid leukemia (AML) is predominant in the elderly, with a median age of onset of 67. A significant proportion of these patients are ineligible for chemotherapy, bone marrow transplantation, and other treatments at the time of onset. Hypomethylating agents (HMAs) can be used to treat these patients. When AML patients receive HMA treatment, leukemia stem cells (LSCs) upregulate CD70 expression, leading to increased CD70 / CD27 signaling and consequently treatment resistance.

[0006] Multiple myeloma (MM) is a plasma cell malignancy and the second most common hematologic malignancy in adults. Major complications for MM patients include tumor-induced bone damage and associated pathological fractures, anemia, renal failure, and immunodeficiency, leading to decreased quality of life and overall survival. Over the past two decades, the treatment options for MM have continued to grow with the advent of proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and monoclonal antibodies (mAbs), as well as autologous hematopoietic stem cell transplantation (auto-HSCT). However, despite recent advances in MM treatment paradigms, the disease remains incurable, and most patients inevitably relapse due to heterogeneity, immune evasion, and the evolution of drug-resistant clones. Conventional therapies offer limited benefit in high-risk patients, with particularly dismal outcomes for patients with MM who are double-refractory to insulin resistance and imipenem or who have relapsed after more than three prior lines of therapy. The 5-year overall survival rate for patients with MM who have high-risk cytogenetics or are frail, elderly patients is less than 50%. Therefore, new treatment modalities with minimal drug toxicity and good tolerability, as well as demonstrating deep and durable responses, are urgently needed.

[0007] The severity of kidney cancer depends on the type and stage of the disease. Surgery is the primary and most effective treatment. Chemotherapy and radiation therapy are generally not used as primary treatments for kidney cancer because, in most cases, patients do not respond to these treatments. However, surgery is not always possible. For example, the patient may have other medical conditions that prevent surgery, or the cancer may have spread to other organs, making it impossible for doctors to remove it.

[0008] Summary of the Invention

[0009] In the first aspect, in order to obtain an antibody or antigen-binding fragment that targets CD70 that is different from the prior art and to obtain a new drug independently developed, the present invention provides an antibody or antigen-binding fragment that targets CD70, and the specific scheme thereof is as follows:

[0010] An antibody targeting CD70 or an antigen-binding fragment thereof that binds to CD70, capable of specifically binding to CD70, and comprising:

[0011] a1) three heavy chain complementarity determining regions—HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are as shown in the amino acid sequences encoded by SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, or as shown in the amino acid sequences encoded by SEQ ID NO: 3, SEQ ID NO: 16, and SEQ ID NO: 5, respectively; and

[0012] a2) Three light chain complementary determining regions - LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively:

[0013] The antibody targeting CD70 is a polyclonal antibody or a monoclonal antibody;

[0014] The antigen-binding fragment that binds CD70 is:

[0015] b1) single-chain Fv; or

[0016] b2) disulfide-linked Fv; or

[0017] b3) Fab fragment; or

[0018] b4) F(ab')2 fragment; or

[0019] b5) Fab' fragment.

[0020] In some embodiments, it comprises a heavy chain variable region and a light chain variable region, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively; the heavy chain variable region comprises the three heavy chain complementary determining regions; the light chain variable region comprises the three light chain complementary determining regions.

[0021] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID NO: 9 and SEQ ID NO: 12, respectively, or as SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0022] In some embodiments, the antibody further comprises an Fc region, the amino acid sequence of the Fc region being shown in SEQ ID NO: 14 or SEQ ID NO: 15.

[0023] In some embodiments, the antibody or antigen-binding fragment is monospecific, bispecific, or multispecific.

[0024] In a second aspect, the present invention also discloses a nucleic acid comprising a nucleotide sequence encoding the antibody targeting CD70 or the antigen-binding fragment that binds to CD70 as described above.

[0025] In a third aspect, the present invention also discloses a composition comprising the antibody targeting CD70 or the antigen-binding fragment binding to CD70 as described above.

[0026] In a fourth aspect, the present invention discloses a biomaterial, which is:

[0027] c1) a vector, host cell or microorganism comprising the nucleic acid according to the second aspect; or

[0028] c2) the expression product, suspension or supernatant of c1).

[0029] In a fifth aspect, the present invention discloses an antibody-drug conjugate (ADC) comprising the antibody targeting CD70 or the antigen-binding fragment binding to CD70 as described above.

[0030] In a sixth aspect, the present invention discloses a protein degradation targeting chimera (PROTAC) molecule comprising an E3 ubiquitin ligase ligand, a target protein ligand, and a linker. The target protein ligand is a protein or polypeptide comprising an antibody targeting CD70 or an antigen-binding fragment that binds CD70 as described above. The E3 ubiquitin ligase ligand and the linker are conventional components of PROTAC molecules in the prior art and can be any combination of E3 ubiquitin ligase ligands and linkers in the prior art.

[0031] In a seventh aspect, the present invention discloses a method for producing an antibody or an antigen-binding fragment, which comprises culturing a host cell or a microorganism comprising the nucleic acid according to the second aspect and recovering the antibody or antigen-binding fragment from the culture.

[0032] In an eighth aspect, the present invention discloses the use of the antibody targeting CD70 or the antigen-binding fragment binding to CD70 as described above, or the nucleic acid as described above, or the composition as described above, or the biomaterial as described above, or the ADC as described above, or the PROTAC molecule as described above in the preparation of a medicament for treating tumors.

[0033] In some embodiments, the tumor is a hematological tumor and / or a solid tumor; the hematological tumor is selected from one or more of acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, diffuse large B-cell lymphoma, T-cell lymphoma, and chronic myelomonocytic leukemia; the solid tumor is renal cancer and / or ovarian cancer.

[0034] In a ninth aspect, the present invention discloses the use of the antibody targeting CD70 or the antigen-binding fragment that binds to CD70 as described above, or the nucleic acid as described above, or the composition as described above, or the biomaterial as described above, or the ADC as described above, or the PROTAC molecule as described above in the preparation of a preparation for blocking CD70-expressing positive cells.

[0035] In a tenth aspect, the present invention discloses the use of the above-mentioned antibody targeting CD70 or the antigen-binding fragment that binds CD70, or the above-mentioned nucleic acid, or the above-mentioned composition, or the above-mentioned biomaterial, or the above-mentioned ADC, or the above-mentioned PROTAC molecule and one or more other cancer therapeutic agents in the combined preparation of a drug for treating tumors or autoimmune diseases.

[0036] In an eleventh aspect, the present invention discloses that the antibody targeting CD70 or the antigen-binding fragment binding to CD70 as described above is administered by bolus injection, infusion, injection, epithelial absorption or skin and mucosal absorption.

[0037] CD70 is transiently expressed on activated T cells, B cells, and dendritic cells, but is almost completely absent in normal non-lymphoid tissues. It is highly expressed in a variety of hematologic malignancies and solid tumors, including B-cell lymphoma, renal cell carcinoma, and ovarian cancer. Currently, a wide range of drug types are being developed globally, including monoclonal antibodies, ADCs, CAR-T cells, and vaccines. Cusatuzumab, a drug developed in collaboration between Johnson & Johnson and Argenx (for which Johnson & Johnson paid a $300 million upfront payment in December 2018), has achieved a complete remission rate of 67% in a Phase II AML clinical trial, with no evidence of dose-limiting toxicities. Currently, no CD70-targeting products are available on the market.

[0038] CD70 is a cell surface antigen and a member of the tumor necrosis factor (TNF) family, typically present as a trimer. Screening for antibodies with high affinity for CD70 and the ability to block its biological activity is challenging. We optimized immunization methods, improved cell fusion techniques, and high-throughput screening to identify antibodies with high affinity and biological activity from a large number of hybridoma cells. By modifying the Fc region, we enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). The CD70-targeting antibody or the antigen-binding fragment that binds to CD70 constructed by the present invention can target and block the CD70 / CD27 signal, and kill cancer cells expressing CD70 by enhancing antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis, and complement-dependent cytotoxicity, thereby restoring immune monitoring of solid tumors (but not limited to solid tumors). Preclinical in vivo experimental studies in mice have shown that antibodies targeting CD70 have strong anti-tumor efficacy and good safety. CD70 can develop a variety of drug types. In addition to naked antibodies, various biomaterials such as CD70-ADC and anti-CD70CAR can also be developed, and their in vivo efficacy in highly expressed solid tumors such as renal cancer can be evaluated. It is expected to directly kill tumors that overexpress CD70 and improve the tumor microenvironment.

[0039] definition:

[0040] In the present invention, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies or antibody fragments.

[0041] The term "variable region (Fv)" refers to the domain of the antibody heavy chain variable region and / or light chain variable region that recognizes and specifically binds to an antigen epitope.

[0042] CDR region or claim " complementary determining region " refers to the region in the variable region of antibody that is hypervariable in sequence and forms the loop that determines in structure and / or contains antigen contact amino acid residue.CDR is mainly responsible for the binding of antibody and antigen epitope, determines the specificity of antibody.In a given heavy chain or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or combination of many well-known numbering conventions, and described numbering conventions include, for example, Kabat, Contact, AbM and Chothia.The CDR of the antibody of the present invention can be determined according to any rule or combination of these in the art.

[0043] The light chain variable region (VL) and heavy chain variable region (VH) both include three complementarity determining regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, FR4). The three CDRs in the light chain variable region (VL) are LC-CDR1, LC-CDR2, and LC-CDR3; the three CDRs in the heavy chain variable region (VH) are HC-CDR1, HC-CDR2, and HC-CDR3.

[0044] In the present invention, antibodies targeting CD70 are also referred to as anti-CD70 antibodies or anti-human CD70 antibodies, including murine anti-human CD70 antibodies, humanized anti-CD70 antibodies, and humanized anti-human CD70 antibodies. Antibodies targeting CD70 or antigen-binding fragments that bind to CD70 are anti-CD70 antibodies and antigen-binding fragments thereof.

[0045] The anti-CD70 antibodies or antigen-binding fragments thereof of the present invention comprise substitutions, insertions, or deletions. The anti-CD70 antibodies of the present invention include modifications to the light chain variable region, heavy chain variable region, light chain, or heavy chain, resulting in an amino acid sequence that differs from the amino acid sequence from which the antibody is derived. For example, an amino acid sequence derived from the same designated protein can be similar to the starting sequence, such as having a certain percentage identity, such as 90%, 92%, 96%, or 98% identity to the starting sequence.

[0046] As used herein, "identity" refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned between two peptides or between two nucleic acid molecules. Software programs known in the art can be used to determine the alignment and homology percentage or sequence identity, such as BLASTN and BLASTP.

[0047] "Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies, nanobodies and epitope-binding fragments of any of the above.

[0048] The term "antigen specificity" refers to a specific antigen or its epitope that is selectively recognized by an antigen binding molecule. The terms "treat" or "cure" or "alleviate" or "improve" are used interchangeably herein and refer to methods for obtaining beneficial or desired results (including but not limited to therapeutic benefit and / or preventive benefit). As used herein, therapeutic benefit generally refers to eradicating or alleviating the severity of the underlying condition being treated. In addition, by eradicating, alleviating severity or reducing the incidence of one or more physiological symptoms associated with the underlying condition, so that improvement is observed in the animal (although the animal may still be tortured by the underlying condition) to achieve therapeutic benefit. For preventive benefit, the risk of morbidity of an animal in the risk of developing a specific disease can be reduced. As used herein, the term "therapeutic effect" generally includes therapeutic benefit and / or preventive benefit as described above. Preventive effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of the symptoms of a disease or condition, slowing down, stopping or reversing the progress of a disease or condition, or any combination thereof.

[0049] The term "cell proliferation" generally refers to a phenomenon in which the number of cells changes due to division. For example, cell proliferation can result in an increase in the number of cells. The term also includes cell growth through which the cell morphology has changed (e.g., increased in size), which is consistent with a proliferation signal. As used herein, the term "proliferation inhibition" or "inhibition of cell proliferation" generally refers to a decrease in the growth rate and / or proliferation rate of cancer cells. For example, this can include the death of cancer cells (e.g., by apoptosis). In some embodiments, the term can also refer to inhibiting the growth and / or proliferation of solid tumors and / or inducing a reduction in the size of a tumor or eliminating it.

[0050] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal, including mammals or primates, for whom diagnosis, prognosis, alleviation, prevention, and / or treatment of a disease or condition is desired. Mammalian subjects include humans, livestock animals, farm animals, and zoo or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0051] As used herein, the term "in vivo" generally refers to events that occur within an animal's body.

[0052] As used herein, the term "in vitro" generally refers to an event that occurs outside an animal. For example, an in vitro cell function assay or any in vitro assay in an animal. In vitro assays include cell-based assays that use dead or live cells. In vitro assays also include cell-free assays that do not use intact cells.

[0053] As used herein, the term "administer" refers to delivering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to a subject. Administration can be systemic or local. Administration can be performed using an administration device, such as a syringe. Modes of administration include, but are not limited to, embedding, nasal inhalation, spraying, injection, and the like. Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration, and the like.

[0054] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 shows the blocking activity of anti-human CD70 antibodies on CD70-CD27 interaction.

[0056] Figure 2 shows that anti-human CD70 antibodies inhibit IL-8 secretion from HT1080-huCD27 cells.

[0057] FIG3 shows the ADCC effect of anti-human CD70 antibodies.

[0058] FIG4 shows the ADCP effect of anti-human CD70 antibodies.

[0059] FIG5 shows the CDC effect of anti-human CD70 antibodies.

[0060] Figure 6 shows the tumor volume (mm) of each group at each time point. 3 ).

[0061] Figure 7 is a photo of the tumor at the end of the experiment. DETAILED DESCRIPTION

[0062] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0063] Example 1 Preparation of Mouse Monoclonal Antibody Against Human CD70 - Primary Antibody

[0064] 1.1 Preparation of hybridoma cells producing mouse monoclonal antibodies

[0065] The method for preparing mouse monoclonal antibodies utilizes the hybridoma preparation technique invented by Kohler and Milstein in 1975 (Nature, 1975, 256:495-497). First, a recombinant human CD70 protein with a mouse Fc tag (Sino Biological, #10780-H04H5) was emulsified with Freund's adjuvant, and then multiple strains of mice were immunized. After four rounds of immunization, serum was collected and titered using an enzyme-linked immunosorbent assay (ELISA). Splenocytes from the best mice were then fused with SP2 / 0 myeloma cells. Hybridoma monoclonal cells were screened using HAT, and the cell culture supernatants were tested for binding activity to cell lines overexpressing human CD70, monkey CD70, and mouse CD70. Furthermore, the hybridoma antibodies were tested for their functional activity in inhibiting CD70-stimulated IL-8 secretion from HT1080-huCD27 cells. The final test data (partial data is recorded in Table 1) and the mean fluorescence intensity (MFI) value for human CD70 affinity was set at 2000 as the positive screening condition value, and the MFI value for monkey CD70 affinity was set at 3000 as the positive screening condition value. The hybridoma antibodies finally screened can bind to human and monkey CD70 but not to mouse CD70, and can effectively block the CD70-CD27 functional activity. The best hybridoma monoclonal cell line was selected for sequence analysis.

[0066] Table 1. Hybridoma screening data

[0067] Example 2 Cloning and humanization of anti-CD70 antibody variable region gene sequences

[0068] 2.1 Cloning of variable region genes of antibodies in hybridoma cells

[0069] Total RNA was extracted from mouse hybridoma cells using a QIAGEN RNA extraction kit (Cat. No. 74181) according to the manufacturer's instructions. Based on the principles of TAKARA's 5'RACE technology, cDNA sequences of the mouse antibody variable regions expressed by the hybridoma cell lines were cloned. Briefly, cDNAs specific for the heavy and light chain variable region genes were synthesized using the SMARTer 5'RACE Synthesis Kit (TAKARA, Cat. No. 634859) according to the manufacturer's instructions. The 5' and 3' ends of the cDNA sequences were modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, enabling seamless cloning of the resulting PCR products into the existing recombinant antibody expression vectors pHB-Fc and pHB-CK. The pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence; the pHB-CK vector contains the human κ light chain constant region gene sequence. The PCR amplified products of the heavy and light chain variable regions were cloned into an expression vector using an In-fusion cloning reagent (TAKARA, Catalog No. 639650) to generate a human-mouse chimeric antibody expression vector. This vector was then transformed into E. coli DH5α competent cells (Yisheng Biotechnology, Catalog No. FYE607-80VL). Monoclonal colonies were selected and subjected to Sanger sequencing, which revealed the variable region sequence of hybridoma clone 7C4. The variable region sequence of the resulting anti-CD70 chimeric antibody (Huabo No. 900713) is as follows:

[0070] [Corrected 31.01.2024 according to Rule 91] >900713 HCVR (heavy chain variable region amino acid sequence) SEQ ID NO: 1

[0071] [Corrected 31.01.2024 according to Rule 91] >900713 LCVR (light chain variable region amino acid sequence) SEQ ID NO: 2

[0072] Among them, the underlined region is CDR (Kabat definition), which is listed separately as shown in Table 2:

[0073] Table 2. CDR sequences of murine anti-CD70 antibodies

[0074] 2.2 Expression detection of chimeric antibodies

[0075] 2.2.1 Expression of Chimeric Antibodies: The expression vector obtained in 2.1 was amplified in E. coli and a sufficient amount of plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No. DP117) for transient transfection to express the chimeric antibody. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, Catalog No. R80007). The heavy chain and light chain vectors were mixed with polyetherimide (PEI, Polysciences, Catalog No. 24765-1) to form a liposome complex. The complex was then transfected into CHO-S cells and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain the human-mouse chimeric antibody.

[0076] 2.2.2 Chimeric Antibody Affinity Assay: A Biacore 8k was set to a sample chamber and flow path temperature of 25°C. A Series S Sensor Chip protein A was used to capture 900713 as the ligand and human CD70 (Cat#: CDL-H5246, ACRO Biosystems, Lot#: 2439C-9AWF1-Q9) as the analyte. Multiple dynamic cycle kinetics were performed. The flow rate was 30 μL / min, association: 120 s, dissociation: 600 s, and regeneration was performed in 10 mM glycine, pH 1.5, at a flow rate of 50 μL / min for 30 s. A 1:1 binding model was used, and the kinetic constants ka, kd, and KD were analyzed by local fitting. Table 3 shows these.

[0077] Table 3. Affinity data of chimeric antibodies

[0078] 2.2.3 Cross-species Qualification of Chimeric Antibodies: A Biacore 8k was set with the sample chamber and flow path temperature at 25°C. A CM5 chip with an amino-coupled anti-human Fc antibody was used to capture 900713 as the ligand, and CD70 from different species was used as the analyte. Multi-cycle kinetic analysis was performed. Flow rate: 30 μL / min, Association: 120 s, Dissociation: 180 s. A 1:1 binding model was used with 3 M MgCl2 at a flow rate of 50 μL / min for 30 s. Kinetic constants ka, kd, and KD were analyzed using a local fit. The results are shown in Table 4.

[0079] Table 4. Chimeric Antibody Species Cross-Species Data

[0080] 2.3 Humanization of Murine Anti-Human CD70 Antibodies - Preparation of Humanized Antibodies

[0081] The humanization of antibodies adopts the following method: first use the 3D modeling method to perform antibody structural homology modeling and select the optimal structural model, then analyze the original mouse sequence to clarify the sequence composition of different parts, and finally design the humanized sequence, mutate the mouse sequence into the humanized sequence, and finally express and verify the humanized sequence.

[0082] The heavy chain variable region was humanized using the germline genotype IGHV1, which has the highest homology, as a template. Three humanized sequences, huVH1-huVH3, were designed. The light chain variable region was humanized using the germline genotype IGKV1, which has the highest homology. Two humanized sequences, huVL1-huVL2, were designed. The sequences of huVH1-huVH3 are as follows:

[0083] [Corrected 31.01.2024 according to Rule 91] >huVH1 (SEQ ID NO: 9)

[0084] [Corrected 31.01.2024 according to Rule 91] >huVH2 (SEQ ID NO: 10)

[0085] [Corrected 31.01.2024 according to Rule 91] wherein HC-CDR2: WINTYSGVPTYVDKFKG (SEQ ID NO: 16) differs from SEQ ID NO: 4 by one amino acid.

[0086] [Corrected 31.01.2024 according to Rule 91] >huVH3 (SEQ ID NO: 11)

[0087] [Corrected 31.01.2024 according to Rule 91] The sequences of huVL1-huVL2 are as follows:

[0088] [Corrected 31.01.2024 according to Rule 91]>huVL1 (SEQ ID NO: 12)

[0089] [Corrected 31.01.2024 according to Rule 91]>huVL2 (SEQ ID NO: 13)

[0090] [Corrected 31.01.2024 according to Rule 91] Note: In SEQ ID NO:9, the I at position 2, I at position 20, S at position 28, A at position 49, F at position 68, F at position 70, L at position 72, L at position 81, and I at position 83 are mouse sequences. The A at position 9, V at position 11, AS at positions 16 and 17, K at position 19, R at position 38, Q at position 43, E at position 46, T at position 69, T at position 71, D at position 73, T at position 76, E at position 82, RS at positions 84 and 85, RSD at positions 87-89, V at position 93, Y at position 95, and V at position 115 are mutation sites. NO:3), WINTYSGVPTYVDDFKG (SEQ ID NO:4), and SRDYYYGSSY (SEQ ID NO:5) are CDR regions. [0090.1] [Corrected 31.01.2024 according to Rule 91] In SEQ ID NO: 10, the I at position 2, the S at position 28, the A at position 49, the F at position 68, the F at position 70, the L at position 72, and the L at position 81 are mouse sequences, the A at position 9, the V at position 11, the AS at positions 16 and 17, the KV at positions 19 and 20, the R at position 38, the Q at position 43, the E at position 46, the K at position 63, the T at position 69, the T at position 71, the D at position 73, the T at position 76, the ELRS at positions 82-85, the RSD at positions 87-89, the V at position 93, the Y at position 95, and the V at position 115 are mutation sites, and TYGMS (SEQ ID NO: 10) is a murine sequence. NO:3), WINTYSGVPTYVDKFKG (SEQ ID NO:16), and SRDYYYGSSY (SEQ ID NO:5) are CDR regions. [0090.2] [Corrected 31.01.2024 according to Rule 91] In SEQ ID NO:11, S at position 28 and A at position 49 are mouse sequences, V at position 2, A at position 9, V at position 11, AS at positions 16 and 17, KV at positions 19 and 20, R at position 38, Q at position 43, E at position 46, K at position 63, VTMTTD at positions 68-73, T at position 76, MELRS at positions 81-85, RSD at positions 87-89, V at position 93, Y at position 95, and V at position 115 are mutation sites, and TYGMS (SEQ ID NO:3), WINTYSGVPTYVDKFKG (SEQ ID NO:16), and SRDYYYGSSY (SEQ ID NO:5) are CDR regions. [0090.3] [Corrected 31.01.2024 according to Rule 91] In SEQ ID NO:12, F at position 36, DGTVT at positions 41-45, and I at position 48 are mouse sequences, S at position 7, V at position 15, T at position 22, K at position 39, T at position 72, S at position 77, Q at position 79, F at position 83, and G at position 100 are mutation sites, and RASQDISNYLN (SEQ ID NO:6), YTSKLHS (SEQ ID NO:7), and QQDSKLPLT (SEQ ID NO:8) are CDR regions. [0090.4] [Corrected 31.01.2024 according to Rule 91] In SEQ ID NO:13, F at position 36, V at position 44, and I at position 48 are mouse sequences, S at position 7, V at position 15, T at position 22, K at position 39, GKA at positions 41-43, K at position 45, T at position 72, S at position 77, Q at position 79, F at position 83, and G at position 100 are mutation sites, and RASQDISNYLN (SEQ ID NO:6), YTSKLHS (SEQ ID NO:7), and QQDSKLPLT (SEQ ID NO:8) are CDR regions.

[0091] Humanized point mutation antibody expression plasmids were expressed in CHO-S cells and purified to obtain humanized antibody proteins. Using Biacore, ELISA, and cell bioactivity assays, the humanized antibodies were screened for target affinity, CD70-CD27 interaction blocking activity, and activated cytokine release. Two high-performance humanized anti-CD70 antibodies were obtained, numbered 900819 (comprising huVH1 and huVL1) and 900820 (comprising huVH3 and huVL1).

[0092] Example 3 Detection of anti-human CD70 humanized antibody

[0093] 3.1 Biacore (SPR) detection of antibody affinity: Biacore 8k was set to 25℃ for sample chamber and flow path temperature. Antibody samples were captured using CM5 chips with amino-coupled anti-human Fc antibody as ligand. Serial dilutions of mouse (Mouse) CD70 (ACRO, Cat#: CDL-M5245, Lot#: 2723a-86WF1-JK), rat (Rat) CD70 (Sino Biological Inc, Cat#: 80161-R07H, Lot#: LC13AU1507), cynomolgus monkey CD70 (ACRO, Cat#: CDL-C5244, Lot#: 3870c-20AGF1-TZ), and human CD70 (ACRO, Cat#: CDL-H5246, Lot#: 2439c-9AWF1-Q9) were used as the analyzers for multi-cycle kinetic analysis. Flow rate: 30 μl / min, association: 120 s, dissociation: 600 s, 3 M MgCl2 regeneration at a flow rate of 50 μl / min for 30 s. A 1:1 binding model was used, and the kinetic constants ka, kd, and KD were analyzed using a local fit. The results are shown in Table 5.

[0094] Table 5. Affinity determination of candidate anti-human CD70 humanized antibodies Note: 900562 is a humanized anti-CD70 antibody developed by Argenx. Huabo independently expressed the gene based on the patented sequence. NA indicates not detected, and NB indicates no binding.

[0095] 3.2 Detection of blocking activity of anti-human CD70 humanized antibody on CD27-CD70 interaction

[0096] Anti- M2 mouse antibody (Sigma Aldrich, Cat#45-F3165) was diluted to 7 μg / mL and added to a 96-well microtiter plate (NUNC, Cat#442404) at 100 μL per well. The plate was incubated at 4°C overnight for 24 h. After coating, the plate was washed once with PBS-0.05% Tween. 200 μL of Assay Diluent (Biolegend, Cat#4331503) was added to each well of the plate and blocked for 2 h at room temperature with shaking. The plate was then washed three times with PBS-0.05% Tween. CD70-FLAG-His protein (Acro, Cat#CDL-H52Da) was diluted to 500 ng / mL with Assay Diluent and added to each well of the plate at 100 μL. The plate was incubated for 1 h at room temperature with shaking. The plate was then washed six times with PBS-0.05% Tween. The antibody to be tested was diluted to 60 μg / mL with Assay Diluent, and then diluted 3-fold to 11 concentrations, for a total of 12 concentration gradients. The diluted antibody was added to a 96-well plate at 50 μL per well. Then, 50 μL of recombinant human CD27 (R&D, Cat#382-CD-100) with a final concentration of 1 μg / mL was added to each well and incubated at room temperature for 1 hour while shaking. The plate was washed 6 times with PBS-0.05% Tween. A 1:500 dilution of biotin anti-CD27 (eBioscience, Cat#13-0271-82) was added to the 96-well plate, 100 μL per well, and incubated at room temperature for 1 hour while shaking. The plate was washed 6 times with PBS-0.05% Tween. Streptavidin-HRP (CST, Cat#3999S) diluted 1:1000 was added to each well of a 96-well plate, and incubated at room temperature for 1 hour while shaking. The plate was washed 6 times with PBS-0.05% Tween. TMB (Biolegend, Cat#421101) was added to each well of the 96-well plate, and then color was developed for 10 minutes. 100 μL of 2M H2SO4 was added to each well and the plate was analyzed on a microplate reader (MD OD450 and OD570 were measured on a 3x plate.

[0097] The results are shown in Figure 1 , and the humanized antibody retained CD27-CD70 blocking activity comparable to that of the murine parent antibody 900713.

[0098] 3.3 Detection of the inhibitory effect of humanized anti-human CD70 antibody on IL-8 secretion by HT1080-huCD27 cells

[0099] After CD70 and CD27 bind, CD27 cells will secrete IL-8. This experiment detected the IL-8 secretion of CD27-expressing cells to examine the effect of anti-CD70 antibodies on the level of CD27 signaling induced by CD70.

[0100] Raji cells (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Cat#TCHu 44) were collected and resuspended in RPMI1640 (Gibco Cat#A1049101) containing 10% FBS (Gibco Cat#10099141C) and diluted to 1×10 6 HT1080-huCD27-2C3 cells [human fibrosarcoma cells expressing human CD27 (ATCC, catalog number CCL-121), clone number 2C3, self-constructed by Huabo] were collected and resuspended in RPMI1640 containing 10% FBS and diluted to 2×10 cells / mL. 5 cells / mL.

[0101] The test antibody was diluted to 90 μg / mL in RPMI1640 containing 10% FBS and then diluted five-fold into eight concentrations, for a total of nine concentrations. Raji cells were plated at a 1:1 ratio with the antibody at each concentration, and 50 μL of each was added to a 96-well plate (Corning Cat#3599). After incubation at room temperature for 60 minutes, 50 μL of HT1080-huCD27-2C3 cells were added to each well.

[0102] After incubation at 37°C, 5% CO2 for 18-20 hours, the 96-well plate was removed and centrifuged at 1000 rpm (Thermo Fisher SCIENTIFIC, Sorvall ST16 / 16R) for 5 minutes to collect the supernatant. ELISA detection was then performed using an IL-8 ELISA kit (Biolegend, Cat#4331503), and OD450 and OD570 were measured on a microplate reader.

[0103] The results are shown in Figure 2. The inhibitory effect of the humanized antibody on cell activation was enhanced compared to the mouse parent antibody 900713 and was close to that of the control antibody 900562.

[0104] 3.4 In vitro ADCC experiments of humanized anti-human CD70 antibodies on Raji cells

[0105] Antibody-mediated cytotoxicity (ADCC) was detected using a reporter gene assay. ADCC FcγRIIIa (158V) Jurkat cells (Jiman Biotechnology, Cat#GM-C05619) were used as effector cells, and Raji cells were used as target cells. Raji cells were harvested, resuspended in RPMI1640 (Gibco, Cat#A1049101) containing 0.5% FBS (Gibco, Cat#10099141C), and diluted to 1.6 × 10 6 ADCC FcγRIIIa (158V) Jurkat cells were collected and resuspended in RPMI1640 containing 0.5% FBS, and the cells were diluted to 5×10 6 cells / mL.

[0106] Dilute the test antibody to 1.5 μg / mL in RPMI1640 with 0.5% FBS, and then dilute it three-fold into eight concentrations, for a total of nine concentrations. Add 30 μL of Raji cell suspension to each well of a 96-well white plate (Corning, Cat#3917), followed by 30 μL of the diluted test antibody and 30 μL of ADCC FcγRIIIa (158V) Jurkat cell suspension. Incubate in a 37°C, 5% CO2 incubator for 60 minutes.

[0107] Take out the 96-well white plate, equilibrate at room temperature for 15 minutes, and add Bio-Glo to the 96-well plate. TM Luciferase Assay System (Promega, Cat# G7940), 90 μL per well, react at room temperature in the dark for 5 minutes, and immediately use microplate reader (MD i3x) for full wavelength detection.

[0108] The results are shown in Figure 3. Antibodies 900823 and 900827 exhibit significantly enhanced ADCC activity compared to their parental antibodies 900819 and 900820, and are also stronger than the control antibody 900562. Note: 900823 is an Fc-enhanced version of 900819, and 900827 is an Fc-enhanced version of 900820. Both antibodies have Fc mutations F243L / R292P / Y300L / V305I / P396L (LPLIL) to enhance ADCC / ADCP / CDC activity. These antibodies were independently expressed and synthesized by Huabo.

[0109] >Fc sequence of 900819, 900820 (SEQ ID NO: 14):

[0110] [Corrected 31.01.2024 according to Rule 91] > Fc sequence of 900823 and 900827 (SEQ ID NO: 15), wherein L at position 126 / P at position 175 / L at position 183 / I at position 188 / L at position 279 is F243L / R292P / Y300L / V305I / P396L (LPLIL) mutation:

[0111] 3.5 In vitro ADCP assay of humanized anti-human CD70 antibody against CHOK1-huCD70-3G8 cells

[0112] Antibody-mediated cellular phagocytosis (ADCP) was detected using a reporter gene assay. ADCP FcγRIIa Jurkat cells (Jiman Biotechnology, GM-C09467) were used as effector cells, and CHOK1-huCD70-3G8 cells (Chinese hamster ovary cells expressing human CD70, clone 3G8, Huabo self-constructed) were used as target cells. CHOK1-huCD70-3G8 cells were harvested and resuspended in RPMI1640 (Gibco Cat#A1049101) containing 0.5% FBS (Gibco Cat#10099141C) and diluted to 4.5×10 6 ADCP FcγRIIa Jurkat cells were collected and resuspended in RPMI1640 containing 0.5% FBS, and the cells were diluted to 6×10 6 cells / mL.

[0113] Dilute the test antibody to 4 μg / mL in RPMI1640 with 0.5% FBS, then dilute it 2-fold into eight concentrations, for a total of nine concentrations. Add 100 μL of the CHOK1-huCD70-3G8 cell suspension to each well of a 96-well white plate (Corning Cat#3917), followed by 50 μL of the diluted test antibody and 50 μL of the Jurkat-CD32a-NFAT cell suspension. Incubate in a 37°C, 5% CO2 incubator for 6.5 hours.

[0114] Take out the 96-well white plate, equilibrate at room temperature for 15 minutes, place the 96-well white plate in a centrifuge (Beckman, ALLegra X-12), and centrifuge at 300g for 5 minutes. After using a pipette to remove and discard 100μL of the culture medium supernatant, add Bio-Glo to the 96-well plate. TM Luciferase Assay System (Promega, Cat# G7940), 100 μL per well, react at room temperature in the dark for 5 minutes, and immediately use microplate reader (MD i3x) for full wavelength detection.

[0115] As shown in FIG4 , the ADCP activities of antibodies 900823 and 900827 were significantly enhanced compared to the parent antibodies 900819 and 900820, and were both stronger than the control antibody 900562.

[0116] 3.6 In vitro CDC experiment of humanized anti-human CD70 antibody on Raji cells

[0117] Raji cells were collected and resuspended in RPMI1640 (Gibco, Cat#A1049101) without FBS, and the cells were diluted to 1×10 6 cells / mL and set aside. Dilute the test antibody to 12 μg / mL in RPMI1640 without FBS, then serially dilute by 2-fold for a total of 10 concentrations. Dilute the stock solution of Normal Human Serum Complement (Quidel, Cat#A112) 2.5-fold in the above medium.

[0118] The complement dilution solution and the antibody dilution solution of each concentration gradient were added in a 1:1 ratio to a 96-well cell culture plate (Corning, Cat#3599), and 25 μL of each was mixed. Subsequently, the treated Raji cell suspension was plated at 5×10 4 cells (i.e., 50 μL per well) were added to a 96-well cell culture plate and cultured in a 37° C., 5% CO 2 incubator for 3 hours.

[0119] After the incubation, 10 μL of lysis solution (Tongren Chemical, Cat#CK12) was added to the control wells containing only cells. The cells were incubated in a 37°C, 5% CO2 incubator for 30 minutes. Then, 100 μL of working solution (Tongren Chemical, Cat#CK12) was added to all wells. The cells were shaken at room temperature in the dark for 10 minutes. Then, 50 μL of stop solution (Tongren Chemical, Cat#CK12) was added to all wells. The OD490 reading was detected using a microplate reader.

[0120] The percentage of cell lysis was determined by the following equation: lysis % = (OD490 of experimental group - OD490 of natural release group) / (OD490 of maximum release group - OD490 of natural release group) x 100%.

[0121] As shown in FIG5 , the CDC activities of antibodies 900823 and 900827 were significantly enhanced compared to the parent antibodies 900819 and 900820, and were comparable to the CDC activity of the control antibody 900562.

[0122] Example 3.7 Non-specific binding test of anti-human CD70 humanized antibody

[0123] The Biacore 8k sample chamber and flow path temperature were set at 25°C. Lysozyme solution from chicken egg (Lysozyme) and trypsin inhibitor type 1-S from Glycine max (Trypsin inhibitor) were amino-coupled to a CM5 chip as ligands. The antibody was diluted to 1 μM, the flow rate was 10 μL / min, the association time was 900 s, and the dissociation time was 1200 s. Regeneration was performed first with 0.85% phosphoric acid at a flow rate of 50 μL / min for 30 s, followed by regeneration with 50 mM sodium hydroxide at a flow rate of 50 μL / min for 30 s. Binding stability signals were collected to analyze nonspecific electrostatic binding of the antibody.

[0124] Quality control of lysozyme solution from chicken egg and trypsin inhibitor type 1-S from Glycine max using polyclonal rabbit anti-lysozyme (Rb pAb to Lysozyme) and anti-trypsin inhibitor antibody (Anti-Trypsin Inhibitor), respectively, confirmed the normal activity of egg white lysozyme and soybean trypsin inhibitor after amino-coupling immobilization on the CM5 chip, indicating that the immobilized ligand activity was maintained throughout the experiment. The PI of trypsin inhibitor type 1-S from Glycine max was 4.5, and that of lysozyme solution from chicken egg was 11.3. In the HBS-EP buffer system at pH 7.4, trypsin inhibitor type 1-S from Glycine max has a strong negative charge, while lysozyme solution from chicken egg has a strong positive charge.

[0125] Table 6 Non-specific binding data of anti-human CD70 humanized antibodies

[0126] It can be seen from Table 6 that the binding response values ​​of all anti-human CD70 humanized antibody test samples (900819 and 900820) with Lysozyme and Trypsin are less than 20RU, which indicates that the anti-human CD70 humanized antibody test samples have no nonspecific electrostatic binding effect.

[0127] Example 4 Evaluation of the efficacy of humanized antibodies in mice

[0128] Containing 5×10 6 100 μL of PBS suspension of U266B1 cells (ATCC, catalog number TIB196) was mixed with an equal volume of Matrigel and inoculated subcutaneously into 5-6 week old female SCID mice (Shanghai Slake Laboratory Animal Co., Ltd.) at the right side of the mouse's back near the armpit. When the tumor grew to an average of approximately 200 mm 3 Around 14 days after the last administration, 36 tumor-bearing mice were selected and randomly divided into 6 groups of 6 mice each according to tumor volume and body weight using E-WorkBook software. The day of group administration was defined as day 0, and the weight of the mice was measured and recorded twice a week during the administration period. Tumor volume and body weight were measured twice a week. At the end of the experiment (7 days after the last administration), all animals were euthanized using CO2, and then all mouse tumor tissues were collected, weighed and photographed. The dosing schedule is shown in Table 7:

[0129] Table 7. Grouping and Dosage Regimen

[0130] Note: 900201 is a negative control antibody; 900562-afu is a positive control antibody synthesized in-house by Huabo based on the cusatuzumab sequence in WHO-INN-PL_118 (WHO Drug Information, Vol. 31, No. 4, 2017); HB0050 is an antibody produced in a stable CHO-K1 cell line constructed by Huabo (derived from humanized molecule 900823); and HB0051 is an antibody produced in a stable CHO-K1 cell line constructed by Huabo (derived from humanized molecule 900827). ivQW*4W refers to intravenous administration once weekly for four consecutive weeks.

[0131] During the experiment, most animals in each group were in good spirits and their weights were relatively stable.

[0132] As shown in Figure 6, this experiment successfully established a subcutaneous transplant tumor model of human multiple myeloma U266B1 cell line in SCID mice. The tumor volume of mice in the G1 900201 negative control group continued to grow, with an average tumor volume of 200.80 mm on day 0. 3At the end of the experiment (day 28), the mean tumor volume reached 652.58 mm 3 , the relative tumor volume reached 3.25.

[0133] Compared with the 900201 negative control group, the tumor volume and relative tumor volume of the HB0050 and HB0051 anti-CD70 antibody administration groups were significantly reduced from day 4 to day 28 (p<0.01).

[0134] As shown in Figure 6 and Table 8, compared with the negative control group (G1 900201), test group G2 to G6 showed significant reductions in tumor volume (TV) and relative tumor volume (RTV) during the dosing period (p < 0.01 or p < 0.05). On day 28, the relative tumor inhibition rates were 17.54%, 89.23%, 96.62%, 90.77%, and 92.62%, respectively, and the tumor weight inhibition rates were 20.37%, 88.89%, 96.30%, 93.52%, and 92.59%, respectively.

[0135] Table 8. Relative tumor inhibition rate TGI (%)

[0136] Figure 7 shows tumor photos collected at the end of the experiment. Compared with the negative control group during the same period, the tumor volume in the HB0050 and HB0051 anti-CD70 antibody administration groups was significantly reduced.

[0137] In summary, the test substances HB0050 and HB0051 anti-CD70 antibodies can both exert significant tumor inhibitory effects.

[0138] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. An antibody targeting CD70 or an antigen-binding fragment binding to CD70, characterized in that: Capable of specifically binding to CD70 and comprising: a1) three heavy chain complementary determining regions - HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, or shown in the amino acid sequences encoded by SEQ ID NO:3, SEQ ID NO:16, and SEQ ID NO:5, respectively; and a2) three light chain complementary determining regions, LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively: The antibody targeting CD70 is a polyclonal antibody or a monoclonal antibody; The antigen-binding fragment that binds CD70 is: b1) single chain Fv; or b2) disulfide-linked Fv; or b3) Fab fragment; or b4) F(ab')2 fragment; or b5) Fab' fragment.

2. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 1, characterized in that: It comprises a heavy chain variable region and a light chain variable region, and its amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively; the heavy chain variable region comprises the three heavy chain complementary determining regions; the light chain variable region comprises the three light chain complementary determining regions.

3. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 2, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID NO: 9 and SEQ ID NO: 12, respectively, or as shown as SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

4. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 1, characterized in that: It also comprises an Fc region, the amino acid sequence of the Fc region is shown in SEQ ID NO: 14 or SEQ ID NO:

15.

5. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 1, characterized in that: It is a monospecific, bispecific or multispecific antibody or antigen-binding fragment.

6. A nucleic acid, characterized in that It comprises a nucleotide sequence encoding the antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to any one of claims 1 to 5.

7. A composition, characterized in that It comprises the antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to any one of claims 1 to 5.

8. A biomaterial, characterized in that for: c1) a vector, a host cell or a microorganism comprising the nucleic acid according to claim 6; or c2) the expression product, suspension or supernatant of c1).

9. An antibody-drug conjugate, characterized in that: Contains the antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to any one of claims 1 to 5.

10. A PROTAC molecule comprising an E3 ubiquitin ligase ligand, a target protein ligand and a linker, characterized in that: The target protein ligand is a protein or polypeptide containing the CD70-targeting antibody or the CD70-binding antigen-binding fragment according to any one of claims 1 to 5.

11. A method for producing an antibody or antigen-binding fragment, characterized in that The host cell or microorganism comprising the nucleic acid according to claim 6 is cultured and the antibody or antigen-binding fragment is recovered from the culture.

12. Use of an antibody targeting CD70 or an antigen-binding fragment binding to CD70 according to any one of claims 1 to 5, or a nucleic acid according to claim 6, or a composition according to claim 7, or a biomaterial according to claim 8, or an antibody-drug conjugate according to claim 9, or a PROTAC molecule according to claim 10 in the preparation of a medicament for treating tumors.

13. The use according to claim 12, characterized in that: The tumor is a blood tumor and / or a solid tumor; the blood tumor is selected from one or more of acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, diffuse large B-cell lymphoma, T-cell lymphoma, and chronic myelomonocytic leukemia; the solid tumor is renal cancer and / or ovarian cancer.

14. Use of an antibody targeting CD70 or an antigen-binding fragment that binds CD70 according to any one of claims 1 to 5, or a nucleic acid according to claim 6, or a composition according to claim 7, or a biomaterial according to claim 8, or an antibody-drug conjugate according to claim 9, or a PROTAC molecule according to claim 10 in the preparation of a preparation for blocking CD70-expressing positive cells.

15. Use of an antibody targeting CD70 or an antigen-binding fragment binding to CD70 according to any one of claims 1 to 5, or a nucleic acid according to claim 6, or a composition according to claim 7, or a biomaterial according to claim 8, or an antibody-drug conjugate according to claim 9, or a PROTAC molecule according to claim 10 and one or more other cancer therapeutic agents in the combined preparation of a drug for treating tumors or autoimmune diseases.

16. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to any one of claims 1 to 5 is administered by bolus injection, infusion, injection, epithelial absorption or skin and mucosal absorption.

Images