Bispecific antibody molecule targeting CD70 and nkp46 and use thereof

Abstract

Provided are a bispecific antibody targeting CD70 and NKp46, a nucleic acid molecule encoding the bispecific antibody, a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector. Also provided are a method for preparing and purifying the bispecific antibody and a use of the bispecific antibody.

Description

Bispecific antibody molecules targeting CD70 and NKp46 and their applications

[0001] Related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. CN 202510386984.X, filed on March 28, 2025, entitled “Bispecific Antibody Molecule Targeting CD70 and NKp46 and Its Application Thereof”, which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This application generally relates to the fields of immunology and antibody drugs; more specifically, it relates to bispecific antibody molecules targeting CD70 and NKp46, and more specifically, to bispecific antibody molecules comprising a binding domain that recognizes CD70 and an NKp46 binding domain that specifically binds to NK cells, as well as methods for their preparation and uses. Background Technology

[0004] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem cells, characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection, and extramedullary tissue and organ infiltration. Currently, the commonly used "3+7" intensive induction chemotherapy regimen and allogeneic hematopoietic stem cell transplantation can effectively treat young patients and improve their survival rate. However, elderly patients and those with multiple comorbidities often cannot tolerate high-intensity chemotherapy, resulting in high early mortality and low long-term survival rates, necessitating the development of new treatment options.

[0005] Natural killer (NK) cells are a component of the innate immune system and form the body's first line of defense. They roam the body and are recruited to tumor sites to destroy tumor cells.

[0006] Currently, there are technologies and reports on the use of bispecific antibodies targeting hematologic tumor cell surface antigens and NK cell surface antigens to treat hematologic malignancies, and further research in this area is needed in the field.

[0007] Invention Overview

[0008] In a first aspect, this application provides a bispecific antibody against CD70 and NKp46, the bispecific antibody comprising a first antigen-binding region that binds to CD70 and a second antigen-binding region that binds to NKp46.

[0009] In some embodiments of the first aspect, the first antigen-binding region and the second antigen-binding region are independently in Fab form, VHH form, or single-chain antibody (scFv) form. For example, the first antigen-binding region may be in Fab form and the second antigen-binding region may be in VHH form. The bispecific antibody may also comprise an Fc fragment, and the first antigen-binding region and the second antigen-binding region are linked to the Fc fragment.

[0010] In some embodiments of the first aspect, the first antigen-binding region comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:14, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:15, LCDR2 as shown in SEQ ID NO:16, and LCDR3 as shown in SEQ ID NO:17;

[0011] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0012] In some embodiments of the first aspect, the second antigen-binding region is in the form of VHH and includes a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:19, HCDR2 as shown in SEQ ID NO:20, and HCDR3 as shown in SEQ ID NO:21.

[0013] The amino acid sequence of HCDR is defined according to Kabat.

[0014] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:10.

[0015] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:11.

[0016] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region is shown in SEQ ID NO:10.

[0017] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region is shown in SEQ ID NO:11.

[0018] In some embodiments of the first aspect, the second antigen-binding region is in the form of VHH and includes a heavy chain variable region having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with SEQ ID NO:18.

[0019] In some embodiments of the first aspect, the second antigen-binding region is in the form of VHH and includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:18.

[0020] In some embodiments of the first aspect, the first antigen-binding region and the second antigen-binding region are linked to an Fc fragment of the antibody heavy chain constant region. The Fc fragment of the antibody heavy chain constant region may include a first Fc fragment and a second Fc fragment.

[0021] In some embodiments of the first aspect, the Fc fragment of the antibody heavy chain constant region is the Fc fragment of the IgG1 subtype.

[0022] In some embodiments of the first aspect, one of the first Fc fragment and the second Fc fragment is linked to the first antigen-binding region, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen-binding region.

[0023] In some embodiments of the first aspect, both the first antigen-binding region and the second antigen-binding region are connected to one of the first Fc fragment and the second Fc fragment. For example, the first antigen-binding region and the second antigen-binding region are respectively connected to the N-terminus and C-terminus of the first Fc fragment or the second Fc fragment.

[0024] In some preferred embodiments of the first aspect, the bispecific antibody comprises a first Fc fragment and a second Fc fragment, wherein the first antigen-binding region is connected to the first Fc fragment, preferably to the N-terminus of the first Fc fragment; and the second antigen-binding region is connected to the second Fc fragment, preferably to the N-terminus of the second Fc fragment.

[0025] In some preferred embodiments of the first aspect, the bispecific antibody comprises a first Fc fragment and a second Fc fragment, wherein the first antigen-binding region and the second antigen-binding region are respectively linked to one end of the first Fc fragment;

[0026] Preferably, the first antigen-binding region is connected to the N-terminus of the first Fc fragment, and the second antigen-binding region is connected to the C-terminus of the first Fc fragment.

[0027] In some embodiments of the first aspect, the first Fc segment includes a 354-bit S354C substitution and a 366-bit T366W substitution; and / or

[0028] The second Fc segment contains a 349-bit Y349C substitution, a 366-bit T366S substitution, a 368-bit L368A substitution, and a 407-bit Y407V substitution. Preferably, the second Fc segment also contains a 435-bit H435R substitution.

[0029] The amino acid positions in the antibody constant region are determined according to EU numbering.

[0030] In some embodiments of the first aspect, the bispecific antibody is capable of binding to the NKp46 protein of humans and / or the CD70 protein of humans and / or cynomolgus monkeys, or the extracellular region of the NKp46 protein of humans and / or the CD70 protein of humans and / or cynomolgus monkeys. Preferably, the bispecific antibody is capable of binding to the following proteins or their extracellular regions: human NKp46 protein as shown in SEQ ID NO:1, cynomolgus monkey NKp46 protein as shown in SEQ ID NO:2, human CD70 protein as shown in SEQ ID NO:3, and / or cynomolgus monkey CD70 protein as shown in SEQ ID NO:4.

[0031] Secondly, this application provides a nucleic acid molecule that encodes the bispecific antibody described in the first aspect.

[0032] Thirdly, this application provides a vector containing the nucleic acid molecules described in the second aspect.

[0033] In some embodiments of the third aspect, the vector further comprises a regulatory sequence, and the nucleic acid molecule is operatively linked to the regulatory sequence.

[0034] Fourthly, this application provides a host cell that contains the nucleic acid molecules described in the second aspect or the vector described in the third aspect.

[0035] Fifthly, this application provides a method for preparing the bispecific antibody described in the first aspect, comprising culturing the host cell of the fourth aspect and recovering the bispecific antibody from the culture.

[0036] Sixthly, this application provides the use of the bispecific antibody described in the first aspect in the preparation of a medicament for the prevention or treatment of CD70-positive tumors.

[0037] In a seventh aspect, this application provides a method for preventing or treating CD70-positive tumors in a patient, comprising administering an effective amount of the bispecific antibody described in the first aspect to the patient.

[0038] In some embodiments of the sixth and / or seventh aspects, the CD70-positive tumor is selected from acute myeloid leukemia, monocytic leukemia, and Burkitt lymphoma. Attached Figure Description

[0039] The accompanying drawings further illustrate the features of this application. Referring to these drawings will provide a better understanding of the features and advantages disclosed in this application; however, it should be understood that these drawings are only for illustrating specific embodiments of the principles disclosed in this application and are not intended to limit the scope of the appended claims.

[0040] Figure 1A shows the results of verifying human NKp46 expression on CHO-K1 cells using flow cytometry with an anti-NKp46 positive control antibody; the dashed line in the figure represents human NKp46 expression on CHO-K1 parent cells that have not been transfected with any plasmid, and the solid line in the figure represents human NKp46 protein overexpression after CHO-K1 parent cells are transfected with human NKp46 plasmid.

[0041] Figure 1B shows the results of flow cytometry analysis using an anti-NKp46 positive control antibody to verify the expression of cynomolgus monkey NKp46 in CHO-K1 cells; the dashed line in the figure represents the expression of cynomolgus monkey NKp46 in CHO-K1 parent cells that have not been transfected with any plasmid, and the solid line in the figure represents the overexpression of cynomolgus monkey NKp46 protein in CHO-K1 parent cells after transfection with the cynomolgus monkey NKp46 plasmid.

[0042] Figure 2A shows the results of verifying human CD70 expression on CHO-K1 cells using flow cytometry with an anti-CD70 positive control antibody; the dashed line in the figure represents the expression of human CD70 on CHO-K1 parent cells that have not been transfected with any plasmid, and the solid line in the figure represents the overexpression of human CD70 protein after CHO-K1 parent cells are transfected with human CD70 plasmid.

[0043] Figure 2B shows the results of flow cytometry using an anti-CD70 positive control antibody to verify the expression of cynomolgus CD70 in CHO-K1 cells; the dashed line in the figure represents the expression of cynomolgus CD70 in CHO-K1 parent cells that have not been transfected with any plasmid, and the solid line in the figure represents the overexpression of cynomolgus CD70 protein in CHO-K1 parent cells after transfection with the cynomolgus CD70 plasmid.

[0044] Figure 3A shows the binding results of antibody clone 70CH-20-H4L3-M, which binds to the CD70 antigen, with the stable human CD70-CHO-K1 cell line expressing human CD70. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0045] Figure 3B shows the binding results of antibody clone 70CH-20-H4L3-M, which binds to the CD70 antigen, with the stable transgenic cynomolgus monkey CD70-CHO-K1 cell line. The isotype control in the figure is a human IgG1-κ isotype control antibody.

[0046] Figure 4A shows the binding results of the antibody clone H2-NKp46-PA27-M, which binds to the NKp46 antigen, with the stable human NKp46-CHO-K1 cell line expressing human NKp46. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0047] Figure 4B shows the binding results of the antibody clone H2-NKp46-PA27-M, which binds to the NKp46 antigen, with the stable transgenic cynomolgus monkey NKp46-CHO-K1 cell line expressing NKp46. The isotype control in the figure is a human IgG1-κ isotype control antibody.

[0048] Figure 5 shows the structures of two exemplary bispecific antibodies (biantibody-1 and biantibody-2, respectively).

[0049] Figure 6A shows the binding results of bispecific antibodies 1 and 2 to the stable human CD70-CHO-K1 cell line expressing human CD70. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0050] Figure 6B shows the binding results of bispecific antibodies 1 and 2 to the stable transgenic cynomolgus monkey CD70-CHO-K1 cell line expressing CD70. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0051] Figure 6C shows the binding results of bispecific antibodies 1 and 2 to the stable human NKp46-CHO-K1 cell line expressing human NKp46. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0052] Figure 6D shows the binding results of bispecific antibodies 1 and 2 to the stable cynomolgus monkey NKp46-CHO-K1 cell line expressing NKp46. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0053] Figure 6E shows the binding of bispecific antibodies 1 and 2 to CD70-expressing tumor cells MOLM-13. The isotype control in the figure is a human IgG1-κ isotype control antibody.

[0054] Figure 7A shows the results of ADCC experiments mediated by bispecific antibody 1 and bispecific antibody 2 against THP-1 tumor cells. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0055] Figure 7B shows the results of ADCC experiments mediated by bispecific antibody 1 and bispecific antibody 2 against MOLM-13 tumor cells. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0056] Figure 8 shows the IL-6 release levels induced by bispecific antibodies 1 and 2 under co-incubation conditions in PBMCs and THP-1 tumor cells. The isotype control in the figure is a human IgG1-κ isotype control antibody.

[0057] Figure 9 shows the efficacy results of bispecific antibodies 1 and 2 in a mouse model reconstituted with human immune cells. The isotype control in the figure is the human IgG1-κ isotype control antibody.

[0058] Sequence Description

[0059] SEQ ID NO:1-2 show the full-length amino acid sequences of human (Homo sapiens) NKp46 (see Uniprot ID: O76036) and cynomolgus monkey (Macaca fascicularis) NKp46 (see Uniprot ID: Q95JB9), respectively.

[0060] SEQ ID NO:3-4 show the full-length amino acid sequences of human (Homo sapiens) CD70 (see Uniprot ID: P32970) and cynomolgus monkey (Macaca fascicularis) CD70 (see Uniprot ID: A0A2K5WTU9), respectively.

[0061] SEQ ID NO:5-6 show the amino acid sequences of the "pestle" heavy chain (containing CD70 VH-CH1-IgG1 Fc(knob)) and the "mortar" heavy chain (containing NKp46 VHH-IgG1 Fc(hole)) of the bispecific antibody, respectively.

[0062] SEQ ID NO:7 shows the amino acid sequences of the light chains of bispecific antibody 1 and bispecific antibody 2.

[0063] SEQ ID NO:8-9 show the amino acid sequences of the "pestle" heavy chain (containing CD70 VH-CH1-IgG1 Fc(knob)-(G4S)3-NKp46 VHH) and the "mortar" heavy chain (containing IgG1 Fc(hole)) of the bispecific antibody, respectively.

[0064] SEQ ID NO:10-11 show the amino acid sequences of the heavy chain variable region and light chain variable region of the CD70 binding portion of bispecific antibody 1 and bispecific antibody 2, respectively.

[0065] SEQ ID NO:12-14 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the CD70 binding part of bispecific antibody 1 and bispecific antibody 2, respectively.

[0066] SEQ ID NO:15-17 show the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the CD70 binding portion of bispecific antibody 1 and bispecific antibody 2, respectively.

[0067] SEQ ID NO:18 shows the amino acid sequence of the heavy chain variable region of the portion of bispecific antibody 1 and bispecific antibody 2 that binds NKp46.

[0068] SEQ ID NO:19-21 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the NKp46 binding part of bispecific antibody 1 and bispecific antibody 2, respectively.

[0069] Invention Details

[0070] Analysis of AML patient samples revealed high expression of CD70 in both AML blast cells and leukemia stem cells (LSCs). High expression of CD70 was also detected in other tumor cells, such as monocytic leukemia cells and Burkitt lymphoma cells. Furthermore, treatment of AML patients with the demethylating drug azacitidine induced CD70 expression in LSCs, promoting LSC proliferation and leading to treatment failure.

[0071] NKp46 (NCR1, CD335) is an activating receptor on the surface of NK cells, present in quiescent and activated NK cells as well as a small subset of T lymphocytes.

[0072] Bispecific antibodies containing the NKp46 binding domain can bring NK cells closer to tumor cells and activate the cytotoxicity of NK cells against tumor cells. These antibodies are called NK cell engagers.

[0073] The inventors of this application have obtained novel bispecific antibodies against CD70 and NKp46 through antibody engineering technology. In various aspects of this application, novel bispecific antibodies against CD70 and NKp46 (e.g., simultaneously binding to CD70 and NKp46 proteins) are provided, along with nucleic acid molecules encoding said bispecific antibodies, vectors containing said nucleic acid molecules, host cells containing said nucleic acid molecules or vectors, methods for preparing and purifying said bispecific antibodies, and medical and biological uses of said bispecific antibodies. Based on the amino acid sequence of the variable region of the bispecific antibody provided in this application, full-length antibody molecules can be constructed as drugs for the prevention or treatment of CD70-positive tumors (e.g., acute myeloid leukemia, monocytic leukemia, Burkitt's lymphoma, etc.) and related diseases.

[0074] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0075] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0076] definition

[0077] In a broad sense, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. "Antibody" includes not only complete (i.e., full-length) antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), their variants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity, including glycosylated variants, amino acid sequence variants, and covalently modified antibodies. When "antibody" and "antigen-binding region / fragment / part" appear in the same context, "antibody" can be understood as the complete entity relative to the "antigen-binding region / fragment / part," both corresponding to the broad concept of antibody.

[0078] Typically, a full-length or complete antibody consists of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody's amino acid sequence of the heavy chain constant domain. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0079] As used herein, the terms "antigen-binding region" or "antigen-binding fragment" or "antigen-binding part" are used interchangeably and refer to a portion or region of the complete antibody molecule responsible for binding the antigen. An antigen-binding domain may contain a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0080] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the variable regions that have the greatest impact on antibody affinity and specificity. There are two common definitions for the CDR amino acid sequence of VH or VL: the Chothia definition and the Kabat definition. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. (1991). [1] ;A1-Lazikani et al., J.Mol.Biol.273:927-948 (1997) [2] ; and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989) [3]For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can be determined according to the Chothia or Kabat definition. In embodiments of this application, the Kabat definition of the CDR amino acid sequence is used. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, based on the sequences herein, humanized antibodies containing one or more CDRs derived from any nomenclature system are explicitly kept within the scope of this document.

[0081] For a given antibody's variable region amino acid sequence, the CDR amino acid sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0082] Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments having a single arm of antibody with VL and VH domains; (4) single-chain Fv(scFv), which may be a single multipeptide chain consisting of VH and VL domains connected by peptide linkers; and (5) (scFv)2, which may contain two VH domains and two VL domains connected by peptide linkers, the two VL domains being combined with the two VH domains via disulfide bridges.

[0083] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0084] As used in this article, “monoclonal antibody” refers to an antibody obtained from a population of essentially homogeneous antibodies, that is, the individual antibodies that make up the population are identical except for the possibility of naturally occurring mutations in a small number of individuals.

[0085] As used herein, the term "bispecific antibody" refers to an antibody that simultaneously binds to two antigenic epitopes. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two binding moieties fused to them (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding moieties can be in the form of Fab fragments, VHH-type antibodies, or single-chain antibodies (scFv), etc. For example, the two different binding moieties of a bispecific antibody may each bind to the N-terminus of an Fc fragment, or the two different binding moieties of a bispecific antibody may bind to the same Fc fragment (e.g., one binding moieties bind to the N-terminus of the Fc fragment, and the other binds to the C-terminus of the Fc fragment). The two antigen-binding moieties can be independently configured as Fab fragments, VHH-type antibodies, or single-chain antibodies (scFv), etc. The Fc fragments can contain mutations that ensure heavy chain heteropolymerization; KIH technology (knob-in-hole, KIH) is a strategy to address heavy chain heteropolymerization. Typically, KIH (kidney-in-heap) technology refers to modifying the amino acid sequence of the CH3 region to create a structure that facilitates the pairing of heterologous half-antibodies, thus forming bispecific antibodies while preserving as much of the normal antibody structure as possible. For guidance on KIH technology, see, for example, "An efficient route to human bispecific IgG," A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998. [4] The full text of this article is incorporated herein by reference. Furthermore, the structure of a bispecific antibody can be such that the antibody binding the first antigen (e.g., in the form of a natural antibody) extends from the C-terminus of the CH3 region to an antigen-binding region capable of binding the second antigen (via a flexible linker). Bispecific antibodies can also be dual-variable-domain immunoglobulin (DVD-Ig) type bispecific antibodies, wherein the first antibody binding the first antigen (e.g., in the form of a natural antibody) can extend from the N-terminus of its heavy chain to a variable region of the heavy chain of the second antibody capable of binding the second antigen (via a flexible linker), and the first antibody can extend from the N-terminus of its light chain to a variable region of the light chain of the second antibody (via a flexible linker). For information on DVD-Ig structured bispecific antibodies, see the reference (Wu C, Ying H, Grinnell C, et al. Simultaneous targeting of multipledisease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol. 2007 Nov).[5] The full text of the reference is incorporated into this paper by way of citation.

[0086] As used herein, the term "nanobody," also known as VHH antibody or single-domain antibody, can be defined as having the following (general) amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Here, FR1-FR4 refer to frames 1-4, and CDR1-CDR3 refer to complementarity-determining regions 1-3. "VHH" refers to the variable antigen-binding domain of heavy chain antibodies derived from camels (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen, 2000 EMBO J., 19, 921-930). [6] ; Muyldermans, 2001, J Biotechnol., 74, 277-302 [7] And a review of Vanlandschoot, 2011, Antiviral Res., 92, 389-407. [8] )

[0087] In some specific embodiments, the bispecific antibody of this application has the structure shown in Figure 5 (i.e., "bispecific antibody one" or "bispecific antibody two").

[0088] In some specific configurations of the bispecific antibody, the bispecific antibody comprises three peptide chains. The first peptide chain comprises a CD70 antibody Fab heavy chain and an IgG1 Fc moiety with a "palm" structure. The second peptide chain comprises an NKp46 nanobody and an IgG1 Fc moiety with a "mortar" structure. The third peptide chain is a CD70 antibody light chain. In some specific embodiments, the amino acid sequence of the first peptide chain of the bispecific antibody is shown in SEQ ID NO:5. In some specific embodiments, the amino acid sequence of the second peptide chain of the bispecific antibody is shown in SEQ ID NO:6. In some specific embodiments, the amino acid sequence of the third peptide chain of the bispecific antibody is shown in SEQ ID NO:7.

[0089] In some specific configurations of the bispecific antibody II, the bispecific antibody II comprises three peptide chains. The first peptide chain comprises a CD70 antibody Fab heavy chain, an IgG1 Fc moiety with a "palm" structure, and an NKp46 nanobody. The second peptide chain comprises an IgG1 Fc moiety with a "mortar" structure. The third peptide chain is a CD70 antibody light chain. In some specific embodiments, the amino acid sequence of the first peptide chain of the bispecific antibody II is shown in SEQ ID NO:8. In some specific embodiments, the amino acid sequence of the second peptide chain of the bispecific antibody II is shown in SEQ ID NO:9. In some specific embodiments, the amino acid sequence of the third peptide chain of the bispecific antibody II is shown in SEQ ID NO:7.

[0090] In some specific embodiments, the IgG1 Fc moiety with the "palm" structure includes amino acid substitutions at sites S354C and T366W. In some specific embodiments, the IgG1 Fc moiety with the "mortar" structure includes amino acid substitutions at sites Y349C, T366S, L368A, and Y407V. In some specific embodiments, to facilitate the purification of the bispecific antibody, the "mortar" structure IgG1 Fc moiety is substituted with H435R. The positions of these amino acid substitutions are determined according to EU numbering.

[0091] The term "therapeutic effective dose" or "effective dose" as used herein refers to a dose sufficient to demonstrate its benefit to the individual to which it is administered. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and severity. Prescribing treatment (e.g., determining the dosage) is ultimately the responsibility of and depends on the general practitioner and other physicians, who typically consider the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0092] The term "CD70-positive tumor" as used in this article refers to tumors whose tumor cells express CD70 molecules. The expression of CD70 molecules can be detected using techniques well-known in the field, such as flow cytometry, ELISA, and nucleic acid probes.

[0093] The term "identity / homology / consistency" in relation to amino acid or nucleic acid sequences is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0094] In a first aspect, this application provides a bispecific antibody against CD70 and NKp46, the bispecific antibody comprising a first antigen-binding region that binds to CD70 and a second antigen-binding region that binds to NKp46.

[0095] In some embodiments of the first aspect, the first antigen-binding region and the second antigen-binding region are independently in Fab form, VHH form, or single-chain antibody (scFv) form. Preferably, the first antigen-binding region can be in Fab form and the second antigen-binding region can be in VHH form. The bispecific antibody may also comprise an Fc fragment, and the first antigen-binding region and the second antigen-binding region are linked to the Fc fragment.

[0096] In some embodiments of the first aspect, the first antigen-binding region comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:14, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:15, LCDR2 as shown in SEQ ID NO:16, and LCDR3 as shown in SEQ ID NO:17;

[0097] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0098] In some embodiments of the first aspect, the second antigen-binding region is in the form of VHH and includes a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:19, HCDR2 as shown in SEQ ID NO:20, and HCDR3 as shown in SEQ ID NO:21.

[0099] The amino acid sequence of HCDR is defined according to Kabat.

[0100] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region has at least 90% identity with SEQ ID NO:10.

[0101] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region has at least 90% identity with SEQ ID NO:11.

[0102] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:10.

[0103] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:11.

[0104] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region differs from the amino acid sequence shown in SEQ ID NO:10 by substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0105] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region differs from the amino acid sequence shown in SEQ ID NO:11 by substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0106] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:10 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of the heavy chain variable region of the first antigen-binding region.

[0107] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 10, and the resulting amino acid sequence still retains the function of the heavy chain variable region of the first antigen-binding region.

[0108] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 10, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the first antigen-binding region.

[0109] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:11 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of the light chain variable region of the first antigen-binding region.

[0110] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:11, and the resulting amino acid sequence still retains the function of the light chain variable region similar to the first antigen-binding region.

[0111] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:11, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the first antigen-binding region.

[0112] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region has at least 90% identity with SEQ ID NO:18.

[0113] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:18.

[0114] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region differs from the amino acid sequence shown in SEQ ID NO:18 by substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0115] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding region differs from the amino acid sequence shown in SEQ ID NO:18 by substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0116] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:18 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of the heavy chain variable region of the second antigen-binding region.

[0117] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:18, and the resulting amino acid sequence still retains the function of the heavy chain variable region of the second antigen-binding region.

[0118] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:18, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the second antigen-binding region.

[0119] In some embodiments of the first aspect, the first antigen-binding region and the second antigen-binding region are linked by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment including a first Fc fragment and a second Fc fragment.

[0120] The Fc fragment of the antibody heavy chain constant region can be an Fc fragment of IgG1, IgG2, IgG3 or IgG4 subtypes, preferably an Fc fragment of IgG1 subtype.

[0121] In some embodiments of the first aspect, one of the first Fc fragment and the second Fc fragment is linked to the first antigen-binding region, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen-binding region.

[0122] In some embodiments of the first aspect, both the first antigen-binding region and the second antigen-binding region are connected to one of the first Fc fragment and the second Fc fragment. For example, the first antigen-binding region and the second antigen-binding region may be connected to the N-terminus or C-terminus of the first Fc fragment or the second Fc fragment, respectively.

[0123] In some preferred embodiments of the first aspect, the bispecific antibody comprises a first Fc fragment and a second Fc fragment, wherein the first antigen-binding region is connected to the first Fc fragment, preferably to the N-terminus of the first Fc fragment; and the second antigen-binding region is connected to the second Fc fragment, preferably to the N-terminus of the second Fc fragment.

[0124] In some preferred embodiments of the first aspect, the bispecific antibody comprises a first Fc fragment and a second Fc fragment, wherein the first antigen-binding region and the second antigen-binding region are respectively linked to one end of the first Fc fragment;

[0125] Preferably, the first antigen-binding region is connected to the N-terminus of the first Fc fragment, and the second antigen-binding region is connected to the C-terminus of the first Fc fragment.

[0126] In some embodiments of the first aspect, the first Fc segment includes a 354-bit S354C substitution and a 366-bit T366W substitution; and / or

[0127] The second Fc segment contains a 349-bit Y349C substitution, a 366-bit T366S substitution, a 368-bit L368A substitution, and a 407-bit Y407V substitution. Preferably, the second Fc segment also contains a 435-bit H435R substitution.

[0128] The amino acid positions in the antibody constant region are determined according to EU numbering.

[0129] In some embodiments of the first aspect, the bispecific antibody is capable of binding to the NKp46 protein of humans and / or cynomolgus monkeys and / or the CD70 protein of humans and / or cynomolgus monkeys.

[0130] In some embodiments of the first aspect, the bispecific antibody is capable of binding to the extracellular region of the NKp46 protein in humans and / or the extracellular region of the CD70 protein in humans and / or cynomolgus monkeys.

[0131] Preferably, the bispecific antibody is capable of binding to the following proteins or their extracellular regions: human NKp46 protein as shown in SEQ ID NO:1, cynomolgus monkey NKp46 protein as shown in SEQ ID NO:2, human CD70 protein as shown in SEQ ID NO:3, and / or cynomolgus monkey CD70 protein as shown in SEQ ID NO:4.

[0132] In some embodiments of the first aspect, the bispecific antibody is capable of simultaneously binding CD70 (e.g., the CD70 protein as shown in SEQ ID NO:3 or 4) and NKp46 protein (e.g., the NKp46 protein as shown in SEQ ID NO:1 or 2).

[0133] Secondly, this application provides a nucleic acid molecule that encodes the bispecific antibody described in the first aspect.

[0134] Thirdly, this application provides combinations of polynucleotides, said combinations comprising a polynucleotide encoding a light chain of an antibody of the application or an antigen-binding moiety thereof and a polynucleotide encoding a heavy chain of an antibody of the application or an antigen-binding moiety thereof.

[0135] Fourthly, this application provides a vector (e.g., an expression vector) containing the nucleic acid molecule described in the second aspect or a combination of polynucleotides described in the third aspect.

[0136] In some embodiments, the nucleic acid molecule is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the vector. The regulatory sequence is well known to those skilled in the art and includes, but is not limited to, promoters, enhancers, terminators, and silencers.

[0137] The polynucleotide can be efficiently linked to a regulatory sequence that allows the polypeptide it encodes to be expressed in a host cell or cell-free expression system. The choice of expression vector depends on the choice of host cell and can be selected to achieve the desired expression and regulatory characteristics in the chosen host cell. The host cell is, for example, a mammalian cell.

[0138] An "expression vector" is a vector that includes one or more expression control sequences. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0139] The nucleic acid in the vector can be operatively linked to one or more expression control sequences. As used herein, "operatively linked" means incorporated into the genetic construct such that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of the DNA molecule typically located within 100 nucleotides upstream of the transcription start site (usually near the start site of RNA polymerase II). For the coding sequence to be under the control of the promoter, the translation start site of the polypeptide translation reading frame must be located between 1 and 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function at different distances from the transcription start site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operatively linked" to and "under the control" of the expression control sequence in the cell.

[0140] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegaloviruses, retroviruses, vaccinia virus, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0141] Expression vectors may include tag sequences. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tags (Kodak, New Haven, CT), maltose E-binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the antibody of this application is contained in a vector of nucleic acid encoding one or more domains of the Ig heavy chain constant region, said domains being, for example, amino acid sequences corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragment).

[0142] This application provides a host cell containing the nucleic acid molecules described in the second aspect, a combination of polynucleotides described in the third aspect, or a vector described in the fourth aspect. In some embodiments of this application, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell may be *Escherichia coli* (e.g., *E. coli* cells well-known in the art such as DH5α and BL21), *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The eukaryotic host cell may be a fungus such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharizoa*, *Trichoderma*, etc.; an insect cell such as *Ardisia crenata*; a plant cell such as tobacco; or a mammalian cell such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cell described in this application is preferably a mammalian cell, more preferably a CHO cell, BHK cell, NSO cell, or COS cell.

[0143] In some embodiments, the method for preparing the bispecific antibody described in the first aspect includes culturing host cells and recovering the bispecific antibody from the culture, wherein the host cells contain the nucleic acid molecule described in the second aspect, the combination of polynucleotides described in the third aspect, or the carrier described in the fourth aspect. Techniques for recovering bispecific antibodies from cultures are well known to those skilled in the art, such as disrupting host cells (e.g., using sonication), extracting and purifying bispecific antibodies (e.g., by affinity chromatography (e.g., utilizing the affinity of the antibody for molecules such as Protein A and Protein G, utilizing the affinity of His tags, GST tags, etc. on the antibody for corresponding affinity ligands, etc.), gel filtration chromatography, etc.).

[0144] In some implementations, the bispecific antibody described in the first aspect can be used to prevent or treat CD70-positive tumors.

[0145] Fifthly, this application provides the use of the bispecific antibody described in the first aspect in the preparation of a medicament for the prevention or treatment of CD70-positive tumors.

[0146] In a sixth aspect, this application provides a method for preventing or treating CD70-positive tumors in a patient, the method comprising administering to the patient an effective bispecific antibody as described in the first aspect.

[0147] In some embodiments of the fifth and / or sixth aspects, the CD70-positive tumor is selected from acute myeloid leukemia, monocytic leukemia, and Burkitt lymphoma. Preferably, the CD70-positive tumor is acute myeloid leukemia.

[0148] In some embodiments of the fifth and / or sixth aspects, the CD70-positive tumor may be a cancer that highly expresses CD70. The expression level of CD70 molecules can be detected using techniques known in the art, such as flow cytometry, ELISA, and nucleic acid probes. For example, by detecting the expression of CD70 molecules on single cells using flow cytometry, the proportion of cells expressing CD70 can be determined, thereby determining the abundance of CD70 expression in tumor cells. In some embodiments, a cancer that highly expresses CD70 refers to a cancer cell population in which at least 10% of the cancer cells express CD70. In some embodiments, a cancer that highly expresses CD70 refers to a cancer cell population in which at least 20% of the cancer cells express CD70. In some embodiments, a cancer that highly expresses CD70 refers to a cancer cell population in which at least 30% of the cancer cells express CD70. In some embodiments, a cancer that highly expresses CD40 refers to a cancer cell population in which at least 40% of the cancer cells express CD70. In some embodiments, a cancer that highly expresses CD70 refers to a cancer cell population in which at least 50% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 60% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 70% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 80% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 90% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 95% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 98% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression refers to a cancer cell population in which at least 99% of the cancer cells express CD70. In some embodiments, cancer with high CD70 expression is selected from acute myeloid leukemia, monocytic leukemia, and Burkitt lymphoma. Preferably, cancer with high CD70 expression is acute myeloid leukemia.

[0149] In other aspects, this application also provides nucleic acid molecules encoding the above-described bispecific antibody or its light or heavy chain, vectors containing said nucleic acid molecules, host cells containing said nucleic acid molecules or said vectors, and methods for generating said bispecific antibodies. In some embodiments, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence that can be recognized by host cells transformed with said vector. In some embodiments, the method of generating bispecific antibodies includes culturing host cells to express nucleic acids. In some embodiments, the method of generating bispecific antibodies further includes recovering antibodies from host cell culture medium.

[0150] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments. Example

[0151] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0152] Example 1: Preparation of cell lines expressing human NKp46 and cynomolgus monkey NKp46

[0153] The nucleotide sequence encoding the human NKp46 amino acid sequence shown in SEQ ID NO:1 was cloned into the pCMV3 (SinoBiological, catalog number CV011) vector to obtain a plasmid for constructing a human NKp46 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 NKp46 (hereinafter referred to as the human NKp46-CHO-K1 cell line).

[0154] The nucleotide sequence encoding the cynomolgus monkey NKp46 amino acid sequence shown in SEQ ID NO.2 was cloned into the pCMV3 vector to obtain a plasmid for constructing a cynomolgus monkey NKp46 cell line. The obtained plasmid was transfected into CHO-K1 cells to obtain a CHO-K1 cell line overexpressing cynomolgus monkey NKp46 (hereinafter referred to as the cynomolgus monkey NKp46-CHO-K1 cell line).

[0155] The expression of human NKp46 in the obtained human NKp46-CHO-K1 cell line and the expression of cynomolgus monkey NKp46-CHO-K1 cell line were detected by flow cytometry (FACS). NKp46 antibody (n1D9) (Santa Cruz, SC-59343) was used as the primary antibody and PE-labeled goat anti-mouse IgG antibody was used as the secondary antibody. The results are shown in Figures 1A and 1B. Both the human NKp46-CHO-K1 cell line (Figure 1A) and the cynomolgus monkey NKp46-CHO-K1 cell line (Figure 1B) showed significant overexpression of the antigen (NKp46), which can be used for subsequent experiments to verify the binding of NKp46 binding molecules at the cellular level.

[0156] Example 2: Preparation of cell lines expressing human CD70 and cynomolgus monkey CD70

[0157] The nucleotide sequence encoding the human CD70 amino acid sequence shown in SEQ ID NO.3 was cloned into the pCMV3 plasmid to obtain a plasmid for constructing a human CD70 cell line. The obtained vector was transfected into CHO-K1 cells to obtain a CHO-K1 cell line overexpressing human CD70 (referred to as the human CD70-CHO-K1 cell line in this paper).

[0158] The nucleotide sequence encoding the cynomolgus CD70 amino acid sequence shown in SEQ ID NO.4 was cloned into the pCMV3 plasmid to obtain a plasmid for constructing a cynomolgus CD70 cell line. The obtained vector was transfected into CHO-K1 cells to obtain a CHO-K1 cell line overexpressing cynomolgus CD70 (hereinafter referred to as the cynomolgus CD70-CHO-K1 cell line).

[0159] 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. Cusatuzumab was used as the primary antibody and PE-labeled goat anti-human IgG antibody was used as the secondary antibody. The results are shown in Figures 2A and 2B. Both the human CD70-CHO-K1 cell line (Figure 2A) and the cynomolgus CD70-CHO-K1 cell line (Figure 2B) showed significant overexpression of the antigen (CD70), which can be used for subsequent experiments to verify the binding of CD70 binding molecules at the cellular level.

[0160] Example 3: Obtaining the anti-CD70 antigen-binding fragment and the anti-NKp46 antigen-binding fragment of the bispecific antibody

[0161] 3.1 Obtaining anti-CD70 antigen-binding fragments via mouse immunization

[0162] The anti-CD70 antigen-binding fragment of the bispecific antibody was generated by immunizing mice. Specifically, Balb / c mice (Charles River) were immunized with human CD70 ECD protein (ACRO, catalog number CDL-H52Da) at 25 μg per immunization at days 0, 14, 28, and 42, with a booster immunization two days before spleen cell fusion. Mice with high antibody titers in their serum that were trending towards a plateau 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 were obtained by fusing cells with human CD70. Using CHO-K1 cells highly expressing human CD70, antibody screening of the hybridoma cell culture supernatant was performed using FACS to obtain positive antibody strains. Further, CHO-K1 cells highly expressing cynomolgus monkey CD70 and blank CHO-K1 cells were used to exclude hybridoma strains that specifically bind to human / cynomolgus monkey CD70, thus selecting hybridomas that specifically bind to human / cynomolgus monkey CD70. The light and heavy chain sequences of the antibodies expressed by these hybridoma cells were obtained by sequencing, and these were used to construct human-mouse chimeric antibodies. Using CHO-K1 cells highly expressing human CD70 and CHO-K1 cells expressing cynomolgus monkey CD70, these chimeric antibodies were screened using FACS. The 70CH-20 antibody was ultimately determined to be the optimal antibody. This antibody was subsequently humanized and sequence optimized to obtain the CD70-binding antibody clone 70CH-20-H4L3-M, whose variable region and CDR amino acid sequences are shown in Table 1. The binding of antibody clone 70CH-20-H4L3-M to the human CD70-CHO-K1 cell line is shown in Figure 3A, and the binding of antibody clone 70CH-20-H4L3-M to the cynomolgus monkey CD70-CHO-K1 cell line is shown in Figure 3B.

[0163] Table 1. Amino acid sequences of the variable region and CDR of antibody clone 70CH-20-H4L3-M

[0164] 3.2 Obtaining anti-NKp46 antigen-binding fragments through alpaca immunization

[0165] The anti-NKp46 antigen-binding fragment of the bispecific antibody was generated by immunizing alpacas. Specifically, alpacas were immunized with human NKp46 ECD protein (Kaikai Biotechnology, NKP-HM146) on days 0, 21, 42, and 63, for a total of four immunizations. After immunization, 50 mL of blood was collected from the alpacas, and 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 processed using Takara PrimeScript.TM cDNA was synthesized using the II reverse transcription kit. Using the cDNA as a template, nested PCR was performed with designed specific primers to amplify the VHH gene fragment. The VHH fragment was recovered and ligated into the pADL-23c phage vector after Sfi I restriction enzyme digestion. An NKp46 alpaca immunotherapy library was constructed using this vector and TG1 electroporated competent cells. The library was amplified and phages were assembled using M13K07 helper phage (NEB, N0315S). After cross-selection using human NKp46 ECD protein (Kaikai Biotechnology, NKP-HM146) and cynomolgus monkey NKp46 ECD protein (Kaikai Biotechnology, NKP-CM146), candidate clones were obtained. Positive clones were obtained after ELISA detection of human NKp46 ECD protein and cynomolgus monkey NKp46 ECD protein. The heavy chain antibody sequences of these positive clones were obtained by sequencing, and these sequences were used to construct human-alpaca chimeric antibodies. Using CHO-K1 cells highly expressing human NKp46 and CHO-K1 cells expressing NKp46 from cynomolgus monkeys, these chimeric antibodies were screened using FACS experiments, and the NKp46-PA27 antibody was ultimately determined to be the optimal one. This antibody was subsequently humanized and its sequence optimized to design an antibody clone H2-NKp46-PA27-M that binds to the NKp46 antigen. The amino acid sequences of its variable region and CDR are shown in Table 2. The binding of antibody clone H2-NKp46-PA27-M to the human NKp46-CHO-K1 cell line is shown in Figure 4A, and the binding of antibody clone H2-NKp46-PA27-M monoclonal antibody to the cynomolgus monkey NKp46-CHO-K1 cell line is shown in Figure 4B.

[0166] Table 2. Amino acid sequence of H2-NKp46-PA27-M antibody

[0167] Example 4: Construction of anti-CD70 and NKp46 bispecific antibodies and their transient transfection expression in eukaryotic cells

[0168] Based on the antibody structures of antibody clones 70CH-20-H4L3-M and H2-NKp46-PA27-M in Example 3, bispecific antibody 1 and bispecific antibody 2 were prepared according to the construction method of "bispecific antibody 1" and "bispecific antibody 2" shown in Figure 5.

[0169] Specifically, the gene fragments of bispecific antibody 1 and bispecific antibody 2 were cloned into the pTT5 expression vector to prepare transfection-level expression plasmids.

[0170] Expi293F cultured in serum-free medium TMCells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) 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 then purified using a Protein A column for affinity purification. 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). After appropriate concentration, the eluted sample was further purified using PBS-equilibrated Superdex 200 (GE) gel chromatography to remove aggregates. The monomer peak was collected, the buffer was changed to PBS, and aliquoted for use. The finally purified antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0171] Example 5: Cellular Affinity Assessment of Anti-CD70 and NKp46 Bispecific Antibodies

[0172] Flow cytometry was used to detect the binding of bispecific antibodies I and II to human CD70-CHO-K1 cells overexpressing human CD70, human NKp46-CHO-K1 cells overexpressing human NKp46, cynomolgus CD70-CHO-K1 cells overexpressing cynomolgus CD70, cynomolgus NKp46-CHO-K1 cells overexpressing cynomolgus NKp46, and MOLM-13 cells (CBP60678).

[0173] Human CD70-CHO-K1 cells, human NKp46-CHO-K1 cells, cynomolgus monkey CD70-CHO-K1 cells, cynomolgus monkey NKp46-CHO-K1 cells, and MOLM-13 cells were cultured. The culture medium for human CD70-CHO-K1 cells, human Kp46-CHO-K1 cells, cynomolgus monkey CD70-CHO-K1 cells, and cynomolgus monkey NKp46-CHO-K1 cells was F12K + 10% FBS + 400 μg / ml hygromycin B, while the culture medium for MOLM-13 cells was RPMI 1640 + 20% FBS. These cells were placed in T75 cell culture flasks and cultured in a 37°C, 5% CO2 incubator. When ready for use, MOLM-13 cells were directly transferred to 50 mL centrifuge tubes without digestion. Human CD70-CHO-K1 cells, human NKp46-CHO-K1 cells, cynomolgus monkey CD70-CHO-K1 cells, and cynomolgus monkey NKp46-CHO-K1 cells were digested with 0.25% trypsin-EDTA, followed by termination of digestion with F12K + 10% FBS + 400 μg / ml hygromycin B.

[0174] Centrifuge the obtained cells at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and resuspend the cells in 1% BSA (in PBS). Count the cells and adjust the cell density to 5E5 / mL, then plate them into 96-well round-bottom plates (corning, catalog number 3799), 100 μL / well. Centrifuge the cells 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 test antibody with 1% BSA (in PBS), starting at 100 nM, and then dilute 5-fold down to 8 concentrations. Resuspend the cells in the diluted antibody, 100 μL / well, and incubate at 4°C for 1 hour. Centrifuge the cells at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Cells were resuspended and washed with 200 μL of 1% BSA (in PBS), centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. Goat anti-human IgG Fc PE secondary antibody (Jackson, 109-115-098) was diluted 1:500 with 1% BSA (in PBS) according to the manufacturer's instructions. Cells were resuspended with the diluted secondary antibody at 100 μL / well and incubated at 4°C for 0.5 hours. Cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. Cells were resuspended and washed with 200 μL of 1% BSA (in PBS), centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. Cells were resuspended in 50 μL of 1% BSA (in PBS), and the average fluorescence intensity of the PE channel in each sample was detected by flow cytometry.

[0175] 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 binding abilities of bispecific antibodies 1 and 2 to various cell lines are shown in Figures 6A-6E and Table 3. The results showed that both bispecific antibodies 1 and 2 exhibited strong binding affinity to cell lines expressing human CD70 antigen and human NKp46 antigen, and also showed significant binding affinity to cell lines expressing cynomolgus monkey CD70 antigen and cynomolgus monkey NKp46 antigen.

[0176] Table 3. Results of binding of anti-CD70 and NKp46 bispecific antibodies to cells expressing tumor antigens.

[0177] Example 6: Protein level affinity assessment of anti-CD70 and NKp46 bispecific antibodies

[0178] This experiment used surface plasmon resonance (SPR) technology to characterize and analyze the affinity between bispecific antibodies 1 and 2 and human CD70 protein (ACRO, CDL-H52Da), cynomolgus monkey CD70 protein (Kaikai Biotechnology, CD7-CM170), human NKp46 protein (Kaikai Biotechnology, NKP-HM146), and cynomolgus monkey NKp46 protein (Kaikai Biotechnology, NKP-CM146).

[0179] Detection was performed using an indirect capture method with a CM5 chip (Cytiva, 29149603). The CM5 chip was first activated with EDC and NHS. Antihistamine (Cytiva, 29234602) was diluted to 10 μg / mL with 10 mM sodium acetate solution at pH 5, immobilized at a flow rate of 10 μL / min for 420 s, and then blocked with ethanolamine. Human CD70 protein, cynomolgus monkey CD70 protein, human NKp46 protein, and cynomolgus monkey NKp46 protein were diluted to 4 μg / mL with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer and captured at a flow rate of 10 μL / min for 60 s. The antibody molecules to be tested were serially diluted twofold to a series concentrations (50 nM - 0.195 nM) using HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer, bound at a flow rate of 50 μL / min for 90 s, and dissociated for 240 s. After each round of experiments, the cells were washed with pH 1.5 glycine solution at a flow rate of 30 μL / min for 60 s to remove antigen protein molecules, thus regenerating the chip. The raw data were evaluated using Biacore Insight software (3.0.12.15655) and fitted with a (1:1) Langmuir model. The obtained bispecific antibody affinity and kinetic experimental data are shown in Tables 4 and 5.

[0180] The experimental results showed that the binding affinity of the first molecule of the bispecific antibody to human CD70 and human NKp46 proteins reached the nanomolar level, with equilibrium dissociation constants (KD) of 2.32 nM and 6.84 nM, respectively. The equilibrium dissociation constants (KD) for the binding affinity of the first molecule of the bispecific antibody to cynomolgus monkey CD70 and NKp46 proteins were 1.55 nM and 13.8 nM, respectively. The affinity difference between the first molecule of the bispecific antibody and human and cynomolgus monkey antigens was within 3-fold. The binding affinity of the second molecule of the bispecific antibody to human CD70 and human NKp46 proteins reached the nanomolar level, with equilibrium dissociation constants (KD) of 2.31 nM and 6.05 nM, respectively. The equilibrium dissociation constants (KD) for the binding affinity of the second molecule of the bispecific antibody to cynomolgus monkey CD70 and NKp46 proteins were 1.6 nM and 11 nM, respectively. The affinity of the two bispecific antibodies for human and cynomolgus monkey antigens differed by less than 3 times.

[0181] Table 4. Binding affinity and kinetics of anti-CD70 and NKp46 bispecific antibodies to human and cynomolgus monkey CD70 protein

[0182] Table 5. Binding affinity and kinetics of anti-CD70 and anti-NKp46 bispecific antibodies to human and cynomolgus monkey NKp46 protein.

[0183] Example 7: Cytotoxicity assay mediated by anti-CD70 and NKp46 bispecific antibodies

[0184] THP-1 cells (human monocytic leukemia cell line, from ATCC, catalog number TIB-202) and MOLM-13 cells (human acute myeloid leukemia cell line, from CARBEN, catalog number CBP60678) were cultured. These cells were placed in T75 cell culture flasks and cultured at 37°C in a 5% CO2 incubator. Before use, cells were collected, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed once with DPBS. Cells were pre-stained for 20 minutes with 30 nM CellTrace far-erythrocyte proliferation kit (Invitrogen, C34572) dissolved in DPBS. Pre-staining was stopped with complete culture medium, and the cells were washed once, resuspended in fresh complete culture medium, and counted. The cell density was adjusted to 1.1E5 / mL, and the cells were seeded into 96-well cell culture plates (corning, 3903), 90 μL / well. Purchased fresh PBMC cells were centrifuged at 400g for 6 minutes, the supernatant was discarded, and the cells were resuspended in fresh RPMI 1640 medium with 10% FBS and counted. The cell density was adjusted to 3.3E6 / mL, and the cells were seeded into 96-well cell culture plates (pre-seeded with target cells) at 90 μL / well (E:T = 30:1). Antibody was prepared using RPMI 1640 blank medium at an initial concentration of 5 nM (final concentration), serially diluted 5-fold, and added to the cell culture plates at 20 μL / well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The cells were then transferred to new 96-well plates (corning 7007), centrifuged to remove the supernatant, washed once with 1% BSA and PBS, stained with DAPI (Thermo, 62248), filtered, and analyzed for cell viability by flow cytometry. Data in FCS3.0 format were exported from the flow cytometer. The FarRed positivity rate and DAPI positivity rate were analyzed using FlowJO. The tumor cell killing rate was calculated using the following formula.

[0185] Cell killing % = (FarRed) + DAPI + Cell ratio) / (FarRed) + Cell percentage) * 100%.

[0186] Import cell killing data into GraphPad Prism, plot cell killing / concentration curves, and calculate EC50. 50 Values. The results are shown in Table 6 and Figures 7A and 7B. Both bispecific antibody 1 and bispecific antibody 2 molecules were able to effectively mediate cytotoxic effects against tumor cells THP-1 and MOLM-13, and were both stronger than the anti-CD70 monoclonal antibody gustuzumab, which was used as a positive control.

[0187] Table 6. Cytotoxicity assays of THP-1 and MOLM-13 cells mediated by bispecific antibodies against CD70 and NKp46.

[0188] Example 8: IL-6 release level induced in anti-CD70 and NKp46 bispecific antibody cell experimental system

[0189] THP-1 cells (ATCC, catalog number TIB-202) were cultured in RPMI 1640 + 10% FBS. Cells were collected, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in fresh complete medium and counted. The cell density was adjusted to 1E5 / mL, and the cells were seeded into 96-well cell culture plates (corning-3799), 90 μL / well. Fresh PBMCs were purchased from Selene Biotech, centrifuged at 400g for 6 minutes, the supernatant was discarded, and the cells were resuspended in fresh RPMI 1640 + 10% FBS (inactivated) medium and counted. The PBMC density was adjusted to 2E6 / mL, and the cells were seeded into 96-well cell culture plates (pre-seeded with target cells), 90 μL / well. Antibodies were prepared using RPMI 1640 blank medium, with an initial concentration of 5 nM (final concentration), and serially diluted 5-fold. The prepared antibodies were added to the cell culture plates, 20 μL / well, and incubated at 37°C in a 5% CO2 incubator for 48 hours. Centrifuge cell culture plates at 1500 rpm and collect the supernatant for cytokine detection. Purchase the CBA kit from BD for multifactorial detection (Cat 551809) and detect IL-6 release according to the kit instructions.

[0190] Import the IL-6 release level data for each sample into GraphPad Prism, plot the IL-6 release level / concentration curve, and calculate the EC50. 50 The results are shown in Figure 8. Both bispecific antibody 1 and bispecific antibody 2 mediated low levels of IL-6 cytokine release, with IL-6 levels in both cases <50 pg / mL.

[0191] Example 9: Stability test of anti-CD70 and NKp46 bispecific antibody

[0192] This embodiment utilizes NanoDSF (micro-difference scanning fluorescence) to detect the thermostability of different antibodies in PBS (pH 7.4) buffer. Sample concentrations were approximately 1 mg / mL, and detection was performed using a Prometheus NT.Plex (nano DSF). Before detection, each sample was centrifuged at 10000g for 10 minutes. 40 μL of sample was added to each well of the sample plate (instrument loading volume was 10 μL, with one replicate for each sample). The scanning temperature ranged from 30℃ to 95℃, with a scan rate of 0.5℃ / min. The experimental results are shown in Table 7. Both bispecific antibodies exhibited good thermostability.

[0193] Table 7. NanoDSF detection results of anti-CD70 and NKp46 bispecific antibodies

[0194] Example 10: Pharmacokinetics of anti-CD70 and NKp46 bispecific antibodies

[0195] Three adult male cynomolgus monkeys (Suzhou Guochen) were administered the drug at a dose of 3 mg / kg, with an administration volume of 10 mL / kg, via intravenous infusion over 30 minutes. Blood was collected at the following times: before administration (0 h), 5 min (±1 min), 4 h (±5 min), 8 h (±10 min), 24 h (±30 min), 36 h (±30 min), 48 h (±30 min), 72 h (±30 min), 96 h (±30 min), 168 h (±1 h), 240 h (±1 h), 336 h (±1 h), 504 h (±1 h), and 672 h (±1 h). 1–2 ml of whole blood was collected from suitable sites such as the cephalic vein or saphenous vein of the cynomolgus monkeys. The whole blood was incubated at 4°C for 30 minutes, and the serum was collected by centrifugation at 1000 rpm for 15 minutes at 4°C. The serum was then immediately stored at -80°C. The concentration of intact CD70-NKp46 bispecific antibody molecules in serum was detected using the LBA method (DELFIA). 100 μL / well of 1 μg / mL goat anti-human IgG was used to coat the test plate with monkey serum adsorption-UNLB (Southern Biotech-2049-1), which was then refrigerated and allowed to stand overnight. The test plate was washed three times with PBST (0.05% Tween 20), 300 μL each time. 200 μL of casein (Pierce-37528) blocking buffer was added to each well, and the plate was incubated at 37°C for 1 h. The plate was then washed three times. Sample dilution buffer was prepared: the casein reagent was diluted 10-fold with 1x PBS, and 100 μL / well of the diluted sample used to generate the standard curve and serum samples at different time points (MRD = 50) were added. After incubation at 37°C for 1 hour, the test plate was washed 6 times, and 100 μL / well of goat anti-human IgG was added, followed by monkey serum adsorption-BIOT (1:5000, Southern Biotech-2049-08). After incubation at 37°C for 1 hour, the test plate was washed 6 times, and then 100 μL / well of EU-streptavidin (1:1000, PerkinElmer-1244-360) was added. After incubation at 37°C for 1 hour, the test plate was washed 6 times, and finally 100 μL / well of DELFIA enhancement solution (PerkinElmer-4001-0010) was added. After incubation at room temperature for 30 minutes, the fluorescence signal at 620 nm was read using the DELFIA module (laser excitation) on an EnVision microplate reader. The monkey serum concentration of the bispecific antibody was calculated using WinNonlin statistical software and a non-compartmental model method. The pharmacokinetic parameters of the bispecific antibody are shown in Table 8.The results showed that the half-life of bispecific antibody 1 in monkeys was 44.8±19.1hr, and the Cmax was 83.1±10.1μg / mL. The half-life of bispecific antibody 2 in monkeys was 53.2±23.7hr, and the Cmax was 96±21.4μg / mL. This indicates that bispecific antibody 1 and bispecific antibody 2 have good pharmacokinetic properties in monkeys and are consistent with the conventional metabolic characteristics of macromolecules.

[0196] Table 8. Serum drug concentrations and p-values ​​of anti-CD70 and NKp46 bispecific antibodies in monkeys.

[0197] The concentration units in the table above are all in μg / mL, and BLQ indicates below the detection limit.

[0198] Example 11: In vivo efficacy experiment of anti-CD70 and NKp46 bispecific antibody

[0199] Human-derived immune system reconstructed mice (huHSC-NCG-IL2, a model of NCG mice reconstructing the human immune system by transplanting human hematopoietic stem cells into severely immunodeficient NCG mice, exhibiting activated T cells, B cells, and NK cells) were provided by Jicui Pharmaceutical. Logarithmically growing Raji-luc cells (a human Burkitt lymphoma cell line expressing luciferase) were collected, washed twice with DPBS, and injected via tail vein into huHSC-NCG-hIL2 mice 6 weeks after immune reconstruction, at a seeding density of 5 × 10⁶ cells / mL. 5 100 μL / mouse (without matrix gel). On day 2 (D2) after tumor cell inoculation, mice were randomly assigned to six groups of eight mice each, based on tumor photon count and immune reconstitution level (hCD45 × hCD56): isotype control 10 mpk, bispecific antibody-1 3 mpk, bispecific antibody-1 10 mpk, bispecific antibody-2 3 mpk, bispecific antibody-2 10 mpk, and goutuzumab 10 mpk. Grouping was on day 0 (D0). Drug administration began on D0; administration dates were D0, D2, D4, D7, D9, and D11; the drug volume was 10 μL / g × mouse body weight (g). After grouping, tumor photon counts were measured by in vivo imaging on D0 and D13. The TGI was calculated based on the tumor photon count using the following formula. TV :

[0200] The mean RTV item is the average RTV of the administration group, and the mean RTV vehicle is the average RTV of the vehicle group.

[0201] RTV is calculated using the following formula:

[0202] Among them, V nt: The number of tumor photons in mouse number n on day t;

[0203] V n0 : The number of tumor photons in mouse number n on day 0;

[0204] RTV n : The relative number of tumor photons in mouse number n on day t.

[0205] Experimental results are expressed as mean plus standard error. Independent samples t-tests were used to compare the tumor photon counts in different treatment groups on day 13 with the control group to determine if there was a significant difference. P < 0.001 was considered statistically significant and marked with an asterisk (*). Data were analyzed using GraphPad Prism 8.0.2. Experimental results are shown in Figure 9.

[0206] As shown in Figure 9, after six administrations to the Raji-luc cell pharmacodynamic model via tail vein transplantation in huHSC-NCG-hIL2 mice, both bispecific antibodies 1 and 2 significantly inhibited tumor photon counts at both dosage conditions (10 mpk and 3 mpk), demonstrating a significant inhibitory effect on tumor growth (p<0.001). Compared with the positive control anti-CD70 monoclonal antibody goutuzumab (defucosylated form), bispecific antibodies 1 and 2 exhibited better tumor-suppressive effects.

[0207] References

[0208] [1] Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991).

[0209] [2]A1-Lazikani, et al., J. Mol. Biol. 273:927-948 (1997).

[0210] [3]Martin, et al., Proc.Natl.Acad.Sci.USA86:9268-9272(1989).

[0211] [4] "An efficient route to human bispecific IgG", A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998.

[0212] [5]Wu C, Ying H, Grinnell C, et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol. 2007Nov.

[0213] [6]Viet Khong Nguyen et al.,Camel heavy-chain antibodies:diverse germline V H H and specific mechanisms enlarge the antigen-binding repertoire. EMBO J.2000Mar 1;19(5):921-30.

[0214] [7]S Muyldermans, Singledomain camel antibodies:current status.J Biotechnol.2001Jun;74(4):277-302.

[0215] [8]Peter Vanlandschoot et al., new ammunition to battle viruses.Antiviral Res.2011Dec;92(3):389-407.

[0216] Other sequence information

[0217] SEQ ID NO:1

[0218] Note: The bolded and double-lined parts are extracellular regions; the underlined parts are intracellular regions; the italicized parts are transmembrane regions.

[0219] SEQ ID NO:2

[0220] Note: The bolded and double-lined parts are extracellular regions; the underlined parts are intracellular regions; the italicized parts are transmembrane regions.

[0221] SEQ ID NO:3

[0222] Note: The bolded area with double horizontal lines is the extracellular region; the wavy line area is the transmembrane region; the underlined area is the intracellular region.

[0223] SEQ ID NO:4

[0224] Note: The bolded area with double horizontal lines is the extracellular region; the wavy line area is the transmembrane region; the underlined area is the intracellular region.

[0225] SEQ ID NO:5

[0226] SEQ ID NO:6

[0227] SEQ ID NO:7

[0228] SEQ ID NO:8

[0229] SEQ ID NO:9

[0230] SEQ ID NO:10

[0231] SEQ ID NO:11

[0232] SEQ ID NO:12

[0233] SEQ ID NO:13

[0234] SEQ ID NO:14

[0235] SEQ ID NO:15

[0236] SEQ ID NO:16

[0237] SEQ ID NO:17

[0238] SEQ ID NO:18

[0239] SEQ ID NO:19

[0240] SEQ ID NO:20

[0241] SEQ ID NO:21

[0242] All patents, patent application publications, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. Exemplary embodiments of the inventions described above have been described; however, those skilled in the art can modify or improve the exemplary embodiments described herein without departing from the spirit and scope of this application, and such variations or equivalents also fall within the scope of this application.

Claims

1. A bispecific antibody against CD70 and NKp46, said bispecific antibody comprising a first antigen-binding region for binding CD70 and a second antigen-binding region for binding NKp46.

2. The bispecific antibody of claim 1, wherein the first antigen-binding region and the second antigen-binding region are independently in Fab form, VHH form or single-chain antibody (scFv) form, preferably, the first antigen-binding region is in Fab form and the second antigen-binding region is in VHH form; Optionally, the bispecific antibody further comprises an Fc fragment, and the first antigen-binding region and the second antigen-binding region are linked to the Fc fragment.

3. The bispecific antibody as described in claim 1 or 2, wherein... The first antigen-binding region comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:12), HCDR2 (as shown in SEQ ID NO:13), and HCDR3 (as shown in SEQ ID NO:14), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:15), LCDR2 (as shown in SEQ ID NO:16), and LCDR3 (as shown in SEQ ID NO:17); and / or The second antigen-binding region is in the form of VHH and includes a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:19, HCDR2 as shown in SEQ ID NO:20, and HCDR3 as shown in SEQ ID NO:

21. in, The amino acid sequences of HCDR and LCDR are defined according to Kabat.

4. The bispecific antibody according to any one of claims 1-3, wherein the amino acid sequence of the heavy chain variable region of the first antigen-binding region has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:10, or the amino acid sequence of the heavy chain variable region of the first antigen-binding region is as shown in SEQ ID NO:10; and / or the amino acid sequence of the light chain variable region of the first antigen-binding region has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:11, or the amino acid sequence of the light chain variable region of the first antigen-binding region is as shown in SEQ ID NO:11; and / or The amino acid sequence of the heavy chain variable region of the second antigen-binding region has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:18, or the amino acid sequence of the heavy chain variable region of the second antigen-binding region is as shown in SEQ ID NO:

18.

5. The bispecific antibody according to any one of claims 1-4, wherein The first antigen-binding region and the second antigen-binding region are connected to the Fc fragment of the antibody heavy chain constant region, wherein the Fc fragment of the antibody heavy chain constant region includes a first Fc fragment and a second Fc fragment; Preferably, the Fc fragment of the antibody heavy chain constant region is the Fc fragment of the IgG1 subtype; and / or One of the first Fc fragment and the second Fc fragment is connected to the first antigen-binding region, and the other of the first Fc fragment and the second Fc fragment is connected to the second antigen-binding region, or both the first antigen-binding region and the second antigen-binding region are connected to one of the first Fc fragment and the second Fc fragment. More preferably, the first antigen-binding region and the second antigen-binding region are respectively connected to the N-terminus or C-terminus of the first Fc fragment or the second Fc fragment.

6. The bispecific antibody according to any one of claims 1-5, wherein the bispecific antibody comprises a first Fc fragment and a second Fc fragment, the first antigen-binding region being linked to the first Fc fragment, preferably linked to the N-terminus of the first Fc fragment; and the second antigen-binding region being linked to the second Fc fragment, preferably linked to the N-terminus of the second Fc fragment.

7. The bispecific antibody according to any one of claims 1-5, wherein the bispecific antibody comprises a first Fc fragment and a second Fc fragment, and the first antigen-binding region and the second antigen-binding region are respectively connected to one end of the first Fc fragment; Preferably, the first antigen-binding region is connected to the N-terminus of the first Fc fragment, and the second antigen-binding region is connected to the C-terminus of the first Fc fragment.

8. The bispecific antibody according to any one of claims 5-7, wherein The first Fc segment contains a 354-bit S354C substitution and a 366-bit T366W substitution; and / or The second Fc segment contains a 349-bit Y349C substitution, a 366-bit T366S substitution, a 368-bit L368A substitution, and a 407-bit Y407V substitution. Preferably, the second Fc segment also contains a 435-bit H435R substitution. in, The amino acid positions in the antibody constant region are determined according to EU numbering.

9. The bispecific antibody according to any one of claims 1-8, wherein the bispecific antibody is capable of binding to human and / or cynomolgus monkey NKp46 protein and / or human and / or cynomolgus monkey CD70 protein; preferably, the bispecific antibody is capable of binding to human NKp46 protein as shown in SEQ ID NO:1, cynomolgus monkey NKp46 protein as shown in SEQ ID NO:2, human CD70 protein as shown in SEQ ID NO:3 and / or cynomolgus monkey CD70 protein as shown in SEQ ID NO:4; Preferably, the bispecific antibody is capable of binding to the extracellular region of the human and / or cynomolgus monkey NKp46 protein and / or the extracellular region of the human and / or cynomolgus monkey CD70 protein; more preferably, the bispecific antibody is capable of binding to the extracellular regions of the following proteins: human NKp46 protein as shown in SEQ ID NO:1, cynomolgus monkey NKp46 protein as shown in SEQ ID NO:2, human CD70 protein as shown in SEQ ID NO:3, and / or cynomolgus monkey CD70 protein as shown in SEQ ID NO:

4.

10. A nucleic acid molecule encoding a bispecific antibody as described in any one of claims 1-9.

11. A vector comprising the nucleic acid molecule of claim 10, preferably an expression vector.

12. The vector of claim 11, wherein the vector further comprises a regulatory sequence, and the nucleic acid molecule is operatively linked to the regulatory sequence.

13. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11 or 12.

14. A method for preparing the bispecific antibody according to any one of claims 1-9, comprising culturing the host cells of claim 13, and optionally comprising recovering the bispecific antibody from the culture.

15. Use of the bispecific antibody according to any one of claims 1-9 in the preparation of a medicament for the prevention or treatment of CD70-positive tumors, preferably the CD70-positive tumors being selected from acute myeloid leukemia, monocytic leukemia, and Burkitt lymphoma.

16. The bispecific antibody according to any one of claims 1-9, for the prevention or treatment of CD70-positive tumors, preferably the CD70-positive tumors are selected from acute myeloid leukemia, monocytic leukemia and Burkitt lymphoma.

17. A method for preventing or treating CD70-positive tumors in a patient, the method comprising administering to the patient an effective amount of the bispecific antibody according to any one of claims 1-9, preferably the CD70-positive tumor being selected from acute myeloid leukemia, monocytic leukemia, and Burkitt lymphoma.

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