Anti-human CD70 antibody and application thereof

Abstract

The invention discloses an anti-human CD70 antibody, a fusion protein and application of the anti-human CD70 antibody and the fusion protein. The anti-human CD70 antibody can be combined with a recombinant human CD70 antigen in a high-affinity mode, block combination of CD70 and a receptor CD27 of the CD70, induce internalization of an antigen-antibody compound through the CD70 combined on the surface of a cell and activate lymphocytes to kill tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of medicine, specifically, to an anti-human CD70 antibody or an antigen-binding fragment thereof and uses thereof, and more specifically, to an anti-human CD70 antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody, a vector and a cell expressing the antibody, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and use of the antibody in preparing a drug. Background Art

[0002] Nanobodies are currently the smallest antibody molecules, with a molecular weight of 1 / 10 of that of ordinary antibodies. In addition to the antigenic reactivity of monoclonal antibodies, nanobodies also have some unique functional properties, such as small molecular weight, strong stability, good solubility, easy expression, strong targeting, simple humanization, etc., which are especially suitable for the development of antibody and Car-T / M / NK therapies.

[0003] Cancer is one of the diseases that currently causes the highest mortality in humans. The recent clinical and commercial success of anti-cancer antibodies has aroused great interest in antibody-based therapies. Therefore, anti-cancer antibodies for the treatment of cancer need to be further studied. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide an anti-human CD70 antibody and a preparation method and application thereof.

[0005] Studies have found that the CD70-AXL signaling pathway plays a key role in promoting tumor growth and metastasis, tumor immune escape and drug tolerance. The expression levels and interactions of the two are closely related to the poor clinical prognosis of patients. At the same time, in CD70 ectopic and orthotopic syngeneic mouse tumor model studies, compared with wild-type mice, tumor growth and metastasis of CD70-deficient mice were hindered. Therefore, blocking this pathway has been considered an effective strategy for treating cancer.

[0006] CD70 is highly expressed in many tumors. For example, it is highly expressed in 100% of renal clear cell carcinoma, about 30% of hepatocellular carcinoma, and 16.3% of NSCLC. The overexpression rate of CD70 in uterine leiomyosarcoma is 90%. Antibodies targeting CD70 have achieved efficacy in hematological tumors. Although there are multiple candidate drugs targeting CD70 in different research stages (Table 1), no drug targeting CD70 has been approved for clinical use so far.

[0007] According to one aspect of the present invention, the present invention provides an anti-human CD70 antibody. According to an embodiment of the present invention, the anti-human CD70 antibody comprises:

[0008] At least one immunoglobulin single variable domain (ISVD) that binds to human CD70, the ISVD having three complementary determining regions, namely CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence shown in DX2CMA, the CDR2 has the amino acid sequence shown in GIVX3GX4X5X6X7SGATGPTX8X9TX 10 The amino acid sequence shown in SVKG, the CDR3 has DHX 11 X 12 X13X 14 X 15 CTSLATFRX 16 The amino acid sequence represented by X1FGY, wherein X1 is D or H; X2 is G, A or H; X3 is I or H; X4 is G or H; X5 is G or H; X6 is T or H; X7 is H or T; X8 is Y or H; X9 is Y or H; X1 is D or H; X1 is G, A ...1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is G, A or H; X1 is G, A or H; X1 is G, 10 D or H; X 11 W or Y; X 12 A or H; X 13 S or T; X 14 T or H; X 15 G or S; X 16 It is G or H.

[0009] According to the embodiments of the present invention, the anti-human CD70 antibody can bind to the recombinant human CD70 antigen with high affinity, block the binding of CD70 and its receptor CD27, and can induce the internalization of the antigen-antibody complex by binding to CD70 on the cell surface, and activate lymphocytes to kill tumor cells.

[0010] According to an embodiment of the present invention, the CDR1, the CDR2 and the CDR3 have one of the following amino acid sequences:

[0011] (1) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 23, respectively;

[0012] (2) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 23, respectively;

[0013] (3) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 35, respectively;

[0014] (4) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 36, respectively;

[0015] (5) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 25, 22 and 38, respectively;

[0016] (6) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 25, 22 and 39, respectively;

[0017] (7) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 40, respectively;

[0018] (8) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 41, respectively;

[0019] (9) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 22 and 37, respectively;

[0020] (10) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 27 and 23, respectively;

[0021] (11) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 28 and 23, respectively;

[0022] (12) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 29 and 23, respectively;

[0023] (13) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 30 and 23, respectively;

[0024] (14) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 31 and 23, respectively;

[0025] (15) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 32 and 23, respectively;

[0026] (16) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 33 and 23, respectively;

[0027] (17) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 34 and 23, respectively;

[0028] (18) The amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 26, 22 and 23, respectively.

[0029] According to an embodiment of the present invention, the at least one immunoglobulin ISVD binding to human CD70 is a humanized Camelidae VHH.

[0030] According to an embodiment of the present invention, the antibody comprises at least one VHH having 80% identity with an amino acid sequence selected from SEQ ID NO: 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21.

[0031] According to another aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: a first domain, the first domain comprises the aforementioned anti-human CD70 antibody; and a second domain, the second domain comprises a polypeptide for prolonging the half-life in vivo and / or having a binding effect on effector cells.

[0032] According to an embodiment of the present invention, the second domain comprises a combination of one or more of a serum albumin fragment, a polyethylene glycol fragment and a nanobody binding to HSA.

[0033] According to another aspect of the present invention, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the aforementioned anti-human CD70 antibody or encodes the aforementioned fusion protein.

[0034] According to another aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the aforementioned nucleic acid.

[0035] According to another aspect of the present invention, the present invention provides an isolated cell. According to an embodiment of the present invention, the cell comprises the aforementioned vector.

[0036] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned anti-human CD70 antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the method comprises:

[0037] (1) culturing the aforementioned cells under appropriate conditions; and

[0038] (2) Isolate and recover the aforementioned anti-human CD70 antibody.

[0039] According to another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned anti-human CD70 antibody or the aforementioned fusion protein; and a pharmaceutically acceptable carrier.

[0040] According to another aspect of the present invention, the present invention provides use of the aforementioned anti-human CD70 antibody or the aforementioned fusion protein in the preparation of a medicament for treating cancer.

[0041] According to an embodiment of the present invention, the cancer is hematological tumor, kidney cancer, pancreatic cancer, and laryngeal cancer.

[0042] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0044] Figure 1 A schematic diagram showing the results of ELISA detection of the binding of anti-CD70 chimeric antibodies to recombinant human CD70 according to one embodiment of the present invention is shown;

[0045] Figure 2 A schematic diagram showing the results of ELISA detection of the binding of anti-CD70 chimeric antibodies to recombinant cynomolgus monkey CD70 according to one embodiment of the present invention is shown;

[0046] Figure 3 A schematic diagram showing the results of ELISA detection of the binding of anti-CD70 chimeric antibodies to recombinant mouse CD70 according to one embodiment of the present invention;

[0047] Figure 4 A schematic diagram showing the results of ELISA detection of the blocking activity of anti-CD70 chimeric antibodies against recombinant CD27 and CD70 according to one embodiment of the present invention;

[0048] Figure 5 A schematic diagram showing the results of FACS detection of the binding activity of anti-CD70 chimeric antibodies on CCRF cells according to one embodiment of the present invention;

[0049] Figure 6 A schematic diagram showing the results of routine blood test of hCD70 transgenic mice injected intraperitoneally with CD70 antibody according to one embodiment of the present invention. DETAILED DESCRIPTION

[0050] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar components or components with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0051] definition

[0052] The term "antibody" or "immunoglobulin" is used as a general term to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively) whether referring to heavy chain antibodies or conventional 4-chain antibodies herein. In addition, the term "sequence" used herein (e.g., in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0053] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0054] The terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, antibodies that naturally lack the light chain and heavy chain constant region 1 (CH1) are first obtained from alpaca immune serum, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0055] The term "single domain antibody (VHH)" refers to an immunoglobulin domain that is essentially composed of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of a single domain antibody (VHH) can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody (VHH) confers specificity to an antigen by having an antigen binding site.

[0056] The terms “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH antibody fragment”, “VHH antibody” and “Nanobody” (“Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably.

[0057] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and immunofluorescence techniques.

[0058] The term "humanized antibody" refers to a molecule having an antigen binding site substantially derived from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity determining region (CDR) transplanted into an appropriate framework region in a variable domain. The antigen binding site may be wild type, or modified by one or more amino acid substitutions, such as modification to be closer to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., humanized nanobodies containing all three CDRs from alpacas). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0059] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.

[0060] The term "pharmaceutical composition" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional ingredients which would be unacceptably toxic to a subject to which the composition would be administered.

[0061] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0062] The term "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen can be a polypeptide, protein, nucleic acid, cell, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments herein, the target antigen is a cell expressing CLD18A2, more preferably, a portion of a molecule expressing CLD18A2.

[0063] Anti-human CD70 antibodies and fusion proteins

[0064] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to CD70. The anti-human CD70 antibodies according to the embodiments of the present invention can bind to the recombinant human CD70 antigen with high affinity, block the binding of CD70 and its receptor CD27, and can induce the internalization of the antigen-antibody complex by binding to CD70 on the cell surface, and activate lymphocytes to kill tumor cells.

[0065] The anti-human CD70 antibody comprises: at least one immunoglobulin single variable domain (ISVD) that binds to human CD70, wherein the ISVD has three complementary determining regions, namely CDR1, CDR2 and CDR3, wherein the CDR1 has an amino acid sequence shown in DX2CMA, and the CDR2 has an amino acid sequence shown in GIVX3GX4X5X6X7SGATGPTX8X9TX 10 The amino acid sequence shown in SVKG, the CDR3 has DHX 11 X 12 X 13 X 14 X 15 CTSLATFRX 16 The amino acid sequence represented by X1FGY, wherein X1 is D or H; X2 is G, A or H; X3 is I or H; X4 is G or H; X5 is G or H; X6 is T or H; X7 is H or T; X8 is Y or H; X9 is Y or H; X1 is D or H; X1 is G, A ...1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is G, A or H; X1 is G, A or H; X1 is G, 10 D or H; X 11 W or Y; X 12 A or H; X 13 S or T; X 14 T or H; X 15 G or S; X 16 It is G or H.

[0066] According to an embodiment of the present invention, the CDR1, the CDR2 and the CDR3 have one of the following amino acid sequences:

[0067] (1) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 23, respectively;

[0068] (2) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 23, respectively;

[0069] (3) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 35, respectively;

[0070] (4) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 36, respectively;

[0071] (5) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 25, 22 and 38, respectively;

[0072] (6) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 25, 22 and 39, respectively;

[0073] (7) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 40, respectively;

[0074] (8) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 41, respectively;

[0075] (9) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 22 and 37, respectively;

[0076] (10) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 27 and 23, respectively;

[0077] (11) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 28 and 23, respectively;

[0078] (12) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 29 and 23, respectively;

[0079] (13) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 30 and 23, respectively;

[0080] (14) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 31 and 23, respectively;

[0081] (15) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 32 and 23, respectively;

[0082] (16) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 33 and 23, respectively;

[0083] (17) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 34 and 23, respectively;

[0084] (18) The amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 26, 22 and 23, respectively.

[0085] Single variable domain antibodies are antibodies whose complementary determining regions are part of single variable domain polypeptides. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single variable domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single variable domain scaffolds that are different from those derived from antibodies. Single variable domain antibodies can be any single variable domain antibody in the art or any future single variable domain antibody. Single variable domain antibodies can be from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, cattle. According to one aspect of the invention, the single variable domain antibodies used herein are naturally occurring single domain antibodies referred to as heavy chain antibodies without light chains. For illustration, the variable domain derived from a heavy chain antibody naturally lacking a light chain is referred to as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be from antibodies produced in camelid species, such as camels, dromedaries, llamas, alpacas, and draft horses. Species other than Camelidae may produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present invention.

[0086] The embodiments of the present invention provide VHHs that specifically bind to CD70. The VHHs described herein can bind to CTLA4, thereby enhancing the immune response. The present disclosure provides anti-human CD70 nanobody C8 and humanized antibodies thereof.

[0087] According to some embodiments of the invention, as defined by the Kabat numbering, the VHH of C8 has a CDR1 as set forth in SEQ ID NO:42, a CDR2 as set forth in SEQ ID NO:22, and a CDR3 as set forth in SEQ ID NO:23.

[0088] According to some embodiments of the present invention, as defined by the Kabat code, the VHH-derived antibodies (humanized antibodies and mutants thereof) of C8 have a CDR1 shown in SEQ ID NO: 42, 25 or 26, a CDR2 shown in SEQ ID NO: 22, 27, 28, 29, 30, 31, 32, 33 or 34, and a CDR3 shown in SEQ ID NO: 23, 35, 36, 37, 38, 39, 40 or 41.

[0089] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence, wherein the selected CDR1 has the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in DX2CMA, and the selected CDR2 has GIVX3GX4X5X6X7SGATGPTX8X9TX 10 The amino acid sequence shown in SVKG, the selected CDR3 has DHX 11 X 12 X 13 X 14 X 15 CTSLATFRX 16 The amino acid sequence represented by X1FGY, wherein X1 is D or H; X2 is G, A or H; X3 is I or H; X4 is G or H; X5 is G or H; X6 is T or H; X7 is H or T; X8 is Y or H; X9 is Y or H; X1 is D or H; X1 is G, A ...1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is D or H; X1 is G, A or H; X1 is G, A or H; X1 is G, A or H; X1 is G, 10 D or H; X 11 W or Y; X 12 A or H; X 13 S or T; X 14 T or H; X 15 G or S; X 16 It is G or H.

[0090] The amino acid sequence of the anti-CD70 antigen nanobody provided by the present invention may include: a) an amino acid sequence as shown in SEQ ID NO: 3; or, b) an amino acid sequence having a sequence identity of more than 80% with SEQ ID NO: 3 and having the function of the amino acid sequence defined in a); specifically, the amino acid sequence in b) specifically refers to: an amino acid sequence as shown in SEQ ID NO: 3 obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of a polypeptide fragment as shown in one of the amino acid fragments of SEQ ID NO: 3, for example, the ability to specifically bind to CD70. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with SEQ ID NO: 3 or 4. The anti-70 nanobody provided by the present invention can bind to the recombinant human CD70 antigen with high affinity and block the binding of CD70 to its receptor CD27.

[0091] According to another aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: a first domain, the first domain having the aforementioned anti-human CD70 antibody; and a second domain, the second domain comprising a polypeptide for prolonging the half-life in vivo and / or having a binding effect on effector cells. The fusion protein can be a binding molecule that can specifically bind to the recombinant human CD70 antigen.

[0092] In the second domain, the fragment used to extend the half-life in vivo may include a serum albumin fragment, a polyethylene glycol fragment, a domain that binds to HSA (e.g., a nanobody that binds to HSA), etc. In the second domain, the fragment that has a binding effect on effector cells may include an immunoglobulin Fc region, etc., preferably selected from a human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations for changing the effector function mediated by Fc, and the effector function includes a combination of one or more of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG may be specifically selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the nanobody fusion protein can cause the fusion protein to form a dimer, while extending the in vivo half-life of the fusion protein and increasing Fc-mediated related activities. In a specific embodiment of the present invention, the immunoglobulin Fc region can be the Fc region of human IgG1, more specifically, it can be a wild-type IgG1 Fc sequence, and the sequence can be introduced with mutations for changing Fc-mediated effector functions, for example, a) mutations that change Fc-mediated CDC activity; b) mutations that change Fc-mediated ADCC activity; or c) mutations that change Fc-mediated ADCP activity.

[0093] Nucleic acids, vectors and cells

[0094] Embodiments of the present invention also provide a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector has been introduced (i.e., such that the host cell contains the polynucleotide and / or a vector containing the polynucleotide), and a recombinant antibody polypeptide or fragment thereof produced by recombinant technology.

[0095] As used herein, a "vector" is any construct capable of delivering one or more target polynucleotides to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more target polynucleotides as encoded polypeptides in a host cell into which the expression vector has been introduced. Therefore, in an expression vector, the target polynucleotide is positioned in the vector for expression by being operably connected to a regulatory component such as a promoter, an enhancer, and / or a polyadenylic acid tail, and the regulatory component is located at or near or on both sides of the integration site of the target polynucleotide in the vector or in the genome of the host cell, so that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0096] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0097] The present invention provides host cells transformed with the vectors described above. The host cell can be a prokaryotic or eukaryotic cell. The preferred prokaryotic host cell is Escherichia coli. Preferably, the eukaryotic cell is selected from: protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. The preferred fungal cell is Saccharomyces cerevisiae.

[0098] Method for preparing anti-CD70 antibodies

[0099] Isolated fragments of human CD70 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated to an agent that is immunogenic in the species to be immunized. The antigenic peptides or proteins can be injected into animals multiple times.

[0100] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, a recombinantly expressed polypeptide or a chemically synthesized polypeptide (e.g., a fragment of human CD70). The preparation may further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0101] Pharmaceutical compositions, uses and methods of treatment

[0102] The present invention also provides a pharmaceutical composition containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the present invention. Two or more (e.g., two, three, or four) of any antibody or antigen-binding fragment described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0103] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc. that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as sugar, polyols such as mannitol, sorbitol or sodium chloride in the composition. A pharmaceutically acceptable carrier may further include a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative or a buffer, which increases the shelf life or effectiveness of the antibody.

[0104] In one aspect, the present invention provides use of the aforementioned anti-human CD70 antibody or antigen-binding fragment thereof in the preparation of a medicament for treating cancer.

[0105] According to an embodiment of the present invention, the cancer is a blood tumor, a kidney cancer, a pancreatic cancer, a throat cancer. The present invention provides one or more antibodies or antigen-binding fragments thereof that can be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides a method for treating a subject's cancer, a method for reducing the rate at which a subject's tumor volume increases over time, a method for reducing the risk of metastasis, or a method for reducing the risk of another metastasis in a subject. In some embodiments, treatment can stop, slow, delay or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity and / or duration of one or more symptoms of a subject's cancer.

[0106] In one aspect, the disclosure features a method comprising administering to a subject in need thereof (e.g., a subject having, identified as having, or diagnosed as having cancer) a therapeutically effective dose of an antibody or antigen-binding fragment thereof disclosed herein.

[0107] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0108] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0109] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in the art (e.g., reference to "Molecular Cloning Experiment Guide" by J. Sambrook, Huang Peitang, etc., the third edition, Science Press) or the product instructions are followed. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially, such as those that can be purchased from Sigma.

[0110] Example 1: Preparation of anti-human CD70 control antibody

[0111] The variable region gene synthesis was performed for the same target control antibody Cusatuzumab (sequence source: WO2012123586), and the light and heavy chain variable region sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The light and heavy chain sequences were cloned into eukaryotic transient expression vectors containing human lambda / IgG1 light and heavy chain constant regions, respectively, to obtain the control antibody light chain and heavy chain expression plasmids, which were transferred into Escherichia coli for amplification, and a large number of plasmids containing the control antibody light chain and heavy chain were isolated and obtained. The plasmids were extracted and ethanol precipitated, and the light and heavy chain plasmids of the control antibody were transferred into HEK293 cells for recombinant expression according to the operating instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain the control antibody.

[0112] SEQ ID NO: 1: Cusatuzumab heavy chain variable region amino acid sequence

[0113] EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS

[0114] SEQ ID NO: 2: Cusatuzumab light chain variable region amino acid sequence

[0115] QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLGQPK

[0116] Example 2. Preparation of camel nanobodies

[0117] Camels were immunized with recombinant CD70 antigen to construct a camel immune library. Positive clone C8 was obtained by screening recombinant CD70 protein. Primers were designed based on the sequencing results of phage-displayed nanoantibodies, and C8 was cloned into a eukaryotic transient expression vector containing the human Fc (hFc) encoding gene by PCR, and recombinantly expressed in HEK293 cells. 5-6 days after cell transfection, the culture supernatant was taken and the expression supernatant was purified using a ProA affinity chromatography column to obtain chC8 recombinant protein. The variable region sequence is shown in SEQ ID NO: 3, and the constant region sequence is shown in SEQ ID NO: 4.

[0118] SEQ ID NO: 3: chC8 VHH amino acid sequence

[0119] QVQLQESGGGSVQAGGSLLRLSCAASGIIVN DGCMA WFRQVPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNGKNTLYLQMNMLKPEDTATYYCAA DHWASTGCTSLATFRGDFGY WGQGTQVTVSS

[0120] SEQ ID NO: 4: hFc constant region amino acid sequence

[0121] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0122] Example 3: Chimeric Antibody Affinity Detection

[0123] The antibody affinity was determined by using the Octet QKe system instrument of Fortebio, and the capture antibody (AHC) bioprobe of the anti-human antibody Fc segment was used to capture the antibody Fc segment. During the determination, the chC8 chimeric antibody and the control antibody Cusatuzumab were diluted to 4ug / ml with PBS buffer and flowed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120s. CD70 recombinant protein (purchased from ACRO, Cat#CDL-H82D7; lot.#BV3407-9BFF1-QD, active trimer) 60nm; as the mobile phase, the binding time was 300s and the dissociation time was 300s. After the experiment, the blank control response value was deducted, and the 1:1 Langmuir binding mode fitting was performed using the software to calculate the kinetic constants of antigen-antibody binding.

[0124] The kinetic parameters are shown in Table 1 below. The results show that chC8 can bind to CD70 recombinant protein, and the binding activity is comparable to that of the control antibody Cusatuzumab.

[0125] Table 1. Affinity determination results of chimeric antibodies and CD70 recombinant protein

[0126] Sample KD(M) kon(1 / Ms) kdis(1 / s) Cusatuzumab 7.18E-10 3.28E+05 2.35E-04 chC8 6.79E-10 3.28E+05 2.23E-04

[0127] Example 4: ELISA to detect species specificity of anti-CD70 chimeric antibodies

[0128] Human CD70 recombinant protein (purchased from ACRO, Cat#CDL-H82D7; lot.#BV3407-9BFF1-QD, activetrimer), cynomolgus monkey CD70 recombinant protein (purchased from ACRO, Cat#CDL-C5244; lot.#3870c-20AGF1-T2), and mouse CD70 recombinant protein (purchased from ACRO, Cat#CDL-M82Qb; lot.#BV2887b-8AQF1-M7) were coated overnight at 4°C with a coating concentration of 1ug / mL. After washing the plate three times with PBS, 5% BSA was added. PBS, blocked at 37℃ for 60min, washed 3 times with PBST; added chC8 and control antibody Cusatuzumab and antibody isotype control Isotype (starting concentration was 66nm, 3-fold gradient dilution to 12 concentrations), incubated at 37℃ for 60min, washed 4 times with PBST; added HRP-anti-human Fc (Cat: 109-035-098, Jackson Immuno Research) diluted 1:5000, incubated at 37℃ for 45min, washed 4 times with PBST; added TMB substrate for color development, incubated at 37℃ for 10min, and then added 2M HCl to terminate the reaction; with 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at a wavelength of 450nm.

[0129] The experimental results showed that chC8 and the control antibody Cusatuzumab can specifically bind to human and cynomolgus monkey CD70 recombinant proteins ( Figure 1 and Figure 2 ), and the corresponding half effective binding concentration EC50 values ​​are shown in Table 2. chC8 and the control antibody Cusatuzumab have weak cross-reactivity with mouse CD70 at high concentrations ( Figure 3 ).

[0130] Table 2. EC50 values ​​of anti-CD70 chimeric antibodies binding to different species detected by ELISA

[0131] Human CD70 Cynomolgus monkey CD70 Mouse CD70 chC8 0.08839 0.1090 / Cusatuzumab 0.07095 0.09368 /

[0132] Example 5: ELISA detection of the blocking activity of anti-CD70 chimeric antibodies against recombinant CD27 and CD70

[0133] The human CD27 extracellular domain recombinant protein (CD27-His, KN ​​expression) was diluted to 0.2ug / mL with PBS, 100ul / well, coated on the enzyme-linked plate, and coated overnight at 4℃; 5% BSA blocking solution was blocked in a 37℃ constant temperature incubator for 120min, and the plate was washed 3 times with PBST; 50uL chC8 and control antibody Cusatuzumab test samples (starting at 3ug / ml, 12 gradients of 3-fold gradient dilution) were taken, 50ul 50ng / ml CD70-mFc was added, mixed, reacted at 37℃ for 30min, and then added to the CD27 coated wells; duplicate wells; 37℃ constant temperature incubator for 60min PBST washed 4 times; 1:5000 diluted HRP-anti-mouse IgG (Cat: 115-035-071, Jackson Immuno Research) for 45 minutes, wash the plate 4 times with PBST; finally, add TMB substrate for color development, react in a 37°C constant temperature incubator for 15 minutes, terminate the reaction with 2M HCl, and read and record the absorbance of the plate at a wavelength of 450nm. The results show ( Figure 4 and Table 3), both chC8 and the control antibody Cusatuzumab can specifically block the binding of CD27 and CD70, and the blocking activity IC50 of chC8 is comparable to that of the control antibody Cusatuzumab.

[0134] Table 3. IC50 results of ELISA detection of blocking activity of CD70 chimeric antibody against recombinant CD27 and CD70

[0135] chC8 Cusatuzumab IC50(ug / ml) 0.003 0.003

[0136] Example 6: FACS detection of the binding activity of anti-CD70 chimeric antibodies on CCRF cells

[0137] 2E5 cells were used to bind to different concentrations of anti-CD70 antibodies, chC8 and control antibody Cusatuzumab were diluted 3-fold in 11 gradients starting from 66nm. Incubated at 4℃ in the dark for 60min, washed with PBS, added 1:200 diluted FITC-labeled goat anti-human antibody (sigma, F9512), incubated at 4℃ in the dark for 30min, washed with PBS, resuspended in 200ul PBS, and detected by flow cytometry. The results showed ( Figure 5 ), chC8 and the control antibody Cusatuzumab have comparable binding abilities, and the EC50 values ​​are shown in Table 4. chC8 and the control antibody Cusatuzumab have similar binding abilities.

[0138] Table 4. EC50 results of binding activity of anti-CD70 chimeric antibodies on CCRF cells detected by FACS

[0139] chC8 Cusatuzumab EC50(nM) 0.5698 0.7453

[0140] Example 7: Humanization and recombinant expression analysis of anti-CD70 nanobody

[0141] First, a comprehensive analysis of the camel-derived antibody VHH sequence was performed to determine the antigen complementary determining cluster (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conservative three-dimensional conformation of the antibody. Subsequently, based on the homology comparison results, the most similar human antibody template was selected as the basic template, and combined with the full sequence blast results, CDR transplantation was performed to achieve the humanization of the chC8 variable region (VHH) in the Framework region. The amino acid sequence of hzC8 after humanization is shown in SEQ ID NO: 5. A series of mutants of hzC8 were designed and verified. The mutant sequences are shown in Table 5, and the specific sequence information is shown in SEQ ID NO: 6-21.

[0142] The humanized hzC8 variable region and different mutant sequences were fully synthesized and cloned into a eukaryotic expression vector containing the human Fc (hFc) coding gene. After obtaining the expression plasmid with the correct sequence, it was transferred into HEK293 cells for recombinant expression. After 5-6 days of cell transfection, the culture supernatant was taken and purified using a ProA affinity chromatography column to obtain the humanized antibody recombinant protein.

[0143] Table 5. hzC8 mutant sequence design information

[0144]

[0145] Note: For example, W108H means that the amino acid W at position 108 is mutated to H.

[0146] SEQ ID NO.5: hzC8 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 25, 22 and 23 respectively)

[0147] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DACMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0148] Example 8: Affinity detection of humanized CD70 antibody

[0149] The antibody affinity was determined using the Octet QKe system instrument of Fortebio, using the capture antibody (AHC) bioprobe of the anti-human antibody Fc segment to capture the antibody Fc segment. During the determination, chC8, hzC8 and different mutants, and the control antibody Cusatuzumab were diluted to 4ug / ml with PBS buffer and flowed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120s. CD70 recombinant protein (purchased from ACRO, Cat#CDL-H82D7; lot.#BV3407-9BFF1-QD, activetrimer) 60nm; as the mobile phase, the binding time was 300s, and the dissociation time was 300s. After the experiment was completed, the blank control response value was deducted, and the software was used to perform 1:1 Langmuir binding mode fitting and calculate the kinetic constants of antigen-antibody binding. The kinetic parameters are shown in Table 6 below. The results showed that the affinity of the humanized antibody hzC8 was basically consistent with that of the chimeric antibody, and there were differences in affinity between different humanized mutants and the parent. The affinities of the m5, m6, m8, m9, m13, and m14 mutants were equivalent to or better than those of the parents, providing a basis for subsequent research.

[0150] Table 6. Affinity determination results of humanized antibodies and CD70 recombinant protein

[0151]

[0152]

[0153] Example 9: FACS detection of the binding activity of anti-CD70 humanized antibodies on SKOV3 cells

[0154] 2E5 cells were used to bind to different concentrations of anti-CD70 antibodies, chC8, hzC8, different mutants and control antibody Cusatuzumab were diluted 3-fold from 66nm in 9 gradients. Incubated at 4℃ in the dark for 60min, washed with PBS, added 1:200 diluted FITC-labeled goat anti-human antibody (sigma, F9512), incubated at 4℃ in the dark for 30min, washed with PBS, resuspended in 200ul PBS, and detected by flow cytometry. EC50 value was calculated.

[0155] The results showed that the EC50 of hzC8 was weaker than that of the chimeric antibody. The EC50 is shown in Table 7. There are differences between the EC50 of different mutants and the parent molecule, which provides a basis for subsequent studies.

[0156] Table 7. EC50 results of binding activity of different humanized anti-CD70 antibodies on SKOV3 cells detected by FACS

[0157] EC50(nM) Cusatuzumab 0.3445 chC8 0.4469 hzC8 0.7889 hzC8m1 1.314 hzC8m2 0.982 hzC8m3 2.749 hzC8m4 8.428 hzC8m5 1.041 hzC8m6 0.269 hzC8m7 3.752 hzC8m8 0.794 hzC8m9 0.483 hzC8m10 2.996 hzC8m11 3.104 hzC8m12 1.036 hzC8m13 0.428 hzC8m14 0.247 hzC8m15 2.268 hzC8m16 0.415

[0158] Example 10: Anti-CD70 antibody mediated internalization in SKOV3 cells

[0159] Trypsin digested SKOV3 cells, divided into 5×10E5 cells / sample / 100μL, 500μL PBS containing 5% BSA blocked cell surface receptors, incubated on ice for 30min. Then centrifuged at 4℃, 1500rpm for 3min, and discarded the supernatant. The working antibodies hzC8, Cusatuzumab and Isotype were diluted to 10μg / mL with RPMI 1640 (containing 10% FBS) and added to the cells. One group was placed in a 37℃ electric thermostatic incubator, and one group was placed in a 4℃ refrigerator as a negative control; 1mL of ice-cold PBS was added to each tube to wash the cells once, and finally 200μL of secondary antibody Goat Anti human IgG Fc-FITC (1:200) was added, the cells were resuspended, incubated in ice water for 45min, and then centrifuged at 4℃, 1,500rpm for 3min, and the supernatant was discarded. 1mL of ice-cold PBS was added to each tube to wash the cells once, and resuspended in 200μL flow buffer for flow cytometry detection. The internalization rate was calculated according to the following formula:

[0160] %MFI at time point tx = MFI of sample incubated at 37°C × 100 / MFI of control sample incubated at 4°C

[0161] Percentage of endocytosis at tx time point = 100 - %MFI at tx time point

[0162] (tx represents the x time of incubation of primary antibody with cells)

[0163] The experimental results (Table 8) showed that hzC8-CF488 and Cusatuzumab-CF488 were internalized into cells at 37°C and were distributed in a dot-like manner in the cytoplasm.

[0164] Table 8. Analysis of internalization ratio of anti-CD70 antibodies

[0165]

[0166] Example 11: Blood routine test of hCD70 transgenic mice injected intraperitoneally with CD70 antibody

[0167] Three hCD70 humanized mice (Cat: 110770, Biocytogen) were intraperitoneally injected with hzC8 at a dose of 20 mg / kg for a total of three times with an interval of 72 hours. After each administration for 24 hours, anticoagulated whole blood was collected from the tail vein for routine blood tests. The collected blood samples were analyzed by IDEXX ProCyte Dx hematology analyzer. The main observation indicators were RBC (red blood cells), HGB (hemoglobin), PLT (platelets), and RET (reticulocytes). The changes in each observation indicator during the administration process were compared. The experimental results are shown in Figure 6 As shown, no significant changes were found in the platelet count, and the related indicators PDW (platelet distribution width) and MPV (mean platelet volume) showed a downward trend. However, the description of changes in some clinical indicators pointed out that in the case of no changes in the number of red blood cells and platelets, changes in PDW and MPV alone had no clinical significance. The results of routine blood tests showed that no obvious red blood cell toxicity was observed in humanized mice with hzC8, providing a research basis for subsequent molecular development.

[0168] sequence:

[0169] SEQ ID NO: 5: hzC8 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 25, 22 and 23, respectively)

[0170] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DACMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0171] SEQ ID NO: 6: hzC8m1 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 22 and 35, respectively)

[0172] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHYASTGCTSLATFRGDFGY WGQGTLVTVSS

[0173] SEQ ID NO: 7: hzC8m2 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 22 and 36, respectively)

[0174] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWHSTGCTSLATFRGDFGY WGQGTLVTVSS

[0175] SEQ ID NO: 8: hzC8m3 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 25, 22 and 38, respectively)

[0176] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DACMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWATTGCTSLATFRGDFGY WGQGTLVTVSS

[0177] SEQ ID NO: 9: hzC8m4 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 25, 22 and 39, respectively)

[0178] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DACMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASHGCTSLATFRGDFGY WGQGTLVTVSS

[0179] SEQ ID NO: 10: hzC8m5 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 25, 22 and 40, respectively)

[0180] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DACMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTSCTSLATFRGDFGY WGQGTLVTVSS

[0181] SEQ ID NO: 11: hzC8m6 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 25, 22 and 41, respectively)

[0182] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DACMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRHDFGY WGQGTLVTVSS

[0183] SEQ ID NO: 12: hzC8m7 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 22 and 37, respectively)

[0184] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGHFGY WGQGTLVTVSS

[0185] SEQ ID NO: 13: hzC8m8 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 27 and 23, respectively)

[0186] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVHGGGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0187] SEQ ID NO: 14: hzC8m9 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 28 and 23, respectively)

[0188] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGHGTTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0189] SEQ ID NO: 15: hzC8m10 (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 29 and 23, respectively)

[0190] EVQLLESGGGLVQ SEQ ID NO: 15:hzC8m10 amino acid sequencePGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGHTTSGATGPTYYTDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCT SLATFRGDFGY WGQGTLVTVSS

[0191] SEQ ID NO: 16: hzC8m11 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 30 and 23, respectively)

[0192] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGHTSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0193] SEQ ID NO: 17: hzC8m12 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 31 and 23, respectively)

[0194] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTHSGATGPTYYTD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0195] SEQ ID NO: 18: hzC8m13 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 32 and 23, respectively)

[0196] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTHYTD S.V.K.G. RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0197] SEQ ID NO: 19: hzC8m14 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 33 and 23, respectively)

[0198] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYHTD S.V.K.G. RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0199] SEQ ID NO: 20: hzC8m15 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 42, 34 and 23, respectively)

[0200] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DGCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTH S.V.K.G. RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0201] SEQ ID NO: 21: hzC8m16 amino acid sequence (CDRs 1, 2 and 3 are SEQ ID NOs: 26, 22 and 23, respectively)

[0202] EVQLLESGGGLVQPGGSLRLSCAASGIIVN DHCMA WFRQAPGKEREGVA GIVIGGGTTSGATGPTYYTD S.V.K.G. RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA DHWASTGCTSLATFRGDFGY WGQGTLVTVSS

[0203] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0204] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. An anti-human CD70 antibody, characterized in that The invention comprises at least one immunoglobulin single variable domain (ISVD) that binds to human CD70, wherein the ISVD has three complementary determining regions, namely CDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence shown in DX2CMA, and the CDR2 has GIVX3GX4X5X6X7SGATGPTX8X9TX 10 The amino acid sequence shown in SVKG, the CDR3 has DHX 11 X 12 X 13 X 14 X 15 CTSLATFRX 16 The amino acid sequence represented by X1FGY, in, X1 is D or H; X2 is G, A or H; X3 is I or H; X4 is G or H; X5 is G or H; X6 is T or H; X7 is H or T; X8 is Y or H; X9 is Y or H; X 10 is D or H; X 11 is W or Y; X 12 A or H; X 13 is S or T; X 14 is T or H; X 15 is G or S; X 16 It is G or H.

2. The antibody according to claim 1, characterized in that The CDR1, the CDR2 and the CDR3 have one of the following amino acid sequences: (1) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 23, respectively; (2) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 23, respectively; (3) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 35, respectively; (4) the amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 42, 22 and 36, respectively; (5) The amino acid sequences of the CDR1, CDR2 and CDR3 are as shown in SED ID NOs: 25, 22, respectively. and 38; (6) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 39, respectively; (7) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 40, respectively; (8) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 25, 22 and 41, respectively; (9) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 22 and 37, respectively; (10) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 27 and 23, respectively; (11) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 28 and 23, respectively; (12) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 29 and 23, respectively; (13) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 30 and 23, respectively; (14) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 31 and 23, respectively; (15) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 32 and 23, respectively; (16) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 33 and 23, respectively; (17) The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SED ID NOs: 42, 34 and 23, respectively; (18) The amino acid sequences of the CDR1, the CDR2 and the CDR3 are shown in SED ID NOs: 26, 22 and 23, respectively.

3. The antibody according to claim 1, characterized in that The at least one immunoglobulin ISVD that binds to human CD70 is a humanized Camelidae VHH.

4. The antibody according to claim 1, characterized in that It includes at least one VHH having 80% identity with the amino acid sequence shown in SEQ ID NO: 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21.

5. A fusion protein, characterized in that include: A first domain, wherein the first domain comprises the anti-human CD70 antibody according to any one of claims 1 to 4; and The second domain comprises a polypeptide for prolonging the half-life in vivo and / or having a binding effect on effector cells.

6. The fusion protein according to claim 5, characterized in that The second domain comprises a combination of one or more of a serum albumin fragment, a polyethylene glycol fragment and a nanobody that binds to HSA.

7. An isolated nucleic acid, characterized in that The nucleic acid encodes the anti-human CD70 antibody according to any one of claims 1 to 4, or encodes the fusion protein according to claim 5 or 6.

8. A carrier, characterized in that Comprising the nucleic acid of claim 7.

9. An isolated cell, characterized in that Comprising the vector of claim 8.

10. A method for preparing the anti-human CD70 antibody according to any one of claims 1 to 4, characterized in that: include: (1) culturing the cell according to claim 9 under appropriate conditions; as well as (2) Isolate and recover the anti-human CD70 antibody according to any one of claims 1 to 4.

11. A pharmaceutical composition, characterized in that include: The anti-human CD70 antibody according to any one of claims 1 to 4 or the fusion protein according to claim 5 or 6; as well as A pharmaceutically acceptable carrier.

12. Use of the anti-human CD70 antibody according to any one of claims 1 to 4 or the fusion protein according to claim 5 or 6 in the preparation of a medicament for treating cancer.

13. The use according to claim 12, characterized in that The cancers include blood tumors, kidney cancer, pancreatic cancer, and throat cancer.

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