Binding molecule for CD70 and use thereof

Abstract

The present disclosure relates to a binding molecule for CD70 or an antigen-binding fragment thereof, a derivative comprising the binding molecule or the antigen-binding fragment thereof, and a pharmaceutical composition. In addition, the present disclosure further relates to a related use of the binding molecule or the antigen-binding fragment thereof in the treatment of cancer and detection and diagnosis.

Description

Binding molecules targeting CD70 and their applications

[0001] This application claims Chinese Patent Application No. 202411997537X, entitled "A Binding Molecule for CD70 and Its Application Thereto," filed with the Chinese Patent Office on December 31, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of immunology, and more specifically, relates to CD70-binding molecules and their antigen-binding fragments, derivatives comprising said binding molecules or their antigen-binding fragments, pharmaceutical compositions, and their related applications in the treatment of cancer. Background Technology

[0003] CD70, also known as CD27L or TNFSF7, is a member of the tumor necrosis factor (TNF) family. It is a type II membrane protein containing 193 amino acids and a molecular weight of approximately 50 kDa, existing as a homotrimer. Upon binding to its receptor, CD27, a member of the tumor necrosis factor receptor superfamily (TNFRSF), it triggers a co-stimulatory signal. Cytoplasmic residues of CD27 bind to TNF receptor-related factors such as TRAF2 and TRAF5, thereby activating the NF-κB and c-Jun kinase pathways. This regulates and promotes the activation, proliferation, and differentiation of NK cells, T cells, and B cells, playing a crucial role in regulating the immune response. Under physiological conditions, the CD27-CD70 interaction is strictly regulated. CD70 is only transiently expressed on activated immune cells (including T cells, B cells, NK cells, and dendritic cells) to prevent overexpression and subsequent lymphocyte overactivation. However, under pathological conditions, CD70 is highly expressed in various tumor cells, including solid tumors such as renal cell carcinoma, central nervous system glioma, nasopharyngeal carcinoma, ovarian cancer, and lung cancer, as well as hematologic malignancies such as lymphoma, leukemia, and Waldenström macroglobulinemia. Tumor cells express large amounts of CD70, thereby inducing T cell apoptosis and T cell exhaustion through the CD70-CD27 signaling pathway, and significantly increasing the number of regulatory T cells (Tregs), thus promoting their own growth. Summary of the Invention

[0004] The purpose of this disclosure is to provide a binding molecule for CD70 and its antigen-binding fragment, said binding molecule or its antigen-binding fragment being capable of specifically binding to CD70.

[0005] The first aspect of this disclosure provides a CD70 binding molecule or an antigen-binding fragment thereof, said CD70 binding molecule or antigen-binding fragment comprising:

[0006] i) Heavy chain complementarity-determining region 1 (HCDR1), whose amino acid sequence is selected from: SEQ ID NO: 9, 12, 15, 18, 21 or 24;

[0007] ii) Heavy chain complementarity-determining region 2 (HCDR2), the amino acid sequence of which is selected from: SEQ ID NO: 10, 13, 16, 19, 22 or 25; and

[0008] iii) Heavy chain complementarity-determining region 3 (HCDR3), whose amino acid sequence is selected from: SEQ ID NO: 11, 14, 17, 20, 23 or 26.

[0009] In one embodiment, the CD70 binding molecule or its antigen-binding fragment comprises a heavy chain variable region (VH), the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are as follows:

[0010] a) SEQ ID NO: 9, 10 and 11; or

[0011] b) SEQ ID NO: 12, 13 and 14; or

[0012] c) SEQ ID NO: 15, 16 and 17; or

[0013] d) SEQ ID NO: 18, 19 and 20; or

[0014] e) SEQ ID NO: 21, 22 and 23; or

[0015] f)SEQ ID NO: 24, 25 and 26.

[0016] In one embodiment, the heavy chain variable region comprises an amino acid sequence selected from any of SEQ ID NO: 3-8, 28-30, or comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 3-8, 28-30.

[0017] A second aspect of this disclosure provides another CD70 binding molecule or its antigen-binding fragment, said CD70 binding molecule or its antigen-binding fragment comprising:

[0018] i) Heavy chain complementarity-determining region 1 (HCDR1), whose amino acid sequence is selected from: SEQ ID NO: 51, 57, 63, 69, 75, 81, 87, 93, 99, 105 or 137;

[0019] ii) Heavy chain complementarity-determining region 2 (HCDR2), the amino acid sequence of which is selected from: SEQ ID NO: 52, 58, 64, 70, 76, 82, 88, 94, 100, 106 or 138;

[0020] iii) Heavy chain complementarity-determining region 3 (HCDR3), whose amino acid sequence is selected from: SEQ ID NO: 53, 59, 65, 71, 77, 83, 89, 95, 101, 107 or 139;

[0021] iv) Light chain complementarity-determining region 1 (LCDR1), the amino acid sequence of which is selected from: SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108 or 140;

[0022] v) Light chain complementarity-determining region 2 (LCDR2), the amino acid sequence of which is selected from: SEQ ID NO: 55, 61, 67, 73, 79, 85, 91, 97, 103, 109 or 141; and

[0023] vi) Light chain complementarity-determining region 3 (LCDR3), whose amino acid sequence is selected from: SEQ ID NO: 56, 62, 68, 74, 80, 86, 92, 98, 104, 110 or 142.

[0024] In one embodiment, the CD70 binding molecule or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as follows:

[0025] a) SEQ ID NO: 51, 52, 53, 54, 55 and 56; or

[0026] b) SEQ ID NO: 57, 58, 59, 60, 61 and 62; or

[0027] c) SEQ ID NO: 63, 64, 65, 66, 67 and 68; or

[0028] d) SEQ ID NO: 69, 70, 71, 72, 73 and 74; or

[0029] e) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or

[0030] f) SEQ ID NO: 81, 82, 83, 84, 85 and 86; or

[0031] g) SEQ ID NO: 87, 88, 89, 90, 91 and 92; or

[0032] h)SEQ ID NO: 93, 94, 95, 96, 97 and 98; or

[0033] i) SEQ ID NO: 99, 100, 101, 102, 103 and 104; or

[0034] j) SEQ ID NO: 105, 106, 107, 108, 109 and 110; or

[0035] k) SEQ ID NO: 137, 138, 139, 140, 141, 142.

[0036] In one embodiment, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, or 135, or comprises an amino acid sequence related to .... NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 or 135, any amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0037] In one embodiment, the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, or 136, or comprises an amino acid sequence similar to .... NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 or 136, any amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0038] In one embodiment, the heavy chain variable region and the light chain variable region each comprise sequences selected from the group consisting of:

[0039] 1) SEQ ID NO: 31 and 32; or

[0040] 2) SEQ ID NO: 33 and 34; or

[0041] 3) SEQ ID NO: 35 and 36; or

[0042] 4) SEQ ID NO: 37 and 38; or

[0043] 5) SEQ ID NO: 39 and 40; or

[0044] 6) SEQ ID NO: 41 and 42; or

[0045] 7) SEQ ID NO: 43 and 44; or

[0046] 8) SEQ ID NO: 45 and 46; or

[0047] 9) SEQ ID NO: 47 and 48; or

[0048] 10) SEQ ID NO: 49 and 50; or

[0049] 11) SEQ ID NO: 111 and 112; or

[0050] 12) SEQ ID NO: 113 and 114; or

[0051] 13) SEQ ID NO: 115 and 116; or

[0052] 14) SEQ ID NO: 117 and 118; or

[0053] 15) SEQ ID NO: 119 and 120; or

[0054] 16) SEQ ID NO: 121 and 122; or

[0055] 17) SEQ ID NO: 123 and 124; or

[0056] 18) SEQ ID NO: 125 and 126; or

[0057] 19) SEQ ID NO: 127 and 128; or

[0058] 20) SEQ ID NO: 129 and 130; or

[0059] 21) SEQ ID NO: 131 and 132; or

[0060] 22) SEQ ID NO: 133 and 134; or

[0061] 23) SEQ ID NO: 135 and 136.

[0062] The CD70 binding molecule or its antigen-binding fragment disclosed herein further comprises a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region comprises an Fc; more preferably, the Fc is derived from a mouse or a human; even more preferably, the sequence of the Fc is natural or modified.

[0063] The CD70 binding molecule or its antigen-binding fragment disclosed herein may be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a humanized antibody, a chimeric antibody, a modified antibody, a fully human antibody, a full-length antibody, a heavy chain antibody, a nanobody, Fab, Fv, scFv, F(ab')2, a linear antibody, or a single-domain antibody.

[0064] The CD70 binding molecule or its antigen-binding fragment disclosed herein may be in the form of IgG1, IgG2, IgG3 or IgG4.

[0065] This disclosure also provides a conjugate formed by conjugating the CD70 binding molecule or its antigen-binding fragment of this disclosure with a capture marker or a detection marker; preferably, the detection marker includes a radionuclide, a luminescent substance, a colored substance, or an enzyme.

[0066] This disclosure also provides an antibody-drug conjugate (ADC), which is formed by conjugating the CD70 binding molecule or its antigen-binding fragment of this disclosure with other bioactive molecules; preferably, the other bioactive molecules are small molecule drugs; preferably, the CD70 binding molecule or its antigen-binding fragment is connected to the other bioactive molecules through a connector.

[0067] This disclosure also provides a nucleic acid encoding the CD70 binding molecule of this disclosure or its antigen-binding fragment, a recombinant vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector. Preferably, the host cell is a prokaryotic cell (preferably Escherichia coli) or a eukaryotic cell (preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell or a HEK293 cell).

[0068] This disclosure also provides a method for preparing the CD70 binding molecule of the present disclosure or its antigen-binding fragment, the method comprising: culturing the host cells under suitable conditions and purifying the expression product from the cells.

[0069] This disclosure also provides the use of the CD70 binding molecule of this disclosure or its antigen-binding fragment in the preparation of medicaments for treating or alleviating tumors.

[0070] In one embodiment, the drug targets tumor cells that abnormally express CD70.

[0071] In one embodiment, the tumor is selected from: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

[0072] This disclosure also provides the use of the CD70 binding molecule of this disclosure or its antigen-binding fragment in the preparation of detection or diagnostic reagents.

[0073] In one embodiment, the detection reagent is used to detect CD70 expression; the diagnostic reagent is used to diagnose tumors; preferably, the tumors are selected from: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

[0074] This disclosure also provides a method for detecting CD70 expression in a sample, the method comprising:

[0075] (1) Contact the sample with the CD70 binding molecule or its antigen-binding fragment disclosed herein;

[0076] (2) Detect the formation of a complex of the CD70 binding molecule or its antigen-binding fragment with CD70; optionally, the CD70 binding molecule or its antigen-binding fragment is detectably labeled.

[0077] This disclosure also provides a pharmaceutical composition comprising an effective amount of the CD70 binding molecule of this disclosure or its antigen-binding fragment, or an effective amount of the antibody-drug conjugate of this disclosure, or an effective amount of the nucleic acid of this disclosure, or an effective amount of the recombinant vector of this disclosure, or an effective amount of the host cell of this disclosure.

[0078] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0079] Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0080] This disclosure also provides a kit or reagent kit comprising a container and a pharmaceutical composition of this disclosure located within the container.

[0081] This disclosure also provides a method for inducing cell death in cells expressing CD70, the method comprising contacting the cells with a pharmaceutical composition of the present disclosure, wherein the cells expressing CD70 are tumor cells.

[0082] In one embodiment, the tumor cells are selected from the following tumors: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

[0083] This disclosure also provides a method for treating a CD70-related disease in a subject, the method comprising administering to the subject in need a pharmaceutical composition of this disclosure, or the aforementioned kit or reagent kit.

[0084] In one embodiment, the disease is a tumor; preferably, the tumor is selected from: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

[0085] In one embodiment, the method further includes administering an additional therapeutic agent to the subject.

[0086] The technical solution disclosed herein has the following beneficial effects: the CD70 binding molecule and its antigen-binding fragment disclosed herein can specifically bind to CD70. Attached Figure Description

[0087] The accompanying drawings further illustrate the novel features disclosed herein. A better understanding of the features and advantages disclosed herein will be achieved by referring to these drawings; however, it should be understood that these drawings are for illustrating specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0088] Figure 1A shows the expression of human CD70 on CHO-K1 cells as verified by flow cytometry using a positive control antibody against CD70. The dashed line in the figure indicates the expression of human CD70 on CHO-K1 parent cells without any plasmid transfection, while the solid line indicates the overexpression of human CD70 protein after CHO-K1 parent cells were transfected with the human CD70 plasmid.

[0089] Figure 1B shows the expression of cynomolgus CD70 on CHO-K1 cells as verified by flow cytometry using a positive control antibody against CD70. The dashed line in the figure indicates the expression of cynomolgus CD70 on CHO-K1 parent cells without any plasmid transfection, while the solid line indicates the overexpression of cynomolgus CD70 protein after CHO-K1 parent cells were transfected with the cynomolgus CD70 plasmid.

[0090] Figure 2A shows the binding of the alpaca-derived anti-CD70 chimeric antibody of this disclosure to human CD70-expressing cells (human CD70-CHO-K1 cell line).

[0091] Figure 2B shows the binding of the alpaca-derived anti-CD70 chimeric antibody of this disclosure to cynomolgus CD70-expressing cells (cynomolgus CD70-CHO-K1 cell line).

[0092] Figure 3A shows the binding of the alpaca-derived anti-CD70 humanized antibody of this disclosure to human CD70-expressing cells (human CD70-CHO-K1 cell line).

[0093] Figure 3B shows the binding of the alpaca-derived anti-CD70 humanized antibody of this disclosure to cynomolgus CD70-expressing cells (cynomolgus CD70-CHO-K1 cell line).

[0094] Figure 4A shows the binding of the mouse hybridoma-derived anti-CD70 chimeric antibody of this disclosure to human CD70-expressing cells (human CD70-CHO-K1 cell line).

[0095] Figure 4B shows the binding of the mouse hybridoma-derived anti-CD70 chimeric antibody of this disclosure to cynomolgus CD70-expressing cells (cynomolgus CD70-CHO-K1 cell line).

[0096] Figure 5A shows the binding of the mouse hybridoma-derived anti-CD70 humanized antibody of this disclosure to human CD70-expressing cells (human CD70-CHO-K1 cell line).

[0097] Figure 5B shows the binding of the mouse hybridoma-derived anti-CD70 humanized antibody of this disclosure to cynomolgus CD70-expressing cells (cynomolgus CD70-CHO-K1 cell line).

[0098] Figure 6A shows the binding of the mouse hybridoma-derived anti-CD70 humanized antibody mutant of this disclosure to human CD70-expressing cells (human CD70-CHO-K1 cell line).

[0099] Figure 6B shows the binding of the mouse hybridoma-derived anti-CD70 humanized antibody mutant of this disclosure to cynomolgus CD70-expressing cells (cynomolgus CD70-CHO-K1 cell line). Detailed Implementation

[0100] the term

[0101] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each publication, patent or patent application has been specifically and individually indicated to be incorporated herein by reference.

[0102] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0103] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0104] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0105] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0106] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, intact extracellular domains (ECDs), or proteins. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing that antigen, can be used to generate antibodies specific to the antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., used for immunization with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., used for immunization with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells expressing the antigen, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0107] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically exist in a unique spatial conformation and comprise at least 3–15 amino acids. Methods for determining the epitope bound to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0108] The terms "bispecific binding molecule" and "multispecific binding molecule" refer to binding molecules (e.g., antibodies or molecules containing antibody fragments) that are specific to two or more different antigens (or epitopes), with bispecific antibodies being preferred.

[0109] The term "antibody" in this article may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding part) or its single chain, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0110] In one embodiment, an antibody typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have this structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and a constant domain.

[0111] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be assigned to one of two distinctly different types based on the amino acid sequence of its constant domain, termed κ and λ.

[0112] In the case of IgG, IgA, and IgD antibodies, this constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0113] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antibody binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence within the heavy chain variable domain (or light chain variable domain): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0114] The term "Fc" is used to define the C-terminal region of the immunoglobulin heavy chain, which includes at least a portion of the constant region. This term includes both the native sequence Fc region and variant Fc regions.

[0115] In this article, "antibody" can be used in the broadest sense, including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0116] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0117] It should be noted that the division of the CDR and FR in the variable region of the monoclonal antibody disclosed herein is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the monoclonal antibody sequence of this disclosure are explicitly kept within the scope of this disclosure.

[0118] The term "humanized antibody" refers to an antibody in which all or part of the amino acids, except for the CDR (Coefficient of Reduction) of a non-human antibody (such as a mouse antibody), have been replaced by corresponding amino acids derived from human immunoglobulins. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible, as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to the original antibody.

[0119] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0120] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its immunogenicity-determining moiety, or a fusion protein product further derived from such fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0121] The term "VHH" or "VHH fraction" refers to a single-domain antibody form, specifically an antibody fragment containing only the variable region of a single monomeric antibody. Antibodies derived from camels (Bactrian camel, dromedary camel, humpback camel, alpaca) are known to consist only of the light chain and a heavy chain lacking the CH1 domain (heavy chain antibodies). The antigen-binding site of heavy chain antibodies consists solely of the VHH. The VHH contains three CDRs, and the specificity of antigen binding depends on the amino acid sequence of the CDRs.

[0122] When using the variable region of this disclosure to prepare antibodies, binding molecules, bispecific binding molecules or multispecific binding molecules, the constant region is not particularly limited. A constant region known to those skilled in the art or a constant region obtained by oneself can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fcγ receptor or FcRn) can also be introduced into the constant region.

[0123] There are no particular limitations on the methods used to obtain the binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules disclosed herein, and they can be obtained by any method, such as Cold Spring Harbor's Guide to Laboratory Antibody Techniques, Chapters 5-8 and 15. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0124] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated with a therapeutic active substance or active pharmaceutical ingredient (API), thereby enabling the therapeutic active substance or active pharmaceutical ingredient (API) to target the antibody's binding target to exhibit its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent linking of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC.

[0125] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0126] The term "biological activity" refers to the ability of an antibody to bind to an antigen and cause a measurable biological response, which can be measured in vitro or in vivo.

[0127] The binding molecules or antigen-binding fragments disclosed herein can be used in combination with other drugs. The active ingredients can be mixed together to form a single drug delivery unit, or they can be used separately as independent drug delivery units.

[0128] The term "effective dose" refers to a dosage of the pharmaceutical formulation of the antibody or fragment of this disclosure that, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0129] The terms "pharmaceutical kit" or "reagent kit" refer to an effective amount of one or more unit dosage forms of the pharmaceutical composition of this disclosure. In some embodiments, the pharmaceutical kit may contain a sterile container; such a container may be a box, ampoule, bottle, vial, tube, bag, blister pack, or other suitable container form known in the art. This container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the medicine. Furthermore, the pharmaceutical kit includes instructions for administering the pharmaceutical composition of this disclosure to an individual. The instructions typically include methods of using the pharmaceutical composition of this disclosure to treat a disease.

[0130] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals disclosed herein include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0131] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0132] As used in this article, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells.

[0133] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0134] Example

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions (such as those described in "J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002") or according to the manufacturer's recommendations.

[0136] Example 1. Human CD70 and cynomolgus monkey CD70 antigen information

[0137] The full-length amino acid sequence of human CD70 used in the examples (SEQ ID NO: 1) (Uniprot ID: P32970) is shown below.

[0138] The double horizontal lines represent the extracellular region; the wavy lines represent the transmembrane region; and the horizontal lines represent the intracellular region.

[0139] The full-length amino acid sequence (SEQ ID NO: 2) (Uniprot ID: A0A2K5WTU9) of the cynomolgus monkey CD70 used in the examples is shown below.

[0140] The double horizontal lines represent the extracellular region; the wavy lines represent the transmembrane region; and the horizontal lines represent the intracellular region.

[0141] Example 2: Preparation of human CD70 and cynomolgus monkey CD70 cell lines

[0142] The nucleotide sequence encoding the human CD70 amino acid shown in SEQ ID NO.1 was cloned into the pCMV3 (SinoBiological, catalog number CV011) plasmid to obtain a plasmid for constructing a human CD70 cell line. The obtained plasmid was transfected into CHO-K1 cells (ATCC, catalog number CCL-61) to obtain a CHO-K1 cell line overexpressing human CD70 (abbreviated as: human CD70-CHO-K1 cell line).

[0143] The nucleotide sequence encoding the cynomolgus CD70 amino acid shown in SEQ ID NO.2 was cloned into the pCMV3 (SinoBiological, catalog number CV011) plasmid to obtain a plasmid for constructing a cynomolgus CD70 cell line. The obtained plasmid was transfected into CHO-K1 cells to obtain a CHO-K1 cell line overexpressing cynomolgus CD70 (abbreviated as: cynomolgus CD70-CHO-K1 cell line).

[0144] The expression of human CD70 in the obtained human CD70-CHO-K1 cell line and the expression of cynomolgus CD70 in the cynomolgus CD70-CHO-K1 cell line were detected using FACS. The results are shown in Figure 1. Both the human CD70-CHO-K1 cell line (Figure 1A) and the cynomolgus CD70-CHO-K1 cell line (Figure 1B) showed obvious antigen overexpression, which can be used for subsequent experiments to verify the cellular binding of CD70 binding molecules.

[0145] Example 3. Alpaca Immunization: Construction of a Heavy Chain-Only Antibody Immunotherapy Library

[0146] Alpacas were immunized with human CD70 ECD protein (ACRO, catalog number CDL-H52Da) on days 0, 21, 42, and 63, for a total of four immunizations. Blood samples were collected on days 28, 49, and 70 to separate serum, and the immune response in the serum was detected by ELISA. Immunization was terminated when the serum titer reached a plateau. 50 mL blood samples were then collected again from each immunized alpaca. Alpaca PBMCs were isolated using Solarbio lymphocyte separation medium according to the manufacturer's instructions. Total RNA was extracted (using a total RNA extraction kit, OMEGA) and then analyzed using Takara PrimeScript. TM Using cDNA synthesized with the II reverse transcription kit as a template, nested PCR was performed with designed specific primers to amplify the VHH gene fragment. After recovering the VHH fragment, it was ligated into the pADL-23c phage vector via Sfi I restriction enzyme digestion. A CD70 alpaca immunoglobulin library (1.68 × 10⁻⁶ cells) was constructed by electroporation of competent cells with TG1. 9 ).

[0147] Example 4. Screening of positive clones of anti-CD70 derived from alpacas

[0148] To obtain positive antibodies that cross-bind with human CD70 and cynomolgus monkey CD70, the above library was amplified and assembled into phages by adding M13K07 helper phage. After cross-screening with human CD70 ECD protein (ACRO, catalog number CDL-H52Da) and cynomolgus monkey CD70 ECD protein (KACTUS, catalog number CD7-CM170), the resulting candidate clones were detected by ELISA using human CD70 ECD protein and cynomolgus monkey CD70 ECD protein to obtain positive clones. Sequencing of the positive clones yielded the amino acid sequences of the variable regions of the six heavy chain antibodies disclosed in this paper. The results are shown in Table 1.

[0149] Table 1. Amino acid sequences of the variable regions of six heavy chain antibodies against CD70 derived from alpacas.

[0150] Based on the above amino acid sequence, the CDR and FR of the heavy chain variable region were divided using the Kabat numbering rule. The composition of the three CDR sequences of each heavy chain antibody is shown in Table 2 below.

[0151] Table 2. CDR sequences of six heavy chain antibodies against CD70 derived from alpacas.

[0152] Example 5. Construction of alpaca-derived anti-CD70 chimeric antibody and its transient transfection expression in eukaryotic cells.

[0153] The target gene fragment, generated by splicing the nucleic acids encoding the disclosed heavy chain antibody variable region and the human IgG1 constant region after sequencing, was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid. The heavy chain antibody variable region can be linked to the human IgG1 constant region via a linker peptide, forming: heavy chain antibody variable region - linker peptide - human IgG1 constant region. The linker peptide sequence used in this embodiment is GGGGS (SEQ ID NO: 143).

[0154] The introduced human IgG1 constant region sequence (SEQ ID NO: 27) is as follows:

[0155] Expi293F was cultured in Expi293 expression medium (Thermo Fisher Scientific, A1435101). TM Cells (Thermo Fisher Scientific, A14527) were seeded in shake flasks and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and affinity purified using a Mabselect Sure column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated and transferred to PBS for aliquoting. The final purified chimeric antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0156] Example 6. Binding of alpaca-derived anti-CD70 chimeric antibody to CD70-expressing cells.

[0157] Both human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells were cultured in F12K + 10% FBS + 400 μg / mL Hygromycin using T75 cell culture flasks in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0158] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom plates (corning3799), centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and dilute 10-fold down to 7 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash with 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:500 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 50 μL of 1% BSA (in PBS), and analyze the average fluorescence intensity of the PE channel for each sample using flow cytometry.

[0159] The average fluorescence intensity obtained from flow cytometry analysis of each sample was imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the assays and the highest mean fluorescence intensity (Top MFI) are shown in Table 3 and Figures 2A and 2B. The results showed that, at the cellular level, anti-CD70 heavy chain antibodies 70VHH-5, 70VHH-10, and 70VHH-11 all exhibited good binding to human CD70 and cynomolgus monkey CD70.

[0160] Table 3. Binding of anti-CD70 chimeric antibodies to CD70-expressing cells.

[0161] Example 7. Humanization design of alpaca-derived anti-CD70 chimeric antibody

[0162] Humanized sequence design was performed using the chimeric antibody sequence with clone number 70VHH-10. Germline gene sequences with high homology to the candidate heavy chain antibody were selected as VHH transplantation framework templates through sequence alignment. After grafting the candidate antibody CDR region into the selected human antibody variable region framework, individual amino acid reversion mutations were performed to obtain the humanized antibody. The amino acid sequence of the humanized heavy chain variable region is shown in Table 4.

[0163] Table 4. Amino acid sequence of the variable region of humanized anti-CD70 antibody derived from alpacas.

[0164] Example 8. Preparation of humanized anti-CD70 antibody derived from alpaca

[0165] Referring to Example 5, the target gene fragment generated by splicing the nucleic acid encoding the heavy chain variable region of the humanized antibody with the nucleic acid encoding the constant region of human IgG1 was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0166] Expi293F was cultured in Expi293 expression medium (Thermo Fisher Scientific, A1435101). TM Cells (Thermo Fisher Scientific, A14527) were seeded in shake flasks and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and affinity purified using a Mabselect Sure column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated and transferred to PBS for aliquoting. The final purified chimeric antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0167] Example 9. Binding of alpaca-derived anti-CD70 humanized antibody to CD70-expressing cells.

[0168] Both human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells were cultured in F12K + 10% FBS + 400 μg / mL Hygromycin using T75 cell culture flasks in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0169] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom plates (corning3799), centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and dilute 10-fold down to 7 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash with 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 50 μL of 1% BSA (in PBS), and analyze the average fluorescence intensity of the PE channel for each sample using flow cytometry.

[0170] The average fluorescence intensity obtained from flow cytometry analysis of each sample was imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the highest mean fluorescence intensity (Top MFI) and the results are shown in Table 5 and Figures 3A and 3B. The results showed that, at the cellular level, the anti-CD70 humanized heavy chain antibodies 70VHH-10-H2 and 70VHH-10-H3 had good binding to both human CD70 and cynomolgus monkey CD70.

[0171] Table 5. Binding of anti-CD70 humanized antibodies to CD70-expressing cells.

[0172] Example 10. Obtaining anti-CD70 antibodies derived from mouse hybridomas

[0173] Anti-CD70 monoclonal antibodies were generated by immunizing mice. Balb / c mice, female, 6 weeks old (Charles River Company), were used in the experiment. Housing environment: SPF grade. After purchase, mice were housed in a laboratory environment for one week with a 12 / 12-hour light / dark cycle, temperature 20-25℃, humidity 40-60%. The immunizer was human CD70 ECD protein (ACRO, catalog number CDL-H52Da), with 25 μg of protein administered on days 0, 14, 28, and 42, and a booster immunization two days before spleen cell fusion. During this period, mouse serum antibody titers were measured using ELISA. After the booster immunization, mice with high and plateauing antibody titers in their serum were selected for spleen cell fusion. An optimized electrofusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0174] After culturing the fused hybridoma cells for 7-14 days, the culture supernatant was collected. Using CHO-K1 cells highly expressing human CD70, antibody screening was performed on the hybridoma cell culture supernatant using FACS assays to obtain positive antibody strains. Further screening was conducted using CHO-K1 cells highly expressing cynomolgus monkey CD70 and blank CHO-K1 cells to exclude non-specific antibody-binding hybridoma strains, thus selecting hybridomas specifically binding to human / cynomolgus monkey CD70. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara#2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced to obtain the amino acid sequences of the variable regions of the 10 monoclonal antibodies disclosed in this invention, as shown in Table 6.

[0175] Table 6. Amino acid sequences of the variable regions of 10 monoclonal antibodies against CD70 derived from mouse hybridomas.

[0176] Based on the above amino acid sequences, the CDR and FR of the antibody variable region were divided using the Kabat numbering rules. The composition of the 6 CDR sequences of each antibody is shown in Table 7 below.

[0177] Table 7. CDR sequences of 10 monoclonal antibodies against CD70 derived from mouse hybridoma.

[0178] Example 11. Construction of mouse hybridoma-derived anti-CD70 chimeric antibody and its transient transfection expression in eukaryotic cells.

[0179] The target gene fragments generated by splicing the nucleic acids encoding the heavy chain variable region and light chain variable region of the monoclonal antibody, which were obtained after sequencing, with the nucleic acids encoding the heavy chain constant region and κ light chain constant region of IgG1, were cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids.

[0180] Expi293F was cultured in Expi293 expression medium (Thermo Fisher Scientific, A1435101). TM Cells (Thermo Fisher Scientific, A14527) were seeded in shake flasks and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and affinity purified using a Mabselect Sure column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated and transferred to PBS for aliquoting. The final purified chimeric antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0181] Example 12. Binding of mouse hybridoma-derived anti-CD70 chimeric antibody to CD70-expressing cells.

[0182] Both human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells were cultured in F12K + 10% FBS + 400 μg / mL Hygromycin using T75 cell culture flasks in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0183] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom plates (corning3799), centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and dilute 10-fold down to 7 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash with 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 50 μL of 1% BSA (in PBS), and analyze the average fluorescence intensity of the PE channel for each sample using flow cytometry.

[0184] The average fluorescence intensity obtained from flow cytometry analysis of each sample was imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the highest mean fluorescence intensity (Top MFI) and the results are shown in Table 8 and Figures 4A and 4B. The results showed that, at the cellular level, the anti-CD70 chimeric antibodies 70CH-4, 70CH-10, 70CH-20, 70CH-25, and 70CH-27 all had good binding to human CD70 and cynomolgus monkey CD70.

[0185] Table 8. Binding of mouse hybridoma-derived anti-CD70 chimeric antibodies to CD70-expressing cells.

[0186] Example 13. Humanization design of mouse hybridoma-derived anti-CD70 antibody

[0187] Based on the results of expression purification and cellular binding assays, the antibody with clone number 70CH-20 was selected for humanization design.

[0188] Humanization of murine anti-human CD70 monoclonal antibodies was performed using methods published in numerous publications in the field. In short, a human constant domain was used to replace the parental (murine antibody) constant domain, and human antibody sequences were selected based on the homology between the murine and human antibodies. Based on the obtained typical VH / VL CDR structure of the murine antibody, the heavy and light chain variable region sequences were compared with human antibody germline databases to obtain highly homologous human germline templates.

[0189] The CDR region of the murine antibody was transplanted onto a selected humanized template, replacing the humanized variable region, and then recombined with the IgG constant region (preferably IgG1 for the heavy chain and κ for the light chain). Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH, designing antibodies composed of the following humanized light and heavy chain variable region sequences, as shown in Table 9.

[0190] Table 9. Amino acid sequences of the light and heavy chain variable regions of 12 humanized antibodies derived from mouse hybridomas.

[0191] Example 14. Preparation of anti-CD70 humanized antibody derived from mouse hybridoma

[0192] The target gene fragments generated by splicing the nucleic acids encoding the heavy chain variable region and the light chain variable region of the humanized antibody with the nucleic acids encoding the heavy chain constant region and the κ light chain constant region of IgG1 were cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids.

[0193] Expi293F was cultured in Expi293 expression medium (Thermo Fisher Scientific, A1435101). TM Cells (Thermo Fisher Scientific, A14527) were seeded in shake flasks and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and affinity purified using a Mabselect Sure column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated and transferred to PBS for aliquoting. The final purified chimeric antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0194] Example 15. Binding of mouse hybridoma-derived anti-CD70 humanized antibody to CD70-expressing cells.

[0195] The culture medium for human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells was F12K + 10% FBS + 400 μg / mL Hygromycin. Before use, human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells were washed twice with sterile DPBS, digested with 0.25% trypsin EDTA for about 5 minutes, and then the digestion was stopped with complete culture medium.

[0196] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom plates (corning3799), centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and dilute 10-fold down to 7 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash with 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 50 μL of 1% BSA (in PBS), and analyze the average fluorescence intensity of the PE channel for each sample using flow cytometry.

[0197] The average fluorescence intensity obtained from flow cytometry analysis of each sample was imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the assay and the highest mean fluorescence intensity (Top MFI) are shown in Table 10 and Figures 5A and 5B. The results showed that, at the cellular level, the anti-CD70 humanized antibody 70CH-20-H4L3 exhibited good binding to both human CD70 and cynomolgus monkey CD70.

[0198] Table 10. Binding of mouse hybridoma-derived anti-CD70 humanized antibodies to CD70-expressing cells.

[0199] Example 16. Mutants of anti-CD70 humanized antibodies derived from mouse hybridomas

[0200] Sequence analysis of the mouse hybridoma-derived anti-CD70 humanized antibody 70CH-20-H4L3 revealed risks related to stability and immunogenicity. Therefore, the sequence was mutated to reduce these risks. The mutation sites were G56A and L114T in the heavy chain and L51D in the light chain. The variable region amino acid sequence of the resulting mutant 70CH-20-H4L3-M is shown in Table 11.

[0201] Table 11 Amino acid sequences of the 70CH-20-H4L3 antibody mutant

[0202] Referring to Example 11, the above-mentioned mutant was prepared by transient transfection expression in mammalian cell lines. The binding ability of the mutant to human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells was then detected by flow cytometry. The results are shown in Figures 6A and 6B. The binding ability of the mutant 70CH-20-H4L3-M to human CD70-CHO-K1 cells and cynomolgus monkey CD70-CHO-K1 cells was similar to that of the maternal antibody 70CH-20-H4L3.

[0203] The embodiments of the present invention described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for unconventional experimentation. All such equivalents are within the scope of the present invention and are encompassed by the claims.

Claims

1. A CD70 binding molecule or its antigen-binding fragment, characterized in that: The CD70 binding molecule or its antigen-binding fragment includes a heavy chain variable region (VH), which contains HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: a) SEQ ID NO: 9, 10 and 11; or b) SEQ ID NO: 12, 13 and 14; or c) SEQ ID NO: 15, 16 and 17; or d) SEQ ID NO: 18, 19 and 20; or e) SEQ ID NO: 21, 22 and 23; or f) SEQ ID NO: 24, 25 and 26; Preferably, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 3-8, 28-30, or comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 3-8, 28-30.

2. A CD70 binding molecule or its antigen-binding fragment, characterized in that: The CD70 binding molecule or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as follows: a) SEQ ID NO: 51, 52, 53, 54, 55 and 56; or b) SEQ ID NO: 57, 58, 59, 60, 61 and 62; or c) SEQ ID NO: 63, 64, 65, 66, 67 and 68; or d) SEQ ID NO: 69, 70, 71, 72, 73 and 74; or e) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or f) SEQ ID NO: 81, 82, 83, 84, 85 and 86; or g) SEQ ID NO: 87, 88, 89, 90, 91 and 92; or h)SEQ ID NO: 93, 94, 95, 96, 97 and 98; or i) SEQ ID NO: 99, 100, 101, 102, 103 and 104; or j) SEQ ID NO: 105, 106, 107, 108, 109 and 110; or k)SEQ ID NO: 137, 138, 139, 140, 141 and 142; Preferably, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, or 135, or comprises an amino acid sequence similar to SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, or 135, or contains an amino acid sequence similar to SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, or 135. NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, or 135, any amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; and / or The light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, or 136, or contains an amino acid sequence similar to SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 50, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, or 136. NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 or 136, any amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; More preferably, the heavy chain variable region and the light chain variable region each comprise sequences selected from the group consisting of: 1) SEQ ID NO: 31 and 32; or 2) SEQ ID NO: 33 and 34; or 3) SEQ ID NO: 35 and 36; or 4) SEQ ID NO: 37 and 38; or 5) SEQ ID NO: 39 and 40; or 6) SEQ ID NO: 41 and 42; or 7) SEQ ID NO: 43 and 44; or 8) SEQ ID NO: 45 and 46; or 9) SEQ ID NO: 47 and 48; or 10) SEQ ID NO: 49 and 50; or 11) SEQ ID NO: 111 and 112; or 12) SEQ ID NO: 113 and 114; or 13) SEQ ID NO: 115 and 116; or 14) SEQ ID NO: 117 and 118; or 15) SEQ ID NO: 119 and 120; or 16) SEQ ID NO: 121 and 122; or 17) SEQ ID NO: 123 and 124; or 18) SEQ ID NO: 125 and 126; or 19) SEQ ID NO: 127 and 128; or 20) SEQ ID NO: 129 and 130; or 21) SEQ ID NO: 131 and 132; or 22) SEQ ID NO: 133 and 134; or 23) SEQ ID NO: 135 and 136.

3. The CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims, further comprising one or more of the following characteristics: i) It also includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region includes Fc; more preferably, Fc is derived from mice or humans; more preferably, the sequence of Fc is natural or modified; ii) The CD70 binding molecule or its antigen-binding fragment is a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a humanized antibody, a chimeric antibody, a modified antibody, a fully human antibody, a full-length antibody, a heavy chain antibody, a nanobody, Fab, Fv, scFv, F(ab')2, a linear antibody, or a single-domain antibody; and / or iii) It is in the form of IgG1, IgG2, IgG3 or IgG4.

4. A coupling agent, characterized in that: The CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims is coupled with a capture marker or a detection marker to form a sample; preferably, the detection marker includes a radionuclide, a luminescent substance, a colored substance, or an enzyme.

5. An antibody-drug conjugate, characterized in that: The CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims is coupled with other bioactive molecules to form the molecule; preferably, the other bioactive molecules are small molecule drugs; preferably, the CD70 binding molecule or its antigen-binding fragment is connected to the other bioactive molecules through a connector.

6. A nucleic acid encoding the CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims, or a recombinant vector containing the nucleic acid, or a host cell containing the nucleic acid or the recombinant vector; preferably, the host cell is a prokaryotic cell (preferably Escherichia coli), or a eukaryotic cell (preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell or HEK293 cell).

7. A method for preparing the CD70 binding molecule or its antigen-binding fragment according to any of the preceding claims, the method comprising: The host cells of claim 6 are cultured under suitable conditions, and the expression product is purified from the cells.

8. Use of the CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims in the preparation of a medicament for treating or alleviating tumors; Preferably, the drug targets tumor cells with abnormal CD70 expression; preferably, the tumor is selected from: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

9. The use of the CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims in the preparation of detection or diagnostic reagents; Preferably, the detection reagent is used to detect CD70 expression; the diagnostic reagent is used to diagnose tumors; preferably, the tumors are selected from: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

10. A method for detecting CD70 expression in a sample, the method comprising: (1) Contact the sample with the CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims; (2) Detect the formation of a complex of the CD70 binding molecule or its antigen-binding fragment with CD70; optionally, the CD70 binding molecule or its antigen-binding fragment is detectably labeled.

11. A pharmaceutical composition comprising an effective amount of the CD70 binding molecule or its antigen-binding fragment as described in any of the preceding claims, or comprising an effective amount of the antibody-drug conjugate as described in claim 5, or comprising an effective amount of the nucleic acid or recombinant vector or host cell as described in claim 6; Preferably, it further comprises a pharmaceutically acceptable carrier; preferably, it further comprises one or more additional therapeutic agents.

12. A kit or reagent kit comprising a container and a pharmaceutical composition as claimed in claim 11 located within the container.

13. A method for inducing cell death expressing CD70, the method comprising contacting the cells with the pharmaceutical composition of claim 11, wherein the cells expressing CD70 are tumor cells; Preferably, the tumor cells are selected from the following tumors: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors.

14. A method of treating a subject with a disease associated with CD70 expression, the method comprising administering to the subject in need a pharmaceutical composition as described in any one of claims 11 and / or a kit or reagent kit as described in claim 12; Preferably, the disease is a tumor; preferably, the tumor is selected from: pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, head and neck squamous cell carcinoma, renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma, anaplastic large cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, NK / T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and non-small cell lung cancer, as well as metastatic cancers of the above tumors. More preferably, it also includes administering additional therapeutic agents to the subject.

Images