WO2024153016A1 - Antibody targeting cd70 or antigen binding fragment binding to cd70, and preparation method therefor and use thereof - Google Patents

Abstract

An antibody targeting CD70 or an antigen binding fragment binding to CD70, and a preparation method therefor and the use thereof. The antibody targeting CD70 or the antigen binding fragment binding to CD70 can block a CD70/CD27 signal in a targeted manner, and can kill cancer cells expressing CD70 by means of enhancing the antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis and complement-dependent cytotoxicity, thereby restoring immune surveillance for solid tumors.

Description

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

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 technique

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

CD70 protein usually exists on the cell membrane in the form of trimers. Since CD70 is highly precisely regulated by antibodies, co-stimulatory molecules and cytokines under physiological conditions, CD70 is expressed on the surface of activated T cells and B cells for a short time, only for a few days, and is only expressed on the surface of effector lymphocytes to maintain its normal physiological function. In addition to the above cells, CD70 is also limitedly expressed on the surface of mature dendritic cells (DCs) and natural killer cells (NK cells), and is usually absent in non-lymphocyte normal tissues. CD70 is highly expressed in hematogenous tumors, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia and Waldenstroms macroglobulinemia. Multiple myeloma (MM) accounts for 1% of all cancers and about 10% of all hematological malignancies. In the United States, more than 32,000 new cases are diagnosed each year, and about 13,000 patients die from the disease. For decades, the age-adjusted annual incidence 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, high expression also occurs in undifferentiated nasopharyngeal carcinoma, as well as in tumors such as germ cell carcinoma, thymoma, Wilms tumor, glioblastoma, astrocytoma, and ovarian cancer. The expression in renal cancer is particularly significant. CD70 is currently considered a new specific tumor marker for renal cancer. According to statistics in 2020, there are approximately 430,000 new cases of renal cancer worldwide. In terms of histology, renal cell carcinoma (RCC) accounts for the vast majority (90%) of renal cancer cases, 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.

In the CD70 direction, more than 30 clinical trials of new drugs targeting CD70 have been registered, including CAR-T therapy, monoclonal antibodies, small molecule drugs and ADC, and the fastest progress is in clinical phase 2. Cusatuzumab, a CD70 naked antibody, is the fastest-progressing drug targeting CD70. Cusatuzumab's research and development code is ARGX-110, developed by Argenx; in December 2018, Cilag GmbH International, a subsidiary of Janssen, reached an agreement with Argenx and obtained the authorization of ARGX-110, for which Janssen paid an advance payment of US$300 million. The complete remission rate CR of Phase II AML was 67%, and no dose-limiting toxicity was shown. CD70 naked antibodies in China include SEA-CD70, a collaboration between BeiGene and Seagen, which received CDE clinical approval in 2021, and IMM40H of Yiminangke received FDA and CDE clinical approval in August 2022. ARX305, a CD70-ADC jointly developed by Sinopharm Biopharma and Ambrx, was approved for clinical trials by CDE in July 2022, and PRO1160 of Pufang Biopharma is in preclinical development.

The resistance of leukemia stem cells (LSCs) to traditional therapies such as chemotherapy is an important factor in the recurrence of leukemia. Acute myeloid leukemia (AML) is common in the elderly, with a median age of 67 years. A considerable proportion of these patients are unable to receive chemotherapy, bone marrow transplantation and other therapies at the time of onset. Hypomethylating agents (HMAs) can be used to treat these patients. When AML patients are treated with hypomethylating agents (HMAs), leukemia stem cells (LSCs) upregulate CD70 expression, resulting in increased CD70/CD27 signal transduction, which leads to treatment resistance.

Multiple myeloma (MM) is a plasma cell malignancy and the second most common hematological malignancy in adults. Major complications of 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 multiple myeloma 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 considerable progress in the treatment paradigm of multiple myeloma, the disease remains incurable, and most patients will inevitably relapse eventually due to heterogeneity, immune evasion, and persistence of resistant clones. Conventional therapies have limited benefits for high-risk patients, and the results are particularly dismal for multiple myeloma patients who are double refractory to insulin resistance and imipenem or who have relapsed after more than three previous treatments. The 5-year overall survival rate for patients with high-risk cytogenetics multiple myeloma or frail elderly patients is less than 50%. Therefore, new treatment modalities with minimal drug toxicity and good tolerability as well as showing deep and durable responses are urgently needed.

The severity of kidney cancer depends on the type and stage of the disease. The main and most effective treatment is surgery. Chemotherapy and radiation therapy are generally not used as the main treatment 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 diseases that prevent surgery from being performed, or the cancer may have spread to other organs and it is no longer possible for doctors to remove it.

Summary of the invention

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

An antibody targeting CD70 or an antigen-binding fragment binding to CD70, which can specifically bind to CD70 and comprises:

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 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.

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 includes the three heavy chain complementary determining regions; the light chain variable region includes the three light chain complementary determining regions.

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 shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

In some embodiments, 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.

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

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

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.

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

c1) a vector, a host cell or a microorganism comprising the nucleic acid as described in the second aspect; or

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

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.

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

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 as described in the second aspect and recovering the antibody or the antigen-binding fragment from the culture.

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

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.

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

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

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.

CD70 is transiently expressed on activated T cells, B cells, and dendritic cells, but is almost not expressed on normal non-lymphoid tissues. It is highly expressed in a variety of hematological tumors and solid tumors, such as B-cell lymphoma, renal cancer, ovarian cancer, etc. At present, a variety of drug types have been developed in the world, including monoclonal antibodies, ADCs, CAR-T, vaccines, etc. The fastest progress is Cusatuzumab, which was jointly developed by Johnson & Johnson and Argenx (Johnson & Johnson paid an advance payment of US$300 million in December 2018). The clinical complete remission rate CR of Phase II AML is 67%, and no dose-limiting toxic effects have been shown. There are currently no products targeting CD70 on the market.

CD70 is a cell surface antigen and a member of the tumor necrosis factor (TNF) family. It usually exists in the form of a trimer. It is difficult to screen antibodies with high affinity for CD70 and that can block its biological activity. We screened antibodies with high affinity and biological functional activity from a large number of hybridoma cells by optimizing immunization methods, improving cell fusion technology, and high-throughput screening. By modifying Fc, the activity of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP) was enhanced. The CD70-targeting antibody or the antigen-binding fragment binding to CD70 constructed by the present invention can target and block CD70/CD27 signals, 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.

definition:

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

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.

CDR region or "complementarity determining region" refers to the region in the variable region of an antibody that is highly variable in sequence and forms a loop that is structurally determined and/or contains antigen contact amino acid residues. CDR is primarily responsible for the binding of the antibody to the antigen epitope and determines the specificity of the 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 of many well-known numbering rules or a combination thereof, including, for example, Kabat, Contact, AbM and Chothia. The CDR of the antibody of the present invention can be determined according to any rule or a combination thereof in the art.

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

In the present invention, antibodies targeting CD70 are also referred to as anti-CD70 antibodies, anti-human CD70 antibodies, including mouse anti-human CD70 antibodies, humanized anti-CD70 antibodies, and anti-human CD70 humanized antibodies. Antibodies targeting CD70 or antigen-binding fragments binding to CD70 are anti-CD70 antibodies and antigen-binding fragments thereof.

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 comprise modifications to the light chain variable region, the heavy chain variable region, the light chain or the heavy chain, and the amino acid sequence thereof after modification is different from the amino acid sequence from which the antibody is derived. For example, an amino acid sequence derived from the same specified protein may be similar to the starting sequence, for example, having a certain percentage identity, for example, it may have a percentage identity of 90%, 92%, 96%, 98% with the starting sequence.

In the present invention, "identity" refers to the percentage of bases (or amino acids) 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.

"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.

The term "antigen specificity" refers to a specific antigen or its epitope selectively recognized by an antigen binding molecule. The terms "treatment" or "treatment" or "relief" or "improvement" 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 eradication or mitigation of the severity of the underlying condition being treated. In addition, by eradicating, mitigating severity or reducing the incidence of one or more physiological symptoms associated with the underlying condition, so that improvements are observed in animals (although the animal may still be afflicted with the underlying condition) to achieve therapeutic benefit. For preventive benefit, the risk of morbidity of animals at 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 effects include delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing down, stopping or reversing the progress of a disease or condition, or any combination thereof.

The term "cell proliferation" generally refers to the phenomenon that the number of cells changes due to division. For example, cell proliferation can lead to 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 may also refer to inhibiting the growth and/or proliferation of solid tumors and/or inducing a reduction in the size or elimination of tumors.

The term "subject" or "individual" or "animal" or "patient" used in this application 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, etc.

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

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

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 may be systemic or topical. Administration may be performed by 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.

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

FIG1 shows the blocking activity of anti-human CD70 antibodies on the interaction between CD70 and CD27.

Figure 2 shows that anti-human CD70 antibody inhibits IL-8 secretion by HT1080-huCD27 cells.

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

FIG. 4 shows the ADCP effect of anti-human CD70 antibodies.

FIG. 5 shows the CDC effect of anti-human CD70 antibodies.

FIG6 shows the tumor volume (mm ³ ) of each group at each time point.

Figure 7 is a photo of the tumor at the end of the experiment.

Detailed ways

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.

Example 1 Preparation method of mouse monoclonal antibody against human CD70 - original antibody

1.1 Preparation of hybridoma cells producing mouse monoclonal antibodies

The method for preparing mouse monoclonal antibodies uses the hybridoma preparation technology invented by Kohler and Milstein in 1975 (Nature, 1975, 256: 495-497). First, the human CD70 recombinant 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 the titer was tested by enzyme-linked immunosorbent assay (ELISA). The best mice were selected to obtain spleen cells and fused with SP2/0 myeloma cells. After HAT screening of hybridoma monoclonal cells, the cell culture supernatant was tested for binding activity in cell lines overexpressing human CD70, monkey CD70, and mouse CD70, as well as the functional activity of hybridoma antibodies in inhibiting CD70-stimulated HT1080-huCD27 cells from secreting IL-8. The final test data (part of the data is recorded in Table 1) and the mean fluorescence intensity (MFI) value of human CD70 affinity are 2000 as the positive screening condition value, and the MFI value of monkey CD70 affinity is 3000 as the positive screening condition value. The hybridoma antibody finally screened can bind to human and monkey CD70 but not to mouse CD70, and can well block the CD70-CD27 functional activity. The best hybridoma monoclonal cell line selected was sequence analyzed.

Table 1. Hybridoma screening data

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

2.1 Cloning of variable region genes of antibodies in hybridoma cells

The total RNA in mouse hybridoma cells was extracted using the RNA extraction kit (Cat. No. 74181) from QIAGEN according to the manufacturer's instructions. Based on the principle of TAKARA's 5'RACE technology, the cDNA sequence of the variable region of the mouse antibody expressed by the hybridoma cell line was cloned. In brief, the variable region gene-specific cDNA of the heavy chain and light chain was synthesized using the SMARTer 5'RACE synthesis kit (TAKARA, Cat. No. 634859) according to the instructions. The 5' and 3' ends of the cDNA sequence were modified with PCR primers, which were designed to add appropriate leading sequences to the heavy chain and light chain variable region cDNAs, respectively, so that the resulting PCR products could be cloned into the existing recombinant antibody expression heavy chain vector pHB-Fc and light chain vector pHB-Cκ by seamless cloning. The pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence; the pHB-Cκ vector contains the human κ light chain constant region gene sequence. The PCR amplification products of the heavy chain and light chain variable regions were cloned into the expression vector using the In-fusion cloning reagent (TAKARA, catalog number 639650) to obtain the human-mouse chimeric antibody expression vector, and then transformed into E. coli DH5α competent cells (Yisheng Biotechnology, catalog number FYE607-80VL). The variable region sequence of the hybridoma clone 7C4 antibody was obtained by selecting monoclonal colonies for Sanger sequencing. The variable region sequence of the constructed anti-CD70 chimeric antibody (Huabo No. 900713) is as follows:

[Corrected 31.01.2024 in accordance with Article 91]
>900713 HCVR (heavy chain variable region amino acid sequence) SEQ ID NO: 1

[Corrected 31.01.2024 in accordance with Article 91]
>900713 LCVR (light chain variable region amino acid sequence) SEQ ID NO: 2

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

Table 2. CDR sequences of mouse anti-CD70 antibodies

2.2 Expression detection of chimeric antibodies

2.2.1 Expression of chimeric antibodies: The expression vector obtained in 2.1 was amplified by 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 and expression of chimeric antibodies. The host cells used for expression were CHO-S cells (Thermo Fisher, Catalog No. R80007). The prepared heavy chain vector and light chain vector were mixed with polyetherimide (PEI, Polysciences, Catalog No. 24765-1) to form a liposome complex, which 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 by Protein A affinity chromatography to obtain human-mouse chimeric antibodies.

2.2.2 Chimeric antibody affinity detection: Biacore 8k set the sample chamber and flow path temperature to 25°C, used the Series S Sensor Chip protein A to capture 900713 as the ligand, and human CD70 (Cat#: CDL-H5246, ACRO Biosystems, Lot#: 2439C-9AWF1-Q9) as the analyte, and performed multi-dynamic cycle kinetic detection. Flow rate: 30 μL/min, association: 120 s, dissociation: 600 s, 10 mM glycine pH 1.5 regeneration at a flow rate of 50 μL/min for 30 s. 1:1 binding model (Binding Model), independent fitting (Fit Local) analysis of kinetic constants ka, kd and KD are shown in Table 3.

Table 3. Affinity data of chimeric antibodies

2.2.3 Chimeric antibody species cross-identification: Biacore 8k set the sample chamber and flow path temperature to 25°C, using the CM5 chip with amino-coupled anti-human Fc antibody (Anti-Human Fc antibody) to capture 900713 as the ligand, and CD70 of different species as the analyzer, to perform multi-dynamic cycle kinetic detection. Flow Rate: 30μL/min, Association: 120s, Dissociation: 180s. 3M MgCl ₂ was regenerated at a flow rate of 50μL/min for 30s 1:1 Binding Model, Fit local analysis kinetic constants ka, kd and KD. The results are shown in Table 4:

Table 4. Chimeric Antibody Species Crossover Data

2.3 Humanization of mouse anti-human CD70 antibody - preparation method of humanized antibody

The humanization of antibodies adopts the following method: first use the 3D modeling method to perform antibody structure 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.

The heavy chain variable region selected the germline genotype IGHV1 category with the highest homology as the humanization design template, and designed three humanized sequences as huVH1-huVH3; the light chain variable region selected the germline genotype IGKV1 category as the humanization design template, and designed two humanized sequences as huVL1-huVL2. The sequences of huVH1-huVH3 are as follows:

[Corrected 31.01.2024 in accordance with Article 91]
>huVH1 (SEQ ID NO:9)

[Corrected 31.01.2024 in accordance with Article 91]
>huVH2 (SEQ ID NO: 10)

[Corrected 31.01.2024 in accordance with Article 91]
The HC-CDR2: WINTYSGVPTYVDKFKG (SEQ ID NO: 16) differs from SEQ ID NO: 4 by one amino acid.

[Corrected 31.01.2024 in accordance with Article 91]
>huVH3 (SEQ ID NO: 11)

[Corrected 31.01.2024 in accordance with Article 91]
The sequences of huVL1-huVL2 are as follows:

[Corrected 31.01.2024 in accordance with Article 91]
>huVL1 (SEQ ID NO: 12)

[Corrected 31.01.2024 in accordance with Article 91]
>huVL2 (SEQ ID NO: 13)

[Corrected 31.01.2024 in accordance with Article 91]
Note: In SEQ ID NO:9, 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; 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; TYGMS (SEQ ID NO:3), WINTYSGVPTYVDDFKG (SEQ ID NO:4), SRDYYYGSSY (SEQ ID NO:5), and TYGMS (SEQ ID NO:6). NO:5) is the CDR region.

[Corrected 31.01.2024 in accordance with Article 91]
In SEQ ID NO:10, I at position 2, S at position 28, A at position 49, F at position 68, F at position 70, L at position 72, and L at position 81 are mouse sequences, 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, T at position 69, T at position 71, D at position 73, T at position 76, ELRS at positions 82-85, RSD at positions 87-89, V at position 93, Y at position 95, and V at position 115 are mutation sites, TYGMS (SEQ ID NO:3), WINTYSGVPTYVDKFKG (SEQ ID NO:16), SRDYYYGSSY (SEQ ID NO:5) is the CDR region.

[Corrected 31.01.2024 in accordance with Article 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.

[Corrected 31.01.2024 in accordance with Article 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.

[Corrected 31.01.2024 in accordance with Article 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.

The humanized point mutation antibody expression plasmids were expressed in CHO-S cells and purified to obtain humanized antibody proteins. The target affinity, CD70-CD27 interaction blocking activity, and activated cytokine release of humanized antibodies were screened using Biacore, ELISA, and cell biological activity detection methods, and two excellent humanized anti-CD70 antibodies were obtained, numbered 900819 (consisting of huVH1 and huVL1) and 900820 (consisting of huVH3 and huVL1).

Example 3 Detection of anti-human CD70 humanized antibody

3.1 Biacore (SPR) detection of antibody affinity: Biacore 8k set the sample chamber and flow path temperature to 25℃, used amino-coupled CM5 chip with Anti-Human Fc antibody to capture antibody samples as ligand, gradient dilution of mouse (Mouse) CD70 (ACRO, Cat#: CDL-M5245, Lot#: 2723a-86WF1-JK), rat (Rat) CD70 (Sino Biolog gical Inc, Cat#: 80161-R07H, Lot#: LC13AU1507), cynomolgus (Cynomolgus) CD70 (ACRO, Cat#: CDL-C5244, Lot#: 3870c-20AGF1-TZ), human (Human) CD70 (ACRO, Cat#: CDL-H5246, Lot#: 2439c-9AWF1-Q9) as Analyte, multi-dynamic cycle kinetic detection. Flow Rate: 30μl/min, Association: 120s, Dissociation: 600s, 3M MgCl2 regenerated at a flow rate of 50μl/min for 30s. 1:1 Binding Model, Fit local analysis of kinetic constants ka, kd and KD. The results are shown in Table 5:

Table 5. Affinity determination of candidate anti-human CD70 humanized antibodies Note: 900562 is a humanized anti-CD70 antibody developed by Argenx, which was synthesized and expressed by Huabo according to its patented sequence. NA means not detected, and NB means no binding.

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

Use PBS to M2 mouse antibody (Sigma Aldrich, Cat#45-F3165) was diluted to 7 μg/mL, and 100 μL was added to each well of a 96-well ELISA plate (NUNC, Cat#442404), and placed at 4°C for overnight coating. After coating, the 96-well plate was washed once with PBS-0.05% Tween, and 200 μL of Assay Diluent (Biolegend, Cat#4331503) was added to the 96-well plate, and the plate was blocked for 2 hours at room temperature while shaking. The plate was then washed 3 times with PBS-0.05% Tween, and CD70-FLAG-His protein (Acro, Cat#CDL-H52Da) was diluted to 500 ng/mL with Assay Diluent, and added to the 96-well ELISA plate, 100 μL per well, and incubated for 1 hour at room temperature while shaking. The plate was then washed 6 times with PBS-0.05% Tween. Dilute the antibody to be tested to 60 μg/mL with Assay Diluent, and then dilute 3 times to 11 concentrations, for a total of 12 concentration gradients. Add the diluted antibody to a 96-well plate at 50 μL per well. Then, add 50 μL of recombinant human CD27 (R&D, Cat#382-CD-100) with a final concentration of 1 μg/mL to each well and incubate at room temperature for 1 hour while shaking. Wash the plate 6 times with PBS-0.05% Tween. Add 1:500 diluted biotin anti-CD27 Biotin-anti-CD27() (eBioscience, Cat#13-0271-82) to a 96-well plate, 100 μL per well, and incubate at room temperature for 1 hour while shaking. Wash the plate 6 times with PBS-0.05% Tween. Streptavidin-HRP (CST, Cat#3999S) diluted 1:1000 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. TMB (Biolegend, Cat#421101) was added to the 96-well plate, 100 μL per well, and then color was developed for 10 minutes. 100 μL of 2M H ₂ SO ₄ was added to each well and the plate was analyzed on a microplate reader (MD OD450 and OD570 were measured on a microplate reader (i3x).

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.

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

After CD70 and CD27 bind, CD27 cells will secrete IL-8. This experiment detected the effect of anti-CD70 antibodies on the level of CD27 signal transduction induced by CD70 by detecting the IL-8 secretion of CD27-expressing cells.

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) to dilute the cells to 1×10 ⁶ cells/mL. HT1080-huCD27-2C3 cells [human fibrosarcoma cells expressing human CD27 (ATCC, Cat#CCL-121), clone 2C3, Huabo self-constructed] were collected and resuspended in RPMI1640 containing 10% FBS to dilute the cells to 2×10 ⁵ cells/mL.

The antibody to be tested was diluted to 90 μg/mL with RPMI1640 containing 10% FBS, and then diluted 5-fold to 8 concentrations, a total of 9 concentration gradients. Raji cells and different concentrations of antibodies were added to a 96-well plate (Corning Cat#3599) at a ratio of 1:1, and 50 μL of each was added. After incubation at room temperature for 60 minutes, 50 μL of HT1080-huCD27-2C3 cells were added to each well.

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, and then ELISA detection was performed using an IL-8 ELISA kit (Biolegend, Cat#4331503), and OD450 and OD570 were measured on a microplate reader.

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.

3.4 In vitro ADCC experiment of anti-human CD70 humanized antibody on Raji cells

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

The antibody to be tested was diluted to 1.5 μg/mL with 0.5% FBS RPMI1640, and then diluted 3-fold to 8 concentrations, a total of 9 concentration gradients. Take a 96-well white plate (Corning, Cat#3917), add Raji cell suspension, 30 μL per well, add the diluted antibody to be tested, 30 μL per well, and then add ADCC FcγRIIIa (158V) Jurkat cell suspension, 30 μL per well; then incubate in a 37°C, 5% CO ₂ incubator for 60 minutes.

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

The results are shown in Figure 3. The ADCC activity of antibodies 900823 and 900827 is significantly enhanced compared to the parent antibodies 900819 and 900820, and is stronger than the control antibody 900562. Note: 900823 is the Fc-enhanced version of 900819, and 900827 is the Fc-enhanced version of 900820. The antibody Fc has undergone F243L/R292P/Y300L/V305I/P396L (LPLIL) mutations to enhance ADCC/ADCP/CDC activity. They are independently expressed and synthesized by Huabo.

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

[Corrected 31.01.2024 in accordance with Article 91]
>900823, 900827 Fc sequence (SEQ ID NO: 15), wherein the 126th L/175th P/183rd L/188th I/279th L mutation is F243L/R292P/Y300L/V305I/P396L (LPLIL):

3.5 In vitro ADCP experiment of anti-human CD70 humanized antibody on CHOK1-huCD70-3G8 cells

Antibody-mediated cellular phagocytosis (ADCP) was detected using a reporter gene method. 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 collected, resuspended in RPMI1640 (Gibco Cat#A1049101) containing 0.5% FBS (Gibco Cat#10099141C), and the cells were diluted to 4.5×10 ⁶ cells/mL. ADCP FcγRIIa Jurkat cells were collected, resuspended in RPMI1640 containing 0.5% FBS, and the cells were diluted to 6×10 ⁶ cells/mL.

The antibody to be tested was diluted to 4 μg/mL with 0.5% FBS RPMI1640, and then diluted 2-fold to 8 concentrations, a total of 9 concentration gradients. Take a 96-well white plate (Corning Cat#3917), add CHOK1-huCD70-3G8 cell suspension, 100 μL per well, add the diluted antibody to be tested, 50 μL per well, and then add Jurkat-CD32a-NFAT cell suspension, 50 μL per well; then incubate in a 37°C, 5% CO ₂ incubator for 6.5 hours.

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. Use a spray gun to remove and discard 100 μL of the culture medium supernatant, add Bio-Glo ^™ Luciferase Assay System (Promega, Cat#G7940) to the 96-well plate, 100 μL per well, react at room temperature in the dark for 5 minutes, and immediately use an ELISA reader (MD i3x) for full wavelength detection.

The results are shown in Figure 4. The ADCP activities of antibodies 900823 and 900827 were significantly enhanced compared with the parent antibodies 900819 and 900820, and were stronger than the control antibody 900562.

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

Collect Raji cells, resuspend in RPMI1640 without FBS (Gibco, Cat#A1049101), dilute cells to 1×10 ⁶ cells/mL, and set aside. Dilute the antibody to be tested to 12 μg/mL in RPMI1640 without FBS, and then dilute it 2-fold, for a total of 10 concentration gradients. Dilute the stock solution of Normal Human Serum Complement (Quidel, Cat#A112) 2.5 times with the above culture medium.

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

After the incubation, add 10 μL of lysis solution (Tongren Chemical, Cat#CK12) to the control wells with only cells, incubate in a 37°C, 5% _CO2 incubator for 30 minutes, then add working solution (Tongren Chemical, Cat#CK12) to all wells, 100 μL per well, and react at room temperature in the dark for 10 minutes; then add stop solution (Tongren Chemical, Cat#CK12) to all wells, 50 μL per well, and detect the OD490 reading using an enzyme reader.

The cell lysis percentage 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%.

The results are shown in Figure 5. The CDC activities of antibodies 900823 and 900827 were significantly enhanced compared to parent antibodies 900819 and 900820, and were comparable to the CDC activity of control antibody 900562.

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

Biacore 8k set the sample chamber and flow path temperature to 25℃, and amino-coupled egg white lysozyme (Lysozyme solution from chicken egg, referred to as Lysozyme) and soybean trypsin inhibitor 1-S (Trypsin inhibitor type 1-S from Glycine max, referred to as Trypsin inhibitor) as ligands on the CM5 chip. The antibody was diluted to 1μM, flow rate: 10μL/min, Association: 900s, Dissociation: 1200s, first regenerated at a flow rate of 50μL/min, 0.85% phosphoric acid for 30s, and then regenerated at a flow rate of 50μL/min, 50Mm sodium hydroxide for 30s. The binding stability signal was collected to analyze the nonspecific electrostatic binding of the antibody.

Through the quality control results of polyclonal rabbit anti-lysozyme (Rb pAb to Lysozyme) and anti-trypsin inhibitor antibody (Anti-trypsin inhibitor antibody, also known as Anti-Trypsin Inhibitor) on Lysozyme solution from chicken egg and Trypsin inhibitor type 1-S from Glycine max, it can be determined that the activity of egg white lysozyme and soybean trypsin inhibitor was normal after amino coupling and fixation to the CM5 chip, that is, the activity of the fixed ligand remained good during the whole experiment. The PI of Trypsin inhibitor type 1-S from Glycine max is 4.5, and the PI of Lysozyme solution from chicken egg is 11.3. In the HBS-EP buffer system of pH 7.4, Trypsin inhibitor type 1-S from Glycine max has a stronger negative charge, and Lysozyme solution from chicken egg has a stronger positive charge.

Table 6. Non-specific binding data of anti-human CD70 humanized antibodies

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 means that the anti-human CD70 humanized antibody test samples have no nonspecific electrostatic binding effect.

Example 4 Evaluation of the efficacy of humanized antibodies in mice

After mixing 100uL of PBS suspension containing 5×10 ⁶ U266B1 cells (ATCC, catalog number TIB196) and an equal volume of Matrigel, the mixture was inoculated into 5-6 week old female SCID mice (Shanghai Slake Experimental Animal Co., Ltd.) by subcutaneous injection. The inoculation site was the right side of the mouse's back near the armpit. When the tumor grew to an average of about 200mm ³ , 36 tumor-bearing mice were selected and randomly divided into 6 groups of 6 mice in each group using E-WorkBook software based on tumor volume and body weight. 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 CO ₂ , and then all mouse tumor tissues were collected, weighed and photographed. The dosing regimen is shown in Table 7:

Table 7. Grouping and dosing regimen

Note: 900201 is a negative control antibody, 900562-afu is a positive control antibody synthesized by Huabo according to the Cusatuzumab sequence in WHO-INN-PL_118 (WHO Drug Information, Vol.31, No.4, 2017), HB0050 is an antibody produced by the CHO-K1 stable cell line constructed by Huabo (source of humanized molecule 900823), and HB0051 is an antibody produced by the CHO-K1 stable cell line constructed by Huabo (source of humanized molecule 900827). i.v.QW*4W refers to intravenous injection, once a week, for 4 consecutive weeks.

During the experiment, most animals in each group were in good mental state and their body weight was relatively stable.

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 ³ on day 0 and 652.58 mm ³ at the end of the experiment (day 28), and a relative tumor volume of 3.25.

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).

As shown in Figure 6 and Table 8, compared with the negative control group G1 900201, the test substance G2 to G6 groups reduced tumor volume (TV) and relative tumor volume (RTV) during the administration period, with significant differences (p<0.01 or p<0.05). On the 28th day, 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.

Table 8. Relative tumor inhibition rate TGI (%)

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

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

The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims (16)

An antibody targeting CD70 or an antigen-binding fragment binding to CD70, characterized in that it can specifically bind to CD70 and comprises: 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 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. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 1 is characterized in that it comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences thereof are as 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. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 2 is characterized in that the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively as shown in SEQ ID NO: 9 and SEQ ID NO: 12, or respectively as shown in SEQ ID NO: 11 and SEQ ID NO: 12. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 1 is characterized in that it also comprises an Fc region, and the amino acid sequence of the Fc region is shown in SEQ ID NO: 14 or SEQ ID NO: 15. The antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to claim 1 is characterized in that it is a monospecific, bispecific or multispecific antibody or antigen-binding fragment. The nucleic acid is 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. 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. A biomaterial, characterized in that: 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). An antibody-drug conjugate, characterized in that it contains the CD70-targeting antibody or the CD70-binding antigen-binding fragment according to any one of claims 1 to 5. A PROTAC molecule comprises an E3 ubiquitin ligase ligand, a target protein ligand and a linker, wherein the target protein ligand is a protein or polypeptide containing an antibody targeting CD70 or an antigen binding fragment binding to CD70 according to any one of claims 1 to 5. A method for producing an antibody or an antigen-binding fragment, characterized in that a host cell or a microorganism comprising the nucleic acid according to claim 6 is cultivated and the antibody or the antigen-binding fragment is recovered from the culture. 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 a tumor. The use according to claim 12 is 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. Use of the antibody targeting CD70 or the antigen-binding fragment binding to CD70 according to any one of claims 1 to 5, or the nucleic acid according to claim 6, or the composition according to claim 7, or the biomaterial according to claim 8, or the antibody-drug conjugate according to claim 9, or the PROTAC molecule according to claim 10 in the preparation of a preparation for blocking CD70-expressing positive cells. 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. 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.